BioMed Notes

Would you like some E. coli with that?

2012-01-22T11:37:00.006-05:00

An epidemic of bloody stools and failing kidneys, some with hemolytic uremic syndrome (HUS) appeared in Germany in May 2011 and subsequently 15 other countries. By late July when the epidemic had subsided, a total of 3,816 cases - including 54 deaths - were reported in Germany, 845 of which included HUS. Rasko and colleagues cultured E. coli bacteria isolated from a 64 year old woman from Hamburg, Germany, who did not develop HUS. They characterized this bacterium, designated C227-11, as enteroaggregative, which means a gut pathogen that aggregates and forms “biofilms” that are resistant to treatment.

They sequenced the bacterium’s genome and found it was a unique strain of the O104:H4 serotype of E. coli bacteria, distinguished by possession of a prophage (http://en.wikipedia.org/wiki/Prophage ) producing the Shiga toxin. Shiga toxin binds to cells, inhibits protein synthesis, and kills by inducing apoptosis [review]. The O104 serotype is rare; the most frequent cause of HUS worldwide is the shiga-toxin–producing E. coli O157 (Tarr 2005).
Although they isolate only one strain themselves, they analyzed also 3 additional sequences from the current outbreak that had been made public (that’s data mine-ing!) together with 7 other O104:H4 serotype isolates, all from Africa, and 4 other reference strains. The authors conclude that the outbreak was caused by a difficult (enteroaggregative) strain made more virulent by its acquisition of the Shiga toxin gene in addition to antibiotic-resistance and “additional virulence and antibiotic-resistance factors”. Rohde and colleagues reached the same conclusion using "rapid, bench-top DNA sequencing technology, open-source data release, and prompt crowd-sourced analyses".

Where did the E. coli O104:H4 come from? A subsequent publication reported the results of trace-back and –forward investigations by Buchholz and colleagues who analyzed 26 HUS patients and 81 healthy controls. They concluded that despite only about a quarter of the patients recalling in exploratory interviews having eaten bean sprouts during the 14 days before the onset of illness, 100% of these illnesses were attributable to the consumption of sprouts – and not other raw foods such as tomatoes or cucumbers or lettuce – at a particular restaurant, and for other patients, sprouts obtained from a single, common supplier (figure).

N Engl J Med. 2011 Aug 25;365(8):709-17. Origins of the E. coli strain causing an outbreak of hemolytic-uremic syndrome in Germany. Rasko DA, Webster DR, Sahl JW, Bashir A, Boisen N, Scheutz F, Paxinos EE, Sebra R, Chin CS, Iliopoulos D, Klammer A, Peluso P, Lee L, Kislyuk AO, Bullard J, Kasarskis A, Wang S, Eid J, Rank D, Redman JC, Steyert SR, Frimodt-Møller J, Struve C, Petersen AM, Krogfelt KA, Nataro JP, Schadt EE, Waldor MK.

Helpful mutants usually won’t cooperate with each other

2011-08-04T19:08:00.004-05:00

How genes interact – the phenomenon termed “epistasis” – is complex, yet must be understood to accurately interpret the contribution of individual genes to the development and behavior of the organism. Two groups -- Chou et al. and Khan et al. -- recently reported in Science the results of their remarkably similar experiments; they isolated individual, beneficial mutations in bacteria then tested how the mutations interact when possessed by the same bacterium.

Previous studies (cited by Chou) suggested that two deleterious mutations in the same pathway are generally less-than-additive but were greater-than-additive when they were in parallel pathways, which seems intuitive. Interactions between mutations in single genes were shown to depend on the background (other genes). However, epistasis among beneficial mutations in different genes was “unexplored”, and the focus of these new experiments. Evolution in laboratory conditions is initially rapid but quickly slows, which fits a model of mutually antagonistic beneficial mutations. Khan states the deceleration is due to either (1) negative epistasis or (2) because the most beneficial would “tend to be incorporated earlier owing to their faster spread and greater success in the face of competing beneficial mutations” (which sounds suspiciously convenient – the best arrive early – but they give a reference: Gerrish 1998 so you can look it up).

Khan grew E. coli with a glucose supplement for 20,000 generations, when they sequenced a clone and identified 45 mutations. They say other beneficial mutations arose but were lost due to “interference” with more-beneficial mutations (24, 26) (which seems to answer their question), or because they were “less able to evolve than the eventual winners” (33). They took the first 5 “that fixed … and whose spread coincided with the period of fastest adaptation” (arbitrary? How many generations was that, 200? 2,000?), which were, in order of appearance: rbs operon, topA, spoT, glmUS promoter, and pykF. Together, these 5 mutations increased fitness ~30%, accounting for ~80% of the fitness increase over the full 20,000 generations.

Then they produced 32 populations of E. coli, one for each possible combination of the 5 genes (Fig. 1 from Khan, copied here, shows the ancestral genotype at the top and the 32 combinations of mutations, with increasing fitness downward). They found that although each combination improved fitness, improvement was less than expected from a “multiplicative null model” (in which the individual fitness effects are multiplied).

Similarly, Chou selected a bacterium to grow with methanol as its sole carbon source and then replaced a key metabolic pathway with a less efficient pathway from a different bacterium. The resulting bacterium grew only one-third as fast as the original. Eight separate populations improved the efficiency over 600 generations. In the fittest strain, 9 mutations were identified; 3 were clearly related to the pathway while 6 others were deemed “unlikely” to contribute to fitness. To determine the interactions among these 3 genes, they constructed 16 strains, one with each combinations of these 3 mutations plus the WT allele. They also observed that combination strains were much less fit than expected if they were acting independently (our old favorite, the multiplicative null model).

These findings should influence our expectations in genetic studies in humans; e.g., two disease-risk alleles may have little additive or even negative effects (ergo protective?), contradicting the simple expectations of most biologists. [Chou distinguished beneficial from detrimental effects.] I wonder if these experiments might be misleading because they first fixed the individual mutants, thereby eliminating mutants that interact positively during selection.

Khan AI, Dinh DM, Schneider D, Lenski RE, Cooper TF."Negative epistasis between beneficial mutations in an evolving bacterial population. " Science. 2011 Jun 3;332(6034):1193-6.

The same issue has a commentary.

Kidney disease linked to putative autoantigen + HLA

2011-05-19T16:49:00.008-05:00

Idiopathic membranous nephropathy is a progressive disease involving the thickening of the basement membranes in glomeruli, which are key blood filtration units in the kidneys. Membranous nephropathy can be caused by exposure to toxins (gold, mercury, some medicines) or autoimmunity, such as lupus. Deposits of antibody-antigen (immune) complexes with complement components can be observed in the glomerulus.

These authors sought genetic associations in 556 biopsy-proven patients (British, French, & Dutch) by comparison of about 300,000 SNPs with matched healthy subjects. They found strong associations with a membrane protein previously implicated in autoimmunity, M-type phospholipase A2 receptor (PLA2R1, p~10E-28), and a histocompatibility gene (HLA-DQA1, p~10E-92). PLA2R1 is normally expressed in human glomeruli, exactly where immune complexes are found in membranous nephropathy patients. PLA2R1 was implicated only recently in autoantibody studies (Beck 2009) and is now known as the major autoantigen in idiopathic membranous nephropathy. The DQA1 association is not surprising, having been discovered by Vaughn et al using the relatively crude restriction fragment length polymorphism (RFLP) analysis and reported way back in 1989. The odds ratio for a single PLA2R1 risk allele is about 2 and for HLA-DQA1 about 6, modest but typical for such association studies. It is therefore astonishing that the risk to individuals possessing homozygous risk alleles at both loci is practically determinate – a 78.5-fold increased risk! – with 42 patients out of the 55 subjects possessing this combination.

How might this happen? The authors’ model is that perhaps the DQA1 molecule binds the PLA2R1 variant peptide and triggers T lymphocytes to help B cells make anti-PLA2R1 autoantibodies that bind to glomerular cells. They could have looked whether any PLA2R1 peptides has anchor residues that determine whether they can fit one of the 35 different allelic forms of DQA1. However, as Segelmark points out in the accompanying review, the strongest SNPs lie within the first introns for both PLA2R1 and DQA1 and (therefore) do not alter the amino acid sequence! The authors discount this, speculating that either the associated SNP is tightly linked to a variant that does change the protein. They could have sequenced the few subjects to identify any rare variant. Segelmark proposes as “more likely” that the SNP changes a regulatory sequence, such as a transcription factor binding site or a microRNA, that increases production of the proteins. A few more facts could help resolve these possibilities.
Risk HLA-DQA1 and PLA(2)R1 alleles in idiopathic membranous nephropathy. Stanescu HC, Arcos-Burgos M, Medlar A, Bockenhauer D, Kottgen A, Dragomirescu L, Voinescu C, Patel N, Pearce K, Hubank M, Stephens HA, Laundy V, Padmanabhan S, Zawadzka A, Hofstra JM, Coenen MJ, den Heijer M, Kiemeney LA, Bacq-Daian D, Stengel B, Powis SH, Brenchley P, Feehally J, Rees AJ, Debiec H, Wetzels JF, Ronco P, Mathieson PW, Kleta R. N Engl J Med. 2011 Feb 17;364(7):616-26.

HLA is the Major HIV Controller (MHC)

2010-11-28T20:57:00.007-05:00

Upon infection by HIV, there follows a massive but transient increase in viruses in the blood followed by a period of years during which CD4+ T lymphocytes slowly decline. However, after the initial spike, some people have far fewer viruses in their blood and their CD4 counts do not decline. They remain apparently healthy, asymptomatic, for a very long time. These people are called “controllers” because their immune system controls virus replication without medication. The authors of this paper – with an impressive number of collaborators – looked for genetic variations in the controllers that could underlie the differences.

A genome wide association study (GWAS) performed with 1, 974 controllers (cases) and 2,648 progressors revealed a strong link with the HLA (the real human MHC, or major histocompatibility complex) on the short arm of chromosome 6 (figure shown). A weaker correlation with a chemokine receptor CCR5delta32 polymorphism previously identified with HIV resistance was also identified. HLA association is not surprising because practically everything immunological is strongly influenced by the HLA. What the investigators did next, however, was novel, imaginative, and highly illuminating.

From the SNP data, they were able to impute the controllers’ HLA type. Of the hundreds of HLA alleles, controllers tended to possess a remarkably narrow number of particular HLA-B and, to a lesser degree, HLA-A and -C alleles. They identified HLA-B*57:01, B*27:05, B*14/Cw*08:02, B*52, A*25 as protective alleles, and B*35 and Cw*07 as risk alleles. Moreover, they identified specific amino acids within the peptide-binding cleft as key variables in HIV control. These HLA amino acids define the ability of a pathogen to stimulate the cellular immune response.

These results provide a solid, well understood albeit complex mechanistic understanding to HIV progression and control. The next step could identify those HIV peptides that are bound preferentially by the protective and risk allele proteins.

The Major Genetic Determinants of HIV-1 Control Affect HLA Class I Peptide Presentation. The International HIV Controllers Study. Science. 2010 Nov 4.

Why Lupus can be Unresponsive to Glucocorticoid Therapy

2010-10-07T19:02:00.006-05:00

Glucocoriticoids, including the natural hormone cortisol, are powerful immune suppressants. Synthetic glucocorticoids, such as dexamethasone and prednisone, are often taken by mouth to control autoimmune diseases. However, controlling the autoimmune disease systemic lupus erythematosus (SLE, lupus) often requires more aggressive treatments with high doses of more potent glucocorticoids, such as methylpredisolone, given intravenously.

Lupus is characterized by serum antibodies to nucleic acids (anti-nuclear antibodies, ANA), by a pattern of interferon-alpha-induced genes transcribed ("IFN signature”), and by an increase in plasmacytoid dendritic cells (PDC). PDC help produce antibodies and IFN. The IFN signature and PDC levels in patients are reduced to normal levels by high doses of intravenous glucocorticoids (see Figure 1c).

The authors noted that serum nucleic acids, which trigger the production of ANA, also stimulate PDC though specific receptors called toll-like receptors-7 and -9 (TLR-7 & -9) expressed on many cells of the immune system. Further, they hypothesized that TLR stimulation renders the PDC resistant to the suppressive effects of glucocorticoids.

Indeed, purified PDC treated with glucocorticoids (1-10 uM) do not survive overnight in a flask unless they are also treated with a nucleic acid (CpG) that stimulates TLRs (Figure 2a, first panel shown here). Nucleic acid-mediated protection is partially reversed by IRS, a synthetic oligonucleotide inhibitor of TLRs (IRS 954). Nucleic acid-containing immune complexes, isolated from the sera of lupus patients, also protected PDC by triggering TLRs.

Similar results were obtained with PDC from healthy people, suggesting that the PDC of lupus patients are not different but instead PDC are made glucocorticoid resistant by chronic stimulation of TLRs by nucleic acids. The authors conclude that “inhibitors of TLR7 and 9 signaling could prove to be effective corticosteroid-sparing drugs.”

Guiducci C, Gong M, Xu Z, Gill M, Chaussabel D, Meeker T, Chan JH, Wright T, Punaro M, Bolland S, Soumelis V, Banchereau J, Coffman RL, Pascual V, Barrat FJ. “TLR recognition of self nucleic acids hampers glucocorticoid activity in lupus.” Nature. 2010 Jun 17;465(7300):937-41.

Note: Similar results were reported by Lepelletier and colleagues at the Hopital Necker.

Old news: Targeting the Target of Rapamycin (TOR) to Stay Youngish

2010-08-04T16:13:00.002-05:00

There are many strategies to remain healthy and reduce the effects of aging: healthy diet and exercise, yoga, drastically reducing calories, etc... all disappointingly slow and indirect. On the other hand, simply feeding mice the anti-rejection drug rapamycin has been shown to dramatically increase both the average and maximum life span, even when started late in life, probably by postponing death from cancer, by retarding mechanisms of aging, or both. Unfortunately, rapamycin also strongly suppresses the immune system, increasing the risk of a dangerous infection. How can we derive the benefits of rapamycin while avoiding the risks?
An endogenous inhibitor of the “Target Of Rapamycin” (TOR), and thus a candidate for the physiological rapamycin-like agent, are sestrins (Sesns), which are conserved proteins that are induced by cellular stress including by elevated adenosine monophosphate kinase (AMPK) or DNA damage.

Drosophila (fruit flies) have a single form of sestrin (dSesn). To define the activities of dSesn, these authors created flies with either gain- or loss-of-function dSesn mutations. Clever sleuthing with agonists and employing other mutant signaling molecules showed that TOR induces dSesn through reactive oxygen species (ROS, quenched by antioxidants such as vitamin E), jun-N-terminal kinase (JNK), and a transcription factor (FoxO). p53 was implicated but narrowly escaped the round-up of usual suspects. dSesn repaid TOR's attention by suppressing its stimulation of tissue growth. Thus dSesn is induced by TOR and suppress TOR activities, constituting a feedback inhibition system.
These authors go on to show that flies lacking dSesn suffer multiple age-related pathologies, including muscle degeneration and heart malfunction. These pathologies are prevented by activating AMPK with AICAR (5-aminoimidazole-4-carboxamide 1-b-D-ribofuranoside) or inhibiting TOR with rapamycin (Figure 5C shows that heart arrythmias suffered by dSesn-deficient flies are treated by feeding them AICAR, rapamycin, or vitamin E).

Forget caloric restriction, I'll take my ice cream with a statin mixin and Rapa sprinkles. Or a burger with heaping side order of sestrins.

JLee JH, Budanov AV, Park EJ, Birse R, Kim TE, Perkins GA, Ocorr K, Ellisman MH, Bodmer R, Bier E, Karin M. “Sestrin as a feedback inhibitor of TOR that prevents age-related pathologies” Science 2010 Mar 5;327(5970):1210-1.

Cholesterol-retentive Leukocytes

2010-07-19T19:45:00.008-05:00

Elevated white cells in the bloodstream (leukocytosis) was correlated with cardiovascular disease and atherosclerosis nearly a century ago but the cause remains unclear. Elevated high-density lipoprotein (HDL), in contrast, is correlated with protection from cardiovascular disease, in part because HDL carries cholesterol away from macrophages in atherosclerotic plaques. Here, Yvan-Charvet and colleagues report that leukocytosis develops in mice lacking the membrane proteins ABCA1 and ABCG1 that normally transport cholesterol out of macrophages to lipoproteins. These ABCA1/G1-deficient mice suffer a myeloproliferative disorder and display an expansion of particular blood-forming (hematopoietic) stem cells. Although lymphoid (B, T, NK cells) precursor cells were unchanged, myeloid precursor cells, which give rise to granulocytes, macrophages, etc, were doubled. Similar findings in MyD88-knockout mice ruled out innate inflammation as a cause of leukocytosis. Transplantation of bone marrow from ABCA1/G1 transporter-deficient mice into (apoA1-transgenic) mice with elevated HDL blocked or slowed the development of leukocytosis, myeloproliferation, the particular stem cell population, and atherosclerosis. The figure shows that elevated HDL in apoA1-tg mice protected against heart disease (Fig 4B, left side shows the entire hearts and, right side, tissue sections from hosts transplanted with ABCA1/G1-deficient bone marrow; upper panel: diseased heart from a normal recipient mouse showing leukocyte infiltration and, lower panel, healthy heart from a HDL-elevated recipient).

The authors state that “these results suggest that HDL suppresses the proliferation of myeloid progenitor cells by promoting cholesterol efflux”. Thus, these transporters (intrinsically) or elevated HDL (extrinsically) can regulate hematopoiesis and atherosclerosis. They also reported that stimulating ABCA1/G1 expression above normal levels using a transcriptional activator (TO901317) increased cholesterol efflux and suppressed myeloid cell proliferation, suggesting a new therapeutic rationale.

Statins, such as atorvastatin (Lipitor) and rosuvastatin (Crestor), were designed and are prescribed to reduce serum cholesterol, which is associated with cardiovascular disease. Surprisingly, statins were also found to be anti-inflammatory. Could these results help explain statins' anti-inflammatory effects?

Laurent Yvan-Charvet, Tamara Pagler, Emmanuel L. Gautier, Serine Avagyan, Read L. Siry, Seongah Han, Carrie L. Welch, Nan Wang, Gwendalyn J. Randolph, Hans W. Snoeck, Alan R. Tall, "ATP-Binding Cassette Transporters and HDL Suppress Hematopoietic Stem Cell Proliferation" Science 25 June 2010: Vol. 328. no. 5986, pp. 1689 - 1693

Rapamycin-induced Stress Stimulates anti-Tuberculosis Immunity

2010-03-26T20:07:00.008-05:00

Tuberculosis (Tb) is an intracellular mycobacterium that causes 9 million illnesses and nearly 2 million deaths annually world-wide. One third of humans are chronically infected and about 9 million are infected every year, which a vaccine could prevent.

Stressing cells infected by Tb has been shown to induce autophagy ("self-eating", free review), increase presentation of Tb antigens on the infected cell surface, and stimulate specific T lymphocytes. This group found that rapamycin, a pharmaceutical drug used to suppress immunity after solid organ transplantation, induces autophagy and improves immune recognition of Tb. This figure shows that rapamycin treatment of dendritic cells causes a >10-fold increase in their ability to induce anti-Tb immunity in mice (Fig. 5e). Most of the cell culture experiments aimed at dissecting this effect show a much more modest effect of extremely high doses of rapamycin (1 mM!). Oddly, many of the early figures measure IL-2, a hallmark of the T lymphocyte response, even though rapamycin's mode of action inhibiting IL-2 activity would seem to confound the interpretation of the results.

Could this adjuvant effect be translated into a vaccine strategy?

Jagannath C, Lindsey DR, Dhandayuthapani S, Xu Y, Hunter RL Jr, Eissa NT. Autophagy enhances the efficacy of BCG vaccine by increasing peptide presentation in mouse dendritic cells. Nat Med. 2009 Mar;15(3):267-76.

Infectious Fatigue

2009-10-30T19:06:00.007-05:00

Between 1 and 4 million Americans suffer from Chronic Fatigue Syndrome (CFS), which is a “complicated disorder characterized by extreme fatigue that may worsen with physical or mental activity, but doesn't improve with rest.” (Mayo Clinic). The cause is unknown and there is no known cure. An infectious cause has been suspected and sought for decades without success. The recent discovery of XMRV, new retrovirus found in some prostate cancer patients (but not others) prompted Lombardi and colleagues to test for its involvement with CFS. They performed PCR for XMRV gag, which encodes structural viral proteins, on peripheral blood mononuclear cells from CFS patients in a repository at their Whittemore Peterson Institute. Of 101 CFS samples tested, 68 (67%) were positive in contrast to only 8 out of 218 samples (4%) from healthy donors. Another viral gene, env, was also detected in most CFS patients positive for gag as were the proteins encoded by these genes. Oddly, patient sample WPI-1118 is negative for gag and env (Fig 1) but weakly positive by cytometry (Fig 2 A & D) and clearly positive by Western protein blot (Fig 4 A). Both B and T lymphocytes express XMRV proteins. And, for what it's worth, some CFS patient cells make virus that can productively infect other cells (shown, from Figure 3 B & C, electron micrographs of budding virus particles).

The authors note in their introduction that “patients with CFS often have active β herpesvirus infections, suggesting an underlying immune deficiency”. This increases the odds that XMRV is an opportunistic infection. The authors return to this question in their closing discussion, asking “Is XMRV infection a causal factor in the pathogenesis of CFS or a passenger virus in the immunosuppressed CFS patient population?” Stand by.

Lombardi VC, Ruscetti FW, Das Gupta J, Pfost MA, Hagen KS, Peterson DL, Ruscetti SK, Bagni RK, Petrow-Sadowski C, Gold B, Dean M, Silverman RH, Mikovits JA. "Detection of an Infectious Retrovirus, XMRV, in Blood Cells of Patients with Chronic Fatigue Syndrome". Science. 2009 Oct 8.

Infecting the Protectors (fluNK)

2009-10-07T19:37:00.009-05:00

Influenza (flu) is typically transmitted through the air, infecting lung epithelial cells. Natural Killer (NK) cells are lymphocytes that help protect from flu, especially early after infection, by killing infected cells and secreting cytokines and chemokines that inhibit virus replication. Here, Guo and colleagues ask whether NK might themselves be infected by flu. They confirmed that NK express on their surface sialic acids, which serve as flu receptors by binding hemagluttinin (HA). By microscopy, they examined mice infected with PR8, a strain originating from the human flu virus A/PR/8/34 and detected flu virus protein M2 inside of NK cells within the lungs of infected mice. NK possess several activating receptors including NCR1, NKG2D, Ly49D and inhibiting receptors including NKG2A, Ly49A. Flow cytometry demonstrated that PR8 did not modulate these receptors or developmental markers.
However, infection did alter the effector functions of NK cells. Infected NK cells were less capable of killing cells from 3 target lines, cells susceptible because they either express ligands for NK activating receptors or fail to express ligands for NK inhibiting receptors (Figure 5, shown). EL4 cells express a transfected H60, which binds the activating receptor NKG2D; YAC-1 cells express H60 constitutively, and RMA-S cells express very little HLA class I protein, which binds the inhibitory Ly49 receptors. The authors and others had previously shown that the 85 kDa subunit of phosphotidyl inositol 3-kinase (PI3K-p85) was critical for NK effector functions, including killing and production of cytokines/chemokines, and that flu NS1 protein interacted with this kinase. Surprisingly, a PR8 virus with a mutated NS1 protein was even more effective in suppressing NK effector functions, leaving the mechanism of inhibition to be determined.
Guo H, Kumar P, Moran TM, Garcia-Sastre A, Zhou Y, Malarkannan S. “The functional impairment of natural killer cells during influenza virus infection.” Immunol Cell Biol. 2009 Sep 1.

Overreacting to Infection is Dangerous to Your Health

2009-09-21T21:47:00.009-05:00

Contributed by DP "Following soon after a similar paper on anthrax pathogenesis, this paper suggests that the downregulation of a lymphocyte surface protein can single-handedly stem the destruction by the Ebola virus. The authors identified mice expressing about half (62%) the "wild type" levels of the leukocyte membrane tyrosine kinase CD45 are protected from the lethal effects of Ebola infections (compare CD45-62% to control 100% in the top panel), with an overall survival rate of 90% and a complete clearance of the virus 10 days after challenge. CD45-62% mice expressing CD45 without phosphatase activity did not survive viral challenge, highlighting the importance of CD45’s enzymatic function.

The proposed mechanism is via constitutively high levels of activated CD8 T cells and IFN-gamma because antibodies that kill CD8 cells or block IFN-gamma render CD45-62% mice susceptible to the virus (fig 4, panels B & C). Can this protection translate to other infectious pathogens? Can this information be used clinically? Should we worry about modulating surface CD45 or just give IFN-gamma to affected individuals? I like the former, if one can come up with inexpensive small molecules, rather than the huge expense for IFN-gamma. These molecules might be used at higher doses for GVHD." And what is the evolutionary value of having wt levels of CD45 if reduced levels are in fact protective?

Panchal RG, Bradfute SB, Peyser BD, Warfield KL, Ruthel G, Lane D, Kenny TA, Anderson AO, Raschke WC, Bavari S. Reduced levels of protein tyrosine phosphatase CD45 protect mice from the lethal effects of Ebola virus infection. Cell Host Microbe. 2009 Aug 20;6(2):162-73.

Th17 cells have issues with commitment

2009-04-06T10:04:00.005-05:00

Th17 cells, so named because they secrete the protein hormone interleukin-17 (IL-17), belong to a recently-described subset of helper T cells responsible for regulating helper subsets Th1 (cellular) and Th2 (allergic).

Naïve T cells develop into different T helper subsets depending upon culture conditions. Lee and colleagues used an IL-17F reporter mouse (IL17F promoter driving expression of a surface protein Thy1.1) to test the stability of the Th17 phenotype in serial cultures. First, they caused Th17 to develop from naïve CD4+ T cells (OT-II transgene receptors specific for ovalbumin (ova) + MHC class II) by treating them in culture with TGF-beta, IL-6, anti-IFNgamma, and anti-IL-4 in the presence of IL-12p40-deficient antigen-presenting cells (APC). (Culture systems don't get much more manipulated than that!) Then, the surviving cells were phenotyped or cultured with TGNbeta or with IL-23, which stimulates the development of Th1 cells.

Several other groups had reported that Th17 cells required IL-23 for pathogenic autoimmunity. Here, Lee et al. showed that TGFbeta, but not IL-23, is essential for maintaining Th17 commitment (IL-17F expression). However, even after two cultures under Th17 conditions, IL-12 added to subsequent cultures 3 and 4 drove Th1 development and expression of interferon-gamma (IFNg, on the x-axis of figure 2, shown below).

Moreover, this final conversion by Th17 required Th1 factors STAT4 and T-bet, suggesting that Th17 might constitute a prolonged adolescence for adult Th1 cells.

Immunity 30:92-107. “Late developmental plasticity in the T helper 17 lineage”. Lee YK, Turner H, Maynard CL, Oliver JR, Chen D, Elson CO, Weaver CT.

IRF4 etc Required for Th17 Development

2009-02-26T12:38:00.007-05:00

Interleukin-17 (IL-17A) is a proinflammatory protein hormone produced by activated T lymphocytes that binds to a ubiquitous low-affinity receptor IL17RA. A subset of T helper (Th) cells that produce IL17 were dubbed Th17 and shown to be induced by IL23 and suppressed by IFNg or IL4, which support the development of Th1 or Th2 cells, respectively.

Brüstle and colleagues tested the ability of lymphocytes to develop into Th17 cells by treating naïve (CD4+CD62L+) Th in culture for 3 days with anti-CD3 and anti-CD28 to stimulate Th0 differentiation. Some cultures also received (1) IL12 and anti-IL4 to stimulate Th1, or (2) IL4 and anti-IFNgamma to stimulate Th2, or (3) anti-IL4, anti-IFNgamma, TGFbeta, IL6, IL1beta, TNF, and IL23 to stimulate Th17. Cells were then restimulated and analyzed by intracellular staining. They found that IRF4-deficient cells differentiate normally into Th1 cells, contradicting an earlier report by the senior author [they attribute the difference to using specific-pathogen-free mice], but not into Th2 cells, as previously reported. They also found that IRF4-deficient cells did not differentiate into Th17 cells. IRF4 is from a family of transcription factors that are required for the production of interferons alpha and beta as well as innate immunity (TLR) signaling and T helper cell differentiation. Mixing cells from normal 'wild-type' (WT) and deficient mice (IRF4-/- also CD45.2+) demonstrated that the defect was intrinsic to the Th17 cell lineage and not due to suppression (figure 1c shown).
They made several other, interesting observations and pursue the IRF4 pathway, finding that the transcription factor RORgamma, also previously reported to be required for Th17 cells, is largely dependent on IRF4. They also showed that TGFbeta-induced FoxP3, a marker of regulatory T cells, is only weakly suppressed by IL6 in IRF4-deficient cells, suggesting that this alternative differentiation pathway may explain the failure of Th17 to develop.
Brüstle A, Heink S, Huber M, Rosenplänter C, Stadelmann C, Yu P, Arpaia E, Mak TW, Kamradt T, Lohoff M. “The development of inflammatory T(H)-17 cells requires interferon-regulatory factor 4” Nat Immunol. 2007 Sep;8(9):958-66

It’s what inside that counts for TLRs 7 & 9

2008-12-31T10:43:00.003-05:00

Toll-like receptors (TLRs) help mammalian immune systems protect against infections by binding to molecular patterns that are characteristic of many dangerous microbes. The location of TLR7 and TLR9 within the cell, not on the cell surface, appears crucial to their distinguishing viral from host nucleic acids and maintaining self-tolerance.

Ewald and colleagues determined how these TLRs ‘traffic’ within macrophages and dendritic cells, which are dedicated to picking up and destroying matter, including viruses. They found that these TLRs, after their synthesis in the endoplasmic reticulum, move to vesicles called endolysosomes where recently-internalized materials are degraded. There, a portion of the TLR within the lumen of the lysosome, the 'ectodomain', is cut and destroyed. Both the full-length and ectodomain-cleaved TLRs can bind nucleic acids but only the proteolysed (cleaved) TLR can activate downstream signaling by recruiting MyD88 (Figure 4e shown, anti-Flag-tagged-TLR binds to and co-precipitates HA-tagged TLR after stimulation with nucleic acid). The authors propose that TLR proteolysis and activation within the endolysosome serves to restrict its exposure to 'self' molecules and thereby reduce the likelihood of triggering autoimmunity.

Ewald SE, Lee BL, Lau L, Wickliffe KE, Shi GP, Chapman HA, Barton GM. "The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor." Nature. 2008 Dec 4;456(7222):658-62. Epub 2008 Sep 28.

Kill your ECTV!

2008-11-13T10:04:00.006-05:00

It’s a mystery why mice resist the Ectromelia poxvirus (ECTV) better than humans resist smallpox, a similar infection. Previous studies demonstrated a role for natural killer (NK) cells in ECTV resistance. Fang and colleagues show here that in addition to 'opportunity', actual NK killing with perforin is essential to the resolution. Many T cells armed with IFN-g were also found in the spleens of infected mice, suggesting that NK cells involve T cells in the murderous conspiracy.

The proximal motive for NK action is NKG2D (AKA KLRK1), which can be triggered by viruses that increase MICA expression. NK cell depletion or NKG2D blockade, but not the depletion of T cells, stop virus clearance (Figure 4B shown). Fang makes a good case for poxvirus clearance triggered by NKG2D/KLRK1 on NK cells.

Fang M, Lanier LL, Sigal LJ. A role for NKG2D in NK cell-mediated resistance to poxvirus disease. PLoS Pathog. 2008 Feb 8;4(2):e30

Genome Modules Track Disease

2008-10-07T21:17:00.004-05:00

The idea is alluring: take a little blood, measure some gene transcripts, and diagnose disease. The problems have been (1) identifying the genes that signal disease, and (2) overcoming natural and laboratory variations. Chaussabel and colleagues approached these problems with reliable microarray measurements of samples from 239 people and by identifying small sets, “modules”, of genes that are coordinately-expressed across a “wide range” of conditions. These modules are likely to be more reproducible than measurements of individual genes. The (seemingly arbitrary) conditions included juvenile idiopathic arthritis (47 patients), lupus (40), and type I diabetes (20), melanoma (39), immune-suppression after liver transplantation (37), and infections with E. coli (22), Staph aureus (18), and influenza (16). Genes from all 239 samples were clustered using a “K-means algorithm” with K=30 (which yields up to 30 groups) without regard to the magnitude of change in expression. The first round grouped all 8 conditions, the second round, 7, and the third, 6 (procedure). A total of nearly 5,000 transcripts in 28 modules were identified using the sample data. When the data were randomized, no modules were identified in 200 trial clusterings, suggesting that the modules reflect states of health and are not statistical artifacts. Modules range from 22 to 325 transcripts. Genes with known relationships, e.g. particular cell types or pathways, constitute about half the modules, underscoring the functional coherence.

Two examples of how health conditions change these 28 modules are shown: healthy vs. melanoma (top) and healthy vs. lupus (bottom) (from fig. 1B, red=overexpressed, blue=underexpressed). All 8 conditions are clearly distinct from healthy and distinguished from each other. The authors also identified 'biomarker' modules, e.g. M1.2 & M1.8 in melanoma or M1.7 & M3.1 in lupus in the examples shown, and made circular ('spider') graphs that can display several patients or one patient over a course of treatment.

In contrast to typical repository-biomarker analyses, very few patient samples were used to generate these modules. It will be crucial to see how they accommodate more, different samples. Also, I'm curious whether rational groupings of 'conditions', e.g., cancers or infections or autoimmune diseases, might further improve module definition. Finally, in addition to improving patient care, this information should provide invaluable insights into disease origin and progression.
Chaussabel et al. A modular analysis framework for blood genomics studies: application to systemic lupus erythematosus. Immunity. 2008 Jul;29(1):150-64.

Tolerating Aire - Out of the Thymus

2008-08-31T21:10:00.005-05:00

The immune system protects us against infections but it must also tolerate the many different proteins that constitute our bodies. T lymphocytes of the immune system that react against the body’s own proteins could trigger autoimmunity, so they are usually deleted during their maturation in the thymus. A protein called Aire (Autoimmune regulator) turns on expression in the thymus of nearly 2,000 genes that are otherwise expressed in only a few organs and tissues, thereby exposing new T cells to these proteins and avoiding autoimmune disease. Indeed, mutations in Aire result in a broad autoimmunity against many organs and tissues. Previously, Aire was thought by many to be expressed only in the thymus.

Gardner and colleagues detected Aire in rare stromal (non-lymphoid) cells they called extrathymic Aire-expressing cells (eTACs) within the spleen, lymph nodes, and Peyer’s patches (nodes associated with the gut). These cells are located between the T and B cell areas of these organs but are highly mobile. Surprisingly, Aire turned on different genes in eTACs than were turned on in the thymus, suggesting that Aire expression in eTACs is not a ‘safety net’ for T cells that accidentally escaped the thymus but rather acts to delete additional autoreactive T cells. In eTACS, Aire turned on fewer than 200 genes, but several of these are suspected to be involved in autoimmunity, including desmoglein 1a (in pemphigous foliaceus) and the N-methyl-D-aspartate receptor 2C (in lupus). In an animal model of type 1 diabetes, eTACs expressing glucose-6-phosphate-related protein deleted T cells specific for this protein and prevented onset of this autoimmune disease (Fig. 2f shown, open squares represent mice expressing glucose-6-phosphatase-related Adig in eTACs).

These findings suggest that manipulating eTACs is a promising approach to therapy for autoimmune disease. Comparing eTACs in healthy people and autoimmune patients might also help identify deficiencies that can be corrected to prevent or ameliorate disease.

Gardner JM, Devoss JJ, Friedman RS, Wong DJ, Tan YX, Zhou X, Johannes KP, Su MA, Chang HY, Krummel MF, Anderson MS. Deletional tolerance mediated by extrathymic Aire-expressing cells. Science. 2008 Aug 8;321(5890):843-7.

Homozygosity mapping: Autism in the family

2008-08-10T22:00:00.007-05:00

Most genes identified in genome-wide analyses contribute only modestly to disease risk or protection, with already low relative risks changed only ~20%, and taken together probably contribute to a minority of disease cases. Morrow and colleagues took a different approach, believing that analyzing individual families might reveal higher-risk genes for autism spectrum disorders (ASDs). They noted that offspring of first-cousins have twice as many neurological birth defects, including ASDs.

They also noted that a significant involvement of autosomal recessive genes would be 'signaled' by change in the male-to-female ratio (because the M:F ratio is typically 1:1 for autosomal recessive traits vs. male-dominant ASD). Indeed, among the 104 families recruited for the study, the M:F ratio was 2.6 for the offspring of the 88 consanguineous families (cousins) vs. 7.4 for the non-consanguineous.

They genotyped using arrays of SNPs and BACs, the latter validated for comparative genomic hybridization (CGH) detection of copy number variation (CNV) due to deletions, etc. Few de novo (not inherited) CNVs were detected. Large deletions on both DNA strands of the affected child (homozygous), and one strand of each parent (hemizygous), were found in 5 of the 78 consanguineous families but not among any of the other ~400 ASD cases or ~2,400 controls. In the figure (from Figure 1C), the SNPs spanning a large deletion in the AU-3100 pedigree is shown: the parents 3103 & 3104 (top and second) are hemizygous as is one child (3102, third down) but the other child, 3101 (bottom), who is homozygous for the deleted chromosome, has autism and seizures.

The investigators were surprised to find 3 genes linked to the 2 largest deletions were previously identified as being regulated by neural activity, and thus candidates for involvement with learning. Mutations in one of these genes, NHE9, were detected in non-consanguineous families and associated with additional neurological disorders (epilepsy) and learning delays (language acquisition).

EM Morrow et al. Identifying autism loci and genes by tracing recent shared ancestry. Science. 2008 Jul 11;321(5886):218-23.

Therapeutic Interruption of Protein Degradation

2008-06-26T20:38:00.010-05:00

Some antibodies that bind ‘self’ molecules (autoantibodies) can signal and even cause autoimmune diseases. Antibodies are produced by B cells, largely by a differentiated, typically short-lived variety called plasma cells (PC), which dedicate 10-20% of their protein-synthesis capacity to immunoglobulin. PC develop in the germinal centers of lymph nodes and spleen and then migrate to the bone marrow, where a subset endures. Treatments that target B cells, such as Rituxan, might often fail to substantially reduce autoantibodies because PC are resistant.

Neubert and colleagues reasoned that precisely the hallmark of PC – their high rate of protein synthesis – could sensitize them to bortezomib (Bz, Velcade), a proteasome inhibitor that is therapeutic in some cancers. Bz interferes with the ubiquitin protein degradation pathway, thereby blocking NF-kB release and promoting the unfolded protein ‘stress’ response that induces apoptosis. Here, they show that Bz reduced short- and long-lived PC (CD138+ CD25- cells with cytoplasmic immunoglobulin light or heavy chains) ~90% in the bone marrow and spleens of mice after just 48 h treatment. Antibody-secreting cells remained decreased during 8 weeks of treatment. Bz had little or no effect on total B cell numbers or many B cell subsets, although germinal center B cells were reduced (Fig. 1). Cyclophosphamide or dexamethasone were less effective in reducing the numbers of long-lived (BrdU-) PC, suggesting a cellular basis for the failure of these current therapeutics in reducing autoantibodies (Fig. 2). In bone marrow and splenic PC, Bz rapidly induces transient expression of CHOP (20-40 fold within 4 h), a signaling protein previously implicated in the apoptotic response to the stress of protease inhibitors.

Lastly, they treated lupus-prone mice (NZB/W F1) with Bz and found it prevented onset of kidney disease (measured by proteinuria) and death. Remarkably, Bz was therapeutic in early disease, reducing serum levels of the autoantibody associated with kidney disease and preventing proteinuria in this lupus model as well as another (MRL/lpr: shown, dotted line Bz treated, solid line control treated, from Fig. 5a).

As Bz is already approved for use in humans, it should not be long before we know its therapeutic efficacy in lupus and other autoimmune diseases. Also, a new generation of proteasome inihibitors that target specific steps in the process are ready to be tested.

Neubert K, Meister S, Moser K, Weisel F, Maseda D, Amann K, Wiethe C, Winkler TH, Kalden JR, Manz RA, Voll RE. “The proteasome inhibitor bortezomib depletes plasma cells and protects mice with lupus-like disease from nephritis” Nat Med. 2008 Jun 8.

Adjuvants: a little less ‘dirty’, less secret

2008-06-18T13:04:00.007-05:00

How do adjuvants – mixtures that promote immunization/vaccination – work? Malherbe and colleagues applied modern tools and ideas to answer this old question. They analyzed the immune responses of mice (B10.BR strain) to the protein PCC (pigeon cytochrome C), which they had previously shown to be dominated by helper T cells (Th) responding to a single PCC fragment (peptide ‘epitope’ associated with I-Ek) and expressing T cell receptors (TCR) encoded by Valpha11Vbeta3 (a11b3). They concluded from those previous studies that TCR affinity, above a threshold level, does not drive clonal diversity.

Here, they compared Th numbers and diversity after subcutaneous immunization in an aluminum precipitate (Alum), or in an emulsion known as “incomplete Freund’s adjuvant” (IFA), or complete Freund’s adjuvant (CFA = IFA + dead mycobacterium), or with aqueous oligodeoxynucleotide (CpG) that stimulates the innate immunity Toll Like Receptor-9 (TLR-9), or monophosphoryl lipid A (MPL) that stimulates TLR-4.

First, they compared the numbers of TCRa11b3+ Th in draining lymph nodes on day 7 after immunization with or without PCC antigen and found a ~30 fold range among the adjuvants. MPL induced the largest number of PCC-specific Th but also many nonspecific Th. CpG induced the largest differential between PCC-specific and nonspecific Th whereas Alum induced nearly as many nonspecific as specific Th. The response kinetics were all similar, peaking around day 7. Second, they analyzed the TCR sequence “features…that assort with PCC-specificity” in dozens of single Th cells, concluding that clonal dominance occurred with or without TLR agonists or antigen ‘depots’ (IFA, CFA, and Alum). Third, they found lower affinity TCR Jbeta2.5 regions predominated among Th responding to depot-forming adjuvants, whereas higher affinity Jbeta1.2 predominated among Th responding to the non-depot forming adjuvants (CpG and MPL). Also, Vbeta usage depended upon the adjuvant.
Fourth, in affinity tests using PCC-I-Ek tetramers, they found a range of responses similar to those they had found in TCRa11b3+ cells (from Figure 4C, open circles are immunizations without PCC, bars are immunizations with PCC). Also consistent with the sequence analysis, CpG and MPL induced higher levels of binding as measured by mean fluorescence intensity (MFI).
Fifth, upon PCC immunization of mice hosting Th with transgenic high- or low-affinity TCRs, they found that IFA stimulated both populations equally whereas MPL stimulated Th with the high-affinity TCR but not those with the low-affinity TCR. Sixth, they found MPL as effective as the depot-forming IFA in activating transgenic Th transferred 5 days after immunization. Finally, 400 ug PCC in MPL induced as much Th proliferation as did 40 ug without changing the ratio of high- and low-affinity TCR, though 4 ug was suboptimal.

These parameters will be useful guides in determining how adjuvants improve immune protection, the goal of vaccination, especially in humans.

Malherbe L, Mark L, Fazilleau N, McHeyzer-Williams LJ, McHeyzer-Williams MG. “Vaccine adjuvants alter TCR-based selection thresholds”. Immunity. 2008 May;28(5):698-709.

The 3% (sialylated) Solution

2008-05-21T15:25:00.011-05:00

Intravenous Immunoglobulin (IVIG) is an immune modulator and anti-inflammatory agent used to treat a wide range of diseases, including rheumatoid arthritis (see also: 1990 NIH consensus panel report). IVIG contains antibodies (immunoglobulins, Ig) pooled from the blood of hundreds or even thousands of donors; pooling raises the risks of infection and complicates standardization.

Here, Anthony and colleagues tested their idea that sialylation is required for the therapeutic activity of the invariant region of Ig called Fc (fragment crystallizable). Sialic acid can be linked through alpha 2,3 or alpha 2,6, which are distinguished by the location of the hydroxyl and methyl groups on the 6 member ring.
First, they looked for these linkages in a preparation of IVIG by comparing its mass spectrum with standards. Nearly all the sialylated IVIG was alpha 2,6 linked and treatment with an enzyme that selectively removed this linkage abolished the anti-inflammatory activity of IVIG. Of potentially enormous significance was their success in making Fc derived from cloned human IgG therapeutic by alpha 2,6 sialylation. The figure shows the clinical score of mice made arthritic by administration of K/BxN sera alone (top line), or also treated therapeutically with IVIG (second line), 1/30 as much sialylated-enriched Fc from IVIG (0.033 g/kg SNA IVIG, third line), or an equivalent amount of recombinant, sialylated Fc (bottom line). This finding could lead directly to the manufacture of much safer, much more consistent IVIG preparations.
Anthony RM, Nimmerjahn F, Ashline DJ, Reinhold VN, Paulson JC, Ravetch JV. Science. 2008 Apr 18;320(5874):373-6. "Recapitulation of IVIG anti-inflammatory activity with a recombinant IgG Fc".

Microbe cheats death by misdirecting killer enzyme chaperone

2008-04-03T16:50:00.007-05:00

Two million people die each year from tuberculosis and about a third of all humans are infected with the intracellular parasite responsible: Mycobacterium tuberculosis. This microbe overcomes the defenses of the macrophages it infects by inhibiting the fusion of phagosomes with lysosomes and avoiding the activity of reactive oxygen and nitrogen intermediates, including nitric oxide (NO). NO is extremely reactive and consequently short-lived, so the microbe misdirects the enzyme that produces NO, inducible NO synthase (iNOS), away from the phagosome. Davis and colleagues guessed that EBP50, a protein previously implicated in anchoring iNOS and other cellular proteins to the actin cytoskeleton, might be responsible.

They first showed that EBP50 is colocated with iNOS in the phagosomes of activated macrophages but less so after infection by M. tuberculosis. Reducing EBP50 with siRNA also reduced colocalization. Moreover, siRNA knockdown of EBP50 increased the viability of M. tuberculosis in infected macrophages, demonstrating its involvement in controlling this microbe (Figure, coronin is a control cytoskeletal chaperone that does not bind EBP50). However, since EBP50 is involved in many cellular processes, the specificity of the inhibition remains unproven.

Davis AS, Vergne I, Master SS, Kyei GB, Chua J, Deretic V. “Mechanism of Inducible Nitric Oxide Synthase Exclusion from Mycobacterial Phagosomes”. PLoS Pathog. 2007 Dec 7;3(12):e186

Identifying Friend or Foe - Mreg?

2008-03-06T08:52:00.005-05:00

How do the immune cells within the gut distinguish between potentially dangerous pathogens and harmless or beneficial commensal organisms? [Hint: It’s not through PAMPs (pathogen-associated molecular patterns.)]

These investigators identified in mice a novel population of macrophages within the lamina propria, the layer of mucosa beneath the epithelium on the lumenal side of the intestine wall. These macrophages secrete IL-10 and other anti-inflammatory cytokines but no pro-inflammatory cytokines (even when stimulated through their Toll-like receptors by PAMPs). They also stimulate the development of regulatory T cells (Tregs) more potently than splenic macrophages (figure, CD4+ FoxP3+ Treg) and suppress the secretion of IL-17 by DC within the lamina propria. The authors suggest that a “dynamic interaction between these subsets may influence the balance between immune activation and tolerance”. They need to abbreviate the idea... how about 'Mregs'?

Denning et al. "Lamina propria macrophages and dendritic cells differentially induce regulatory and interleukin 17-producing T cell responses". Nat Immunol. 2007 Oct;8(10):1086-94.

Targeting the Plant Inside

2008-03-04T18:13:00.004-05:00

Toxoplasm gondii are intracellular parasites that cause toxoplasmosis, the “third leading cause of death attributed to foodborne illness in the United States. More that 60 million [Americans] carry the Toxoplasma parasite, but very few have symptoms because the immune system usually keeps the parasite from causing illness” (CDC). T.gondii contain organelles or plastids called “apicoplasts” that were probably acquired in evolution along with an algal endosymbiont.

Apicoplasts contain unique proteins that may be effectively targeted by therapeutic agents. These researchers previously demonstrated a calcium control of protein secretion and identified conserved Ca-responsive proteins. Here they show that calcium-dependent development in T. gondii is controlled by a plant hormone produced by the apicoplast, abscisic acid (ABA, named for its role in abscission, the shedding of plant leaves, fruit, etc.). They found that fluridone, an herbicide that specifically inhibits an enzyme on the synthetic pathway producing ABA, blocked T gondii maturation. Finally, they demonstrated that the herbicide also worked as a therapeutic, protecting mice from a lethal dose of T gondii (shown, from Figure 4). Another infamous apicoplast-containing parasite is Plasmodium falciparum, which causes severe malaria, suggesting an exciting new approach to treating this scourge.

Nagamune et al. "Abscisic acid controls calcium-dependent egress and development in Toxoplasma gondii" Nature. 2008 Jan 10;451(7175):207-10.

Protracted IKKb inhibition is dangerous

2008-02-22T16:34:00.008-05:00

Many pro-inflammatory molecules share the intracellular signaling pathway with Nuclear Factor – kappa Binding (NF-kB) for their induction and activity. Cytoplasmic NF-kB is released and transported into the nucleus, where it drives transcription of specific genes, when an inhibitor (IkB) is phosphorylated by the inhibitor-kappa kinase beta (IKKb) The importance of this pathway suggests that IKKb would be a promising target for new anti-inflammatory drugs. Here, Greten and colleages tested this idea using mice in which IKKb was deleted in myeloid cells: macrophages and neutrophils (IKKb-del-myo). [A total IKKb deficiency is an embryonic lethal]. IKKb-del-myo mice were viable and had no obvious changes even within tissues that are rich in myeloid cells, such as lymphoid organs and the gastrointestinal tract. They observed that IKKb-del-myo mice were actually more susceptible to endotoxin- (LPS-) induced toxic shock, which is mediated by TNF and interleukin-1beta (IL-1b). IKKb-deficient cells are more susceptible to apoptosis because some NF-kB-induced genes are protective (e.g., A20).

The authors also studied another conditional knockout mouse, in which IKKb is deleted in all “interferon-inducible” cells (IKKb-del), though this model is complicated by a requisite induction with the pleiotropic agent poly-I:C and deletion probably occurs in many more cell types. These mice develop many more granulocytes and, like the IKKb-del-myo mice, also produced much higher levels of IL-1b after LPS treatment. However, mRNA encoding IL-1 was actually decreased, suggesting a change in IL-1 processing.

Indeed, the authors determined that NF-kB inhibits caspase-1 and serine proteases that are required in macrophages and neutrophils, respectively, to cleave pro-IL-1b and release the active cytokine. Consistent with this interpretation, a serine protease inhibitor protected mice against IL-1b-induced death. Mice exposed to LPS die more quickly and more often when fed ML120B, an inhibitor of IKKb for a protracted period with multiple doses, correlating with an increased processing and release of IL-1b (figure, extracted from Figure 7). Definitely not a lead compound for a daily regimen.
Greten et al. Cell. 2007 Sep 7;130(5):918-31. "NF-kappaB is a negative regulator of IL-1beta secretion as revealed by genetic and pharmacological inhibition of IKKbeta"

Fatter and Sicker: Obesity weakens Immunity

2008-02-18T11:31:00.003-05:00

This paper reminds me of an old joke: when a patient, having received a dubious diagnosis, asks for a second opinion, the doctor replies 'you're also ugly'. But seriously, folks, this paper suggests that the rise of obesity in the US could have unanticipated consequences for infectious disease. To test the effect of obesity on immunity, Amar and colleagues infected fat or lean mice with Porphyromonas gingivalis, a bacterium that causes gum disease. Mice fed with high fat food for 4 months weighed about 30% more than the mice on normal chow.
They found that mice with diet-induced obesity (DIO) suffered a greater loss of alveolar (gum) bone than did lean mice. P. gingivalis infection induced less inflammatory cytokine release in DIO mice than in lean mice. Macrophages cultured from DIO mice showed significantly reduced activation by LPS of the intracellular signaling molecule NF-kappaB (a chromosome immunoprecipitation is shown, from Fig. 8). Oddly, C-reactive protein, a marker of inflammation, is reported elevated in the bloodstream of obese humans. Do you have a 'second opinion'?
Amar et al., Diet-induced obesity in mice causes changes in immune responses and bone loss manifested by bacterial challenge. Proc Natl Acad Sci U S A. 2007 Dec 18;104(51):20466-71.

T cell diversity

2008-01-23T20:57:00.000-05:00

Patrick writes: "Virginal CD8 T cells encountering MHC-associated antigen will undergo activation, proliferation, gaining effector functions, followed by contraction and the development of central and effector T cell memories. A number of models have been proposed to account for the generation of these T cell subsets. The observation of homeostatic proliferation suggests that virginal CD8 T cells may not have been as naïve as one have previously thought. This proliferation may have given rise to intraclonal diversity and pre-programmed the “naïve” precursors to develop into differing subsets when encountering antigens. An alternative “progressive differentiation” model proposes that different signal strength at the time of priming may give rise to the diverse subsets. An extension of this model is that a stronger signal may impact the differentiation pattern of one naïve T cells encountering antigen on a DC in contrast to two or three naïve T cells encountering the antigen on the same DC. A “latecomer effect”, i.e., a naïve T cell encountering the antigen late, also may drive the development of long-living T cells.

In this report (Immunity. 2007 Dec 21;27(6):985-997), Stemberger et al. developed a single cell adoptive transfer system to show one CD8+ CD45+ naïve T cell can give rise to diverse phenotypic subsets with functionality (as measured by degranulation, IL-2, IFNg and TNFα expression), including T effectors (CD127-lo, CD62L-lo); effector memory T cells (CD127-hi, CD62L-lo); and, central memory T cells (CD127-hi, CD62L-hi). The observed phenotypic and functional patterns of these subsets are comparable to those seen in resident CD8+ CD45+ T cells upon antigen activation. This in vivo assay, while technically challenging, may provide a handle to study the factors that promote the differentiation and development of effector and central memories."
Stemberger et al. Immunity. 2007 Dec 21;27(6):985-997. A Single Naive CD8(+) T Cell Precursor Can Develop into Diverse Effector and Memory Subsets.

T cell diversity: E Unum Pluribus

2008-01-20T19:36:00.000-05:00

Infections stimulate the expansion of a few individual progenitor T lymphocytes, perhaps as few as 50, into millions of specific T lymphocytes (Review). These T cell clones are typically diverse, encompassing subsets of short-lived effector cells, long-lived memory cells, and a variety of intermediate subsets. The proposed models of diversification focus on the priming phase because previous observations have shown that an initial, transient encounter with antigen suffices to induce protracted proliferation and complete subset diversity. Differentiation capabilities might be predetermined, with the naive precursor cells programmed to undergo diversification into all subsets. Alternatively, a ‘‘progressive differentiation’’ model proposes that the strength of antigen stimulation and costimulation at priming determines how far the T cell progeny are driven along a differentiation spectrum.

Here, the ability of precursor T cells to differentiate into multiple subsets was tested by transferring a single naive T cell and then analyzing its progeny after immunization. Twelve days after transfer and immunization of the host with intracellular bacteria expressing ovalbumin, (L.m.-ova), the donor (CD45.1+) transgenic, ova-specific T cells were found to generate many subsets of CD8+ T cells, including CD127+ memory T cells detected in spleen and lymph nodes. The figure, derived from fig. 1A, shows 2 host mice, one on each row; progeny of the transferred cell, the CD45.1+ population circled in the left panels, could be detected in about a quarter of the host mice. Progeny T cells were also detected in lungs. Both 'effector' and 'central' memory T cells (low vs. high CD62L) were detected, the former in the lymphoid organs and latter in the lungs. Upon restimulation of the progeny in vitro, production of the inflammatory cytokines interferon-gamma and tumor-necrosis-factor was detected, demonstrating maturation into effector T cells. This is a straightforward test, albeit technically demanding, that provides strong evidence that a single naïve T cell can generate many, and perhaps all, subsets.
Stemberger et al. Immunity. 2007 Dec 21;27(6):985-997. A Single Naive CD8(+) T Cell Precursor Can Develop into Diverse Effector and Memory Subsets.

How bacteria avoid dying alone

2008-01-14T14:51:00.000-05:00

Programmed cell death, PCD or apoptosis, was discovered as the way individual cells altruistically remove themselves during development of a multi-cellular organism (e.g., C. elegans). However, single celled bacteria also undergo PCD to prevent the spread of a bacteriophage infection, for example. Stressed E. coli produce the stable endoribonuclease toxin MazF as well as the labile antitoxin MazE; even a transient interruption of protein synthesis can lead to PCD.

This group previously suggested that mazEF-mediated PCD depends on cell density. Here they show that indeed the mazEF system is only effective above ~3 million cells/ml. Adding supernatant from a dense culture to a diluted culture along with a stress-inducing antibiotic quickly induced PCD in wild-type (WT) cells but not cells in which mazEF is deleted (del-mazEF). This observation led them to identify an “extracellular death factor” (EDF) that is produced during log-phase growth but not during the stationary phase. Here they show that EDF is a pentapeptide (NNWNN) and that adding synthetic EDF to supernatants from stationary phase cultures renders them capable of inducing mazEF-induced PCD (Figure). High EDF (> 200 ng/ml) reduced viability of even del-mazEF strains, which the authors ascribe to perhaps inducing other PCD systems or inactivating essential components. Using mutagenesis, they demonstrated that NNWNN is the optimal sequence for EDF activity. No E. coli genes encode NNWNN but zwf-encoded NNWDN could be amidated to yield NNWNN and deletion of both zwf and ygeO, which encodes a similar NNWN peptide, prevented EDF induction. The authors propose a quorum-sensing role for EDF and point out that synthetic EDF “may be a lead for a new class of antibiotics that specifically trigger bacterial cell death".
Kolodkin-Gal et al. Science. 2007 Oct 26;318(5850):652-5. “A linear pentapeptide is a quorum-sensing factor required for mazEF-mediated cell death in Escherichia coli”.

2007 Flu vaccine: Pain, and gain?

2007-12-24T13:22:00.000-05:00

The influenza vaccine contains three influenza virus strains – A (H1N1), A (H3N2), and B. The effectiveness of the vaccine depends on the match between the strains in the vaccine and those that are circulating in the community.

In years when vaccine and circulating strains were poorly matched, the vaccine can be ineffective (Bridges 2000). (Surprisingly, this study also concluded that even in a year when they were well matched, the cost outweighed benefit for people under 65.) In contrast, other studies have demonstrated some benefit even when the vaccine and circulating strains were not well matched. During the 2003-04 season, for example, vaccine effectiveness among people 50-64 years old was over 50% for protecting against contracting flu and 90% against hospitalization (Herrera 2006). The 2003-04 vaccine was similarly effective in children (Ritzwoller 2005). Children are responsible for the most transmission yet they are vaccinated at a lower rate than older adults.

For the current flu season, the Advisory Committee on Immunization Practices (ACIP) recommended vaccination with the strains A/Solomon Islands/3/2006 (H1N1)-like, A/Wisconsin/67/2005 (H3N2)-like, and B/Malaysia/2506/2004. The first detailed analysis of the current flu season was posted last week by the CDC. Between September 30 and December 1, 2007, World Health Organization and National Respiratory and Enteric Virus Surveillance System tested 24,897 respiratory specimens for influenza viruses; 559 (2.2%) were positive. Of these, 92% were influenza A viruses, and 8% were influenza B viruses. One hundred thirty-five of the influenza A viruses were subtyped; 83% of these were influenza A (H1) viruses, and 17% were influenza A (H3) viruses. CDC characterized 27 isolates to date (see table): 19 were influenza A (H1) isolates, 5 were influenza A (H3) isolates, and 3 were influenza B isolates. All 19 A/H1 viruses were Solomon Islands/3/2006-like. Two A/H3 isolates were Wisconsin/67/2005-like. Three A/H3 isolates were similar to Brisbane/10/2007, a strain recommended in the vaccines for the Southern Hemisphere. The three influenza B viruses characterized all belong to the Yamagata/16/88 lineage, whereas the Malaysia strain in the vaccine belongs to the Victoria lineage.

The fact that all H1 isolates are in the vaccine indicates that ACIP accurately predicted the rise of this strain. Although the efficacy of the vaccine is not established, its good match with the circulating strains suggests it will be beneficial. However, the H3 component matches only a minority of the circulating strains and the B component match none. Fortunately, these strains are less prevalent than the H1 strains.

Reap before sowing: depleting host Hematopoetic Stem Cells improves transplantation

2007-12-13T10:42:00.000-05:00

Hematopoetic stem cells (HSC) can generate all the cells of the blood, including the myeloid (erythrocytes, neutrophils, etc.) and lymphoid (B, T, and NK cells) lineages. HSC transplantation would be a valuable therapy for many conditions, including reconstitution of immune deficiencies and following radiation.

HSCs transplanted by intravenous injection efficiently home to the bone marrow. However, transplanted HSCs are only transiently productive, with donor cell frequencies in the blood reduced to < 1% within months, unless the host is “conditioned” by toxic regimens that are thought to work by depleting host HSC occupying a limiting number of niches in the bone marrow.

Here, Czechowicz and colleagues tested a targeted depletion with ACK2, an antibody that blocks the cytokine stem cell factor (SCF) receptor CD117 (c-kit). They report that ACK2 treatment of immunodeficient mice led to the transient removal of >98% of HSCs. (Using immunodeficient hosts avoided host-vs.-graft immune responses that would complicate the interpretation. Rag2-knockout mice lack mature B and T cell lineages due to failure to recombine V(D)J regions of immunoglobulin and T cell receptor genes. Common-gamma-chain-knockout mice are severely immunodeficient due to the failure to signal IL2, IL4, IL7, IL9, and IL15. ) Moreover, they show that donor HSC in ACK2-treated immunodeficient host mice produced up to 90% of the blood cells months after transplantation (figure 3a). This result demonstrates that host HSC must be depleted from a limiting number of niches to allow donor HSC to stably reconstitute an immunodeficent host.

Czechowicz et al. "Efficient transplantation via antibody-based clearance of hematopoietic stem cell niches". Science. 2007 Nov 23;318(5854):1296-9.

T(H)-17 & Pathology

2007-12-05T13:26:00.000-05:00

A colleague writes: "The recent group of papers in Nature Immunology (Stumhofer, Awasthi, and Fitzgerald) concerning TH-17 cells is driven, at least in part, by a question as old as the finding that some T cell lines or clones can cause a model inflammatory disease, EAE: What distinguishes pathogenic T cells in an EAE model from those cells that cannot cause inflammatory disease? With the emergence of the TH1-TH2 paradigm, it became “clear” that the pathogenic cells were of the proinflammatory, TH1 persuasion. This belief was then shaken by experiments using antibodies to, and knockouts of, to the p35 and p40 subunits of IL-12, which revealed that the “key cytokine” leading to pathogenicity was not the TH1-promoting IL-12 itself, but rather IL-23, which shares the p40 subunit. IL-23 was shown to promote a T cell population that produced TNF, IL-6, and IL-17, among other factors. Further study showed that differentiation of T cells toward this new “TH-17” phenotype was most effectively driven by IL-6 in combination with TGF-beta, whereas TGF-beta alone drove expression of Foxp3 and emergence of regulatory T cells.

This new group of papers incorporates a variety of approaches that all lead toward similar conclusions: TH-17s do not constitute a monolithic proinflammatory population, but can be influenced by either IL-27 or the combination of TGF-beta and IL-6 to include cells that secrete IL-10 in addition to IL-17 and that thus suppress inflammatory disease. As the figure from the paper by McGeachy et al. shows, IL-23 and the combination of TGF-beta and IL-6 both induce secretion of IL-17 by T cells during a recall response, but only the IL-23-treated cells cause EAE when transferred into naïve hosts. Other results in this paper and others show that this protection from disease is attributable to IL-10, which is secreted by T cells in response to IL-27 or the TGF-beta/IL-6 combination. These results may point the way to new modalities for steering otherwise harmful autoimmune responses into more benign channels."

McGeachy et al. "TGF-beta and IL-6 drive the production of IL-17 and IL-10 by T cells and restrain T(H)-17 cell-mediated pathology." Nat Immunol. 2007 Dec;8(12):1390-7.

T cell maintenance: Care and Feeding

2007-11-15T10:37:00.000-05:00

An abstract with the words ‘dynamic’ and ‘interplay’ usually signals a paper to be avoided but this one tells an interesting story about T cell survival. T cells require occasional stimulation through their antigen receptor (TCR) and feeding with interleukin-7 (IL-7), a product of stromal cells, monocytes, and some epithelial cells. T cells seem to share a limited amount of IL-7 through a negative feedback on the receptor gene transcription (IL-7Ra).

Tickling the TCR is trickier: too much could provoke autoimmunity, too little and the T cell dies. Here, Park and colleagues show that IL-7 (and other gamma common chain agonists) also regulates transcription of the CD8 gene encoding the monomorphic co-receptor of the TCR on a large subset of T cells. Moreover, they report that TCR signals from endogenous antigens inhibit CD8 expression, thereby promoting self tolerance.
The balance is illustrated in HY RAG mice, which are are engineered to contain only T cells specific for HY (a tissue antigen encoded on the Y-chromosome and therefore expressed only in males). If no antigen is present (females), then there is no TCR engagement, leading to more CD8 expression and less IL-7Ra expression than normal (Figure, top vs. second line). With more (and more) TCR engagement in males with appropriate H-2b alleles, comes less CD8 and more IL-7Ra expression (bottom 2 lines). They term this dynamic interplay “coreceptor tuning”.
Park JH, et al. Nat Immunol. 2007 Oct;8(10):1049-59. 'Coreceptor tuning': cytokine signals transcriptionally tailor CD8 coreceptor expression to the self-specificity of the TCR.

Adaptive Control of Innate Immunity

2007-10-31T14:52:00.000-05:00

Innate immunity controls infections while the adaptive immune response develops. In the absence of T cells, infections that kill the host were thought to result from an uncontrolled infection. However, Kim and colleagues demonstrate here that the adaptive immune response is also necessary to control the innate immune response. They show that host death can result from an uncontrolled innate response that occurs in the absence of an adaptive immune response.
Triggering the innate immune receptor TLR3 with poly(I:C), a double stranded RNA that mimics viruses, produced a destructive and potentially lethal “cytokine storm”. This fatal reaction required Natural Killer (NK) cells and TNF. The figure is a portion of figure 4 showing the response of immune deficient animals: panel e shows that abrogating TNF signaling but not immunoglobulin signaling is protective and panel f shows that NK cell depletion is protective. As the authors note, these findings might be also important for understanding the responses of “individuals with congenital or acquired immune deficiency”.
Kim et al. Nat Med. 2007 Nov;13(10):1248. "Adaptive immune cells temper initial innate responses".

Shear forces trigger T cell adhesion

2007-10-15T13:03:00.000-05:00

At sites of inflammation, T lymphocytes bind to activated endothelium and move from the bloodstream into lymph nodes. Recruited T cells remain highly motile within the lymph node, scanning many dendritic cells and other antigen-presenting cells for cognate antigen. T cell motility is stimulated by chemokines, which can also promote adhesion by activating integrins.

Woolf and colleagues observed that many cells, including T cells themselves, express the same adhesion molecules and asked how premature arrest and clumping was avoided. To delineate the contributions of chemokines and adhesion molecules, they coated surfaces with CCL21, ICAM-1, or VCAM-1 and observed T cells moving on these surfaces. They found that surface-bound CCL21 stimulated T cell motility but soluble CCL21 did not. Although CCL21 also induced clustering of LFA-1 and VLA-4, these integrins did not mediate adhesion to their ligands ICAM-1 and VCAM-1. However, when T cells were exposed to shear stress, they rapidly developed strong adhesion to ICAM-1 or VCAM-1 (Figure). Video microscopy of T cells and lymph nodes cells obtained from mice deficient in adhesion molecules provided additional support for their conclusion that integrin-mediated adhesion, which is crucial to T cell recruitment from the bloodstream, is 'silenced' within the lymph node due to the absence of shear force.

Woolf et al. Nat Immunol. 2007 Oct;8(10):1076-85. Lymph node chemokines promote sustained T lymphocyte motility without triggering stable integrin adhesiveness in the absence of shear forces

TNF-R family member 4-1BBL keeps TNF production “on”

2007-06-21T10:38:00.000-05:00

The bacterial cell wall component LPS (lipopolysaccharide) binds to Toll-like receptor 4 (TLR4) on macrophages and stimulates them to produce the strong pro-inflammatory cytokine TNF (tumor necrosis factor). To identify potential signaling proteins, Kang and colleagues used the intracellular domain of TLR4 in a two-hybrid screen of binding proteins. They isolated 4-1BBL (CD137L), a transmembrane protein previously best known as a T cell costimulator, and the well-known TLR signaling protein MyD88. 4-1BBL binding was not abrogated by a P712H substitution in the TLR4 TIR (Toll-IL-R) motif that is required for MyD88 binding, indicating that they bind to different parts of TLR4.

LPS induced less TNF production at 24 h from 4-1BBL “knockout” (4-1BBL KO) macrophages than from wild-type (WT) macrophages, though equivalent amounts of IL-1beta were produced. Moreover, kinetic analysis demonstrated that TNF production was equivalent soon after stimulation but ceased after about 6 hours in 4-1BBL-KO macrophages while continuing in WT macrophages (Figure, panel b), accounting for the difference at 24 h. Although 4-1BBL was cloned as the ligand for 4-1BB, which is also expressed by macrophages, it does not contribute to this response (Figure, panel c).

LPS induces a gradual appearance of 4-1BBL on the cell surface, with marked accumulation after 2 hours, which accounts for the delayed influence on TNF production. Indeed, surface 4-1BBL alone at high levels of expression, or cross-linked at lower levels of expression, is sufficient induce TNF expression. Finally, in the ultimate test of relevance, they showed that 4-1BBL-KO mice survived a dose of LPS that killed all WT mice. Their discovery of 4-1BBL's role in sustained TNF production provides a new target for therapeutic intervention in the development of inflammatory diseases.

Young Jun Kang et al. Nat Immunol. 2007 Jun;8(6):601-9 "Cell surface 4-1BBL mediates sequential signaling pathways ‘downstream’ of TLR and is required for sustained TNF production in macrophages"

Micro RNA sets sensitivity of T cell

2007-04-14T07:51:00.000-05:00

MicroRNAs are single-stranded RNA molecules, averaging 22 nucleotides in length and encoded in the genome, that regulate the expression of other genes. MicroRNA and the process of “RNA interference” were discovered only a few years ago. The miRNA miR-181a was known to be hightly expressed in the thymus and to function in T cell development. Li and colleagues investigated the function of this miRNA by manipulating its expression in T cells. Forced (ectopic) expression makes T cells more sensitive to stimulatory peptides, generating a stronger calcium flux with about half as many peptides (Figure, left panel). Even an antagonist peptide, which usually turns off T cells, triggers calcium flux (right panel) and growth factor (IL-2) secretion from T cells overexpressing miR-181a. This apparently occurs by miR-181a repressing transcripts encoding phosphatases, including DUSP5 and 6, SHP-2 (but not SHP-1), and PTPN22. For example, phosphorylated ERK (the substrate for DUSP6) remains high longer after activation of T cells expressing more miR-181a. Knocking down individual phosphatases did not overcome miR-181a-enhanced sensitivity, suggesting that multiple pathways are targeted. However, overexpression of DUSP6 or DUSP5 or SHP-2 (using modified sequences that are not recognized by miR-181a) restored repression in T cells expressing miR-181a. The authors designed an “antagomir” to block miR-181a and found it disrupted selection of antigen-specific T cells in the thymus. This new and unexpected mechanism of regulation may be important in understanding T cell development and activation.
Cell. 2007 Apr 6;129(1):147-61. “miR-181a Is an Intrinsic Modulator of T Cell Sensitivity and Selection.” Li QJ, Chau J, Ebert PJ, Sylvester G, Min H, Liu G, Braich R, Manoharan M, Soutschek J, Skare P, Klein LO, Davis MM, Chen CZ.

Interferon type I mediates resistance to Trypanosomes

2007-03-21T20:28:00.000-05:00

The intracellular parasite Trypanosoma cruzi causes Chagas disease in over 16 million people worldwide, mostly in poor and rural areas in Central and South America. Chagas disease has a variety of debilitating symptoms and kills about 50,000 people annually. Prevention focuses on killing the insect vectors; treatment is expensive and not entirely effective. Resistance to T. cruzi in humans is thought to be initiated by TLRs and effected by interferon (IFN)-gamma and nitric oxide (NO), produced by host T cells and NK cells, which kill the parasite. Here, Koga and colleagues investigated the pathway between TRL activation by the parasite and effector mechanisms. They found that resistance requires TLR signaling because infected macrophages and dendritic cells cultured from mice without the TLR adapter proteins MyD88 and TRIFF support T. cruzi proliferation. Since TRIFF signals the production of type I interferons, namely the IFN-alphas and IFN-beta, the investigators tested cells from mice without a receptor for these IFNs: IFNAR1-/-. As expected, these cells were unable to control T. cruzi in culture (Figure, panel A). Moreover, these mice quickly succumb to infection (panel B). A gene expression analysis found that IRG47 was strongly induced and previous reports had implicated this GTPase in the IFN response. When IRG47 expression was “knocked down” with RNAi, even wild type cells were rendered susceptible to T. cruzi, demonstrating the critical role of type I IFNs and induced IRG47 in resistance to this trypanosome.
"TLR-Dependent Induction of IFN-beta Mediates Host Defense against Trypanosoma cruzi." Koga et al. J Immunol. 2006, 177: 7059

TLR2 Helps Bacteria Weaken Gums

2007-02-28T13:21:00.000-05:00

The bacterium Porphyromonas gingivalis gets most of the blame for periodontal disease, which erodes bone that supports teeth and is the leading cause of tooth loss. P gingivalis adheres to oral surfaces using molecules such as lipopolysaccharides (LPS) and lipoproteins. These molecules are recognized by Toll-like receptors (TLRs) that are expressed on cells of the immune system: TLR2 binds bacterial lipopeptides and TLR4 binds LPS. Burns and colleagues tested how these TLRs are involved in the host mouse response against P gingivalis. They implanted metal coils subcutaneously and injected bacteria into the lumen, and then sampled the space at later times to monitor the immune response in wild-type (WT), TLR2, and -4 knockout (-/-) mice. They observed differences in the induction of cytokines, most dramatically a reduced accumulation of TNF, interferon-gamma, and interleukin-10 in TLR2-/- mice. Surprisingly, TLR2-/- mice cleared the bacteria from the injection site and blood within a day. In contrast, bacteria remained in the blood of WT and TLR4-/- mice for at least 4 days. Moreover, when mice were orally challenged with P. gingivalis, significant bone loss resulted within 6 weeks in WT but not TLR2-/- mice (Figure). Would blocking TLR2 with lipopeptides in a mouthwash or chewing gum help protect teeth?
J Immunol. 2006 Dec 15;177(12):8296-300. "TLR2 is required for the innate response to Porphyromonas gingivalis: activation leads to bacterial persistence and TLR2 deficiency attenuates induced alveolar bone resorption." Burns E, Bachrach G, Shapira L, Nussbaum G.

Flu Vaccine - wheeze now or sick later?

2007-02-15T12:13:00.000-05:00

The prestigious New England Journal of Medicine just published a paper claiming to demonstrate a superiority of attenuated live flu vaccine over the conventional, dead flu vaccine for young children. About half as many kids in the attenuated vaccine group went on to contract flu (figure). USA Today and other publications trumpeted the results with headlines such as "FluMist spray protects kids better than flu shots". They downplayed the concern raised by independent scientists that the live vaccine "comes with a significant risk for medically important wheezing".
This story illustrates some of the problems with reporting such clinical trials. First, it was "sponsored" (paid for) by MedImmune, the producer of the live vaccine. Sponsors have been known to not publish disappointing results, producing a publication bias. Although we can welcome these results, the arrangement raises questions about the "spin". How should parents and pediatricians balance the decrease in disease (from 9% to 4%) with the increase in wheezing (from 3% to 6%)? Second, the description of the trial is misleading. The Methods section begins "children ... were randomly assigned ...to receive either ... live attenuated influenza vaccine ... or inactivated vaccine in a double-blind manner" (emphasis added). This use of the term "double-blind" is nonsensical because random is completely "blind". The wording seems intended to make the reader think the trial was double-blind. Double-blind is the gold standard for trials because it reduces bias by keeping the both the doctor and patient ignorant of which treatment they are assigned. In this trial, however, the treatments could not be "blinded" because one involves a shot and the other involves sniffing the virus, treatments that are clearly distinguishable even by a patient. Finally, journals and journalists like to report positive, exciting news. These results merit a more sober discussion.
N Engl J Med. 2007 Feb 15;356(7):685-96. "Live attenuated versus inactivated influenza vaccine in infants and young children." Belshe RB, Edwards KM, Vesikari T, Black SV, Walker RE, Hultquist M, Kemble G, Connor EM

Tuberculosis Traffic Arrest

2007-02-13T12:04:00.000-05:00

Mycobacterium tuberculosis is a life-threatening pathogen that persists in infected cells after being phagocytosed (eaten). Phagocytosed matter is usually digested but M. tuberculosis avoids this fate by blocking the acidification of the its compartment. The process can be studied in U937 cells because they behave like macrophages ("big eaters") and grow well in culture. Rab proteins form a large family of Ras-like GTPases that mediate vesicle trafficking, budding, etc. A recent report showed that Rab14 participates in trafficking to the early endosome, a stage just after the phagosome. Kyei and colleagues found that Rab14 associated with phagosomes containing live tuberculosis mycobacteria and investigated whether Rab14 was required for the arrested development of the phagosome. Phagosomes of U937 cells contain live tuberculosis bacilli of the strain H37Rv (green label, left panels). The middle panels show the same cells stained with a dye that detects endosomes that have undergone acidification, a step toward digestion and marker of the early endosome. A "knock-down" of Rab14 with siRNA permits the maturation of the phagosome, as demonstrated by the overlap of the acidified (red), bacillus-containing (green) phagosomes, (yellow color in the lower right panel). These findings suggest that inhibiting Rab14 may permit the infected cell to digest M. tuberculosis, thereby offering a new target for therapeutic intervention and potentially reducing the burden of this disease.
Kyei GB, Vergne I, Chua J, Roberts E, Harris J, Junutula JR, Deretic V. "Rab14 is critical for maintenance of Mycobacterium tuberculosis phagosome maturation arrest". EMBO J. 2006 Nov 15;25(22):5250-9.

Infectious Cancer Cells

2007-01-29T19:35:00.000-05:00

Some cancers are caused by infectious agents, such as viruses that cause cancer by infecting and transforming host cells. For example, Epstein-Barr virus (EBV) is implicated in Burkitt's lymphoma, nasopharyngeal carcinoma, and other cancers (review). For canine transmissible venereal tumor (CTVT), however, the tumor cell itself was thought to be the agent. To test this hypothesis, Murgia and colleagues isolated CTVT cells and non-cancerous blood cells from 40 different dogs on 5 continents, and determined their relationships by comparing nuclear and mitochondrial genetic markers. They conclude that all the tumors are clones of a single tumor that arose between 200 and 2,500 years ago. Although the tumor has a very unusual chromosome arrangement (aneuploid), it is stable. They also studied the highly polymorphic histocompatibility genes. Expression of histocompatibility proteins, which would block transplantation between different animals, is reduced but not extinguished on CTVT cells. Another study demonstrated that CTVT cells make TGF-beta1, which could also reduce the immune response. This surprising cell could provide ideas for inducing tolerance of allografts.
Claudio Murgia, Jonathan K. Pritchard, Su Yeon Kim, Ariberto Fassati, and Robin A. Weiss "Clonal Origin and Evolution of a Transmissible Cancer" Cell 126, 477–487, August 11, 2006

How Peptides bind Proteins

2007-01-27T18:46:00.000-05:00

Peptides are short proteins (polypeptides) -- up to about 50 amino acids. They can convey signals and mediate some of the activities of modular proteins. For example, peptides regulate behavior (neuropeptides), mood (substance P), and digestion (glucagon) The rules for peptides binding to proteins are not established, so Hertz and Yanover developed a new “framework” called PepDist for analyzing these interactions computationally. They say that most previous methods used only “binary” (binding/not-binding) classifiers, but a better method would rank peptides and the best would predict affinity. PepDist learns peptide-peptide distance functions and uses these to predict affinity. They trained their model peptides bound to histocompatibility proteins, which constitute a large and diverse family. They claim that PepDist outperforms the (formerly) state of the art models, including SVMHC, NetMHC (these links work, theirs don't!), and RANKPEP. The best part is that you can easily test it yourself – they provide an on-line service.
Hertz T and Yanover C, "PepDist: a new framework for protein-peptide binding prediction based on learning peptide distance functions." BMC Bioinformatics. 2006 Mar 20;7 Suppl 1:S3.

Genes and obesity

2007-01-27T18:38:00.000-05:00

Gnome writes "Obese people, defined as a body mass index (BMI) over 30 (weight/(height)^2 in kg/m^2), are at increased risk of disease (including type 2 diabetes, heart disease, stroke, some types of cancer) and death. Unlike risk that is clearly behavioral, such as smoking, or genetic, such as some BRCA alleles, obesity is itself caused by a combination of factors. Though the recent increase in obesity rates results largely from behavioral changes, BMI is partially heritable. Herbert and colleagues looked through the whole genome for common variants associated with obesity among participants in the Framingham Heart Study. They avoided the statistical problem of multiple comparisons by first screening using parental genotypes to identify SNPs and inheritance models that best predict offspring phenotypes and then performing a family based association test (FBAT, a type of transmission disequilibrium test (TDT)) to test the selected SNPs for their association with BMI. In step 1, they tested 116,204 SNPs in 694 participants. In step 2, the top 10 SNPs were tested using a recessive model, yielding only 1 significant SNP: rs7566605, which defines a single haplotype and is near INSIG2 (insulin-induced gene 2). “For all exams, rs7566605 CC homozygotes are about 1 BMI unit heavier than individuals with GC or GG genotypes (P <0.0001). They replicated this correlation in a cohort from a town near Munich, Germany, in a combination of Caucasian cohorts from Poland and the USA, and in two samples from an African American population from Illinois, USA. However, the rs7566605 SNP was not correlated with BMI among the Nurses Health Study cohort, perhaps because the cohort contained fewer individuals with a high BMI or because of differences in environment and lifestyle. This is one gene among several associated with obesity and leanness.
Herbert A, Gerry NP, McQueen MB, Heid IM, Pfeufer A, Illig T, Wichmann HE, Meitinger T, Hunter D, Hu FB, Colditz G, Hinney A, Hebebrand J, Koberwitz K, Zhu X, Cooper R, Ardlie K, Lyon H, Hirschhorn JN, Laird NM, Lenburg ME, Lange C, Christman MF. "A common genetic variant is associated with adult and childhood obesity." Science 2006; 312:279-83."Science. 2006 Apr 14;312(5771):279-83.

Intermediate Avidity T Cells Regulated Through Qa-1

2007-01-27T18:32:00.000-05:00

Jiang and colleagues have developed a model of immune regulation based on the details of antigen recognition: T cell receptors (TCRs) engaging peptides held by histocompatibility (MHC) molecules on an antigen presenting cell (APC). The strength of multivalent binding, or avidity, such as occurs between T cell and APC, depends on the affinity of individual receptor-ligand pairs (TCR-peptide/MHC) and the number of pairs engaged. They propose that autoimmunity may be avoided by controlling T cells with intermediate avidity for self proteins, since autoreactive T cells with high and low avidity are deleted during T cell maturation in the thymus (positive and negative selection). They suggest that T cells recognizing antigen with intermediate avidity are triggered to express non-classical MHC molecules (class Ib: Qa-1 in mice, HLA-E in humans), which are recognized by regulatory CD8+ T cells (Treg). Previously, they had shown that some pathogenic CD4+ T cells expressed Qa-1 and were suppressed by CD8+ Treg in experimental allergic encephalomyelitis, a mouse model of multiple sclerosis. Here, they analyze the immune response to the protein hen-egg lysozyme (HEL). The susceptibility of the HEL-specific CD4+ T cells to suppression was strikingly dependent on their avidity. (Avidity was estimated by titering HEL into a fixed number of APC and measuring proliferation.) Intermediate-avidity clones, but not high- or low-avidity clones, were suppressed. Though they demonstrate that this suppression can be blocked by Qa-1-specific antibodies, they do not report the correlation with Qa-1 expression. Qa-1 has an established role as a checkpoint for classical MHC molecule expression and regulator of Natural Killer cells but they cite previous studies of a Qa-1 knockout mouse to discount these mechanisms in this model. An intriguing, though incomplete, model.
Jiang H, Wu Y, Liang B, Zheng Z, Tang G, Kanellopoulos J, Soloski M, Winchester R, Goldstein I, Chess L. "An affinity/avidity model of peripheral T cell regulation." J Clin Invest. 2005 Feb;115(2):302-12.

Picosecond Protein Movements

2007-01-27T18:21:00.000-05:00

Protein structure can be studied by crystallography or spectroscopy, which can also probe movement. Mukherjee and colleagues used 2 dimensional infrared spectroscopy (2D IR) to study the motions of a peptide on the scale of picoseconds, or trillionths of a second. (Interactions between proteins occur about a million times slower, on the mico- to millisecond time scales probed by NMR.) The 27 amino acid peptide that they studied spans the transmembrane region of CD3zeta, a signaling subunit of T lymphocyte antigen receptor. The peptide forms an alpha helix within the membrane. They synthesized 11 peptides, each with an amide [13]C labeled at a different position. Using a vibrational echo pulse sequence to resolve fast and slow molecular dynamics, they varied the delay between the pulses to measure motion on the picosecond scale. This revealed that all amino acids along the peptide showed the same relaxation times, which were similar to those observed in other peptides in solution, suggesting that this behavior is intrinsic to peptides. Comparisons with a molecular dynamic simulation confirmed that the peptides formed tetramers. They also found 2 'kinks' in the alpha helix, causing a 'funnel' structure to form in the membrane. Based on their observations of how the polar water molecules and lipid headgroups interact with the labeled amides, they predict that the IR patterns of amino acids lining the pores of transmembrane channel proteins will be easily distinguished from those facing the membrane.
Mukherjee P, Kass I, Arkin I, Zanni MT. "Picosecond dynamics of a membrane protein revealed by 2D IR." Proc. Natl. Acad. Sci. U S A. 2006 Feb 27;

Children helping Strangers

2007-01-27T16:16:00.000-05:00

Altruism is helping unrelated individuals (non-kin) without expectation of reward. Effective helping in simple tasks requires an understanding of the intentions and needs of the other person (empathy). When does the capacity for altruism develop? These investigators tested 24, 18 month old children. In one test, an adult male experimenter who was unknown to the child dropped a marker (or a paper ball, or a clothespin, or a cup) on the floor. The experimenter focused first on the object for 10 s then alternated their gaze between the object and the child while saying something like "my marker". In the control situation, the experimenter threw the object down to the floor and then looked at it with a "neutral facial expression" for 20 s. "No reward or praise" was given. Most children (22 of 24) helped in at least one task. For 6 of the 10 tasks, the children helped in the experimental situation significantly more often than in the control situation. Three young chimpanzees were also tested. All three were raised by humans and tested by their caretakers. All 3 chimps helped in some tasks and helped "reliably" in the tasks involving reaching. This was also the type of task helped most often by children. The authors concluded that young children and, to a lesser degree, chimpanzees can be altruistic.
Warneken F, Tomasello M. "Altruistic helping in human infants and young chimpanzees." Science. 2006 Mar 3;311(5765):1301-3.

Evolution and Extreme Polymorphism

2007-01-27T16:06:00.000-05:00

Many disease-related genes have been mapped to the major histocompatibility complex (MHC), a polymorphic 4 Mbp region on human chromosome 6. The extreme polymorphism of this region stymies some conventional whole genome analysis techniques. Here, Traherne et al. solved this by cloning and sequencing large chromosome fragments using bacterial artificial chromosomes (BACs). They compared in detail two new haplotypes (QBL: HLA-A26-B18-Cw5-DR3-DQ2; and COX: HLA-A1-B8-Cw7-DR3-DQ2), which share the centromeric DR-DQ alleles, to the 'reference' MHC haplotype. This region encompasses more than 259 loci and 20,000 single nucleotide polymorphisms (SNPs). They identified the site where these 2 new haplotypes recombined, probably within the last 3,400 generations. They provide evidence that recombination shuffles haplotype blocks containing certain allelic combinations favored by immunological functions. Moreover, these haplotype blocks appear to spread across haplotypes and populations through recombination suppression, selection, and population expansion.
Traherne JA, Horton R, Roberts AN, Miretti MM, Hurles ME, et al. "Genetic analysis of completely sequenced disease-associated MHC haplotypes identifies shuffling of segments in recent human history." PLoS Genetics 2(1):e9

RNA interference protects worms from viruses

2007-01-27T16:01:00.000-05:00

Caenorhabditis elegans (C. elegans) is a small worm (~1000 cells) that is widely used to study development. There are no known natural viral pathogens of C. elegans. Wilkins and colleagues suspected a protective role for RNA interference (RNAi), an anti-viral system that was discovered in plants and subsequently implicated in insect immunity. RNAi acts through small interfering RNA (siRNA) that matches the target sequence (animation from Nature). They tried infecting primary cell cultures with vesicular stomatitis virus (VSV), which is a negative sense RNA virus with a broad host range. To make the measurement easier, they used a virus marked with green fluorescent protein (VSV-GFP). The system worked: replication competent viruses infected wild-type worm cells. Infection could be monitored by fluorescence or by measuring the virus transcripts. Cells from two different mutants defective in RNAi -- one (eri-1) that reduces siRNA cleavage and a second (rrf-3) that permits siRNA amplification -- supported higher levels of virus infection and amplification. Moreover, they could detect a 20-30 nucleotide endogenous siRNA that matches the VSV nucleoprotein. They conclude that RNAi blocks virus replication and reduces virus infection and expression.
Wilkins C, Dishongh R, Moore SC, Whitt MA, Chow M, Machaca K. "RNA interference is an antiviral defence mechanism in Caenorhabditis elegans." Nature. 2005 Aug 18; 436(7053):1044-7.

Toxoplasmosis is mood altering?

2007-01-27T15:50:00.000-05:00

Recent reports in the popular press suggest that infection by Toxoplasma gondii (toxoplasmosis) may alter human behavior and cause schizophrenia. T. gondii is a common infection of domestic cats. Unfortunately, the full text of this science paper is not available for free. However, we can analyze in depth these findings from a comprehensive study of young children, comparing those who were infected congenitally with those who were not. This study was aimed at addressing understandable “parental concerns and anxiety”. In a prospective study, pregnant women and neonates were screened for toxoplasmosis between 1996 and 2000 in ten European centers. At 3 years of age, parents of children with and without congenital toxoplasmosis were surveyed about their child's development and behavior using a postal questionnaire. Parents of 178/223 (80%) infected, and 527/821 (64%) uninfected children responded. The researchers “found no evidence that impaired development or behavior were more common in infected children”. Well, that's certainly reassuring though it's not nearly as “newsworthy”.
Freeman K, Salt A, Prusa A, Malm G, Ferret N, Buffolano W, Schmidt D, Tan HK, Gilbert RE; European Multicentre Study on Congenital Toxoplasmosis. "Association between congenital toxoplasmosis and parent-reported developmental outcomes, concerns, and impairments, in 3 year old children." BMC Pediatr. 2005 Jul 13;5:23.

T cell changes in chronic infection

2007-01-27T15:35:00.000-05:00

What happens to lymphocytes that are specific for persistent infections? Most adult humans are chronically infected with several viruses, including Epstein-Barr virus and cytomegalovirus. Chronic HIV and Hepatitis C virus infections are large public health concerns. Here, investigators analyzed T cells in mice infected with one of two strains of a virus: one strain is quickly cleared while a nearly identical strain becomes a chronic infection. They found that a protein, programmed cell death 1 or PD-1, was increased on the surface of CD8+ “killer” T lymphocytes in chronically infected mice. PD-1 is previously identified as an inhibitory molecule on T cells that binds PD-L1, which is expressed on most cells in the body. (PD-1 also binds PD-L2, expressed on dendritic cells and macrophages.) PD-1 expression normally increases transiently after infection but remains elevated on specific T cells in chronically infected mice. Could persistent infections be cleared through PD-1 blockade? The final result presented here sounds a cautionary note: these viruses kill mice genetically deficient in PD-1 (knockouts), apparently due to an excessive immune response.
Barber DL, Wherry EJ, Masopust D, Zhu B, Allison JP, Sharpe AH, Freeman GJ, Ahmed R.. "Restoring function in exhausted CD8 T cells during chronic viral infection." Nature. 2006 Feb 9;439:682-7.

Many more than 23,000 Genes?

2007-01-21T22:27:00.000-05:00

A gene, the unit of heredity that encodes a trait, is hard to define in molecular terms. The best known examples of genes encode proteins that contribute to observable phenotypes, such as purple flowers or blue eyes. The genome sequencers have identified about 23,000 genes in the human genome, mostly protein coding. The FANTOM (Functional Annotation of Mouse) Consortium took a broader view, using a number of technologies to identify full length RNA transcripts with unique starts (5’) and stops (3’). They identified 181,047 independent transcripts! Previously unidentified proteins are encoded by 5,154 transcripts. Over one-half of the genome is transcribed on one or both strands without gaps, forming 18,461 transcription “forests” separated by deserts devoid of transcripts. The lengths of these transcripts show 2 major peaks around 2 kb and 20 kb and a minor peak at ~100 Mb. Unbiased analysis of transcription using “tiling arrays”, which probe continuous lengths of the genome exhaustively, have also suggested that there are many more genes than earlier estimates. The authors point out that these results raise concerns about the interpretation of microarray expression studies and gene manipulated mice (knock-ins and -outs). They conclude by estimating the enormous scale of the reevaluated genome code.
Carninci et all FANTOM Consortium; RIKEN Genome Exploration Research Group and Genome Science Group (Genome Network Project Core Group). "The transcriptional landscape of the mammalian genome." Science 2005 309:1559-63.

Neutralizing antibodies drive HIV mutation

2007-01-21T22:19:00.000-05:00

Gnome writes "HIV mutants accumulate rapidly due to a short generation time, a sloppy replication mechanism, and the large number of viruses within the host. The viral envelope (env) gene mutates especially quickly (1-2% per year) because of the selective pressure of neutralizing antibodies. The resulting heterogeneity poses a challenge to vaccine development. Frost and colleagues sought to understand the patterns of rapid mutation and escape by analyzing 13 people recently infected with HIV. They compared the initial env sequence with sequences obtained at yearly intervals and measured neutralizing antibodies in the blood. They found that HIV escape from neutralizing antibodies strongly correlated with changes in the amino acid sequence of env. Escape did not correlate with changes in N-linked glycosylation sites or insertions or deletions (indels). Their data support a simple model for escape from neutralizing antibodies proposed by Haraguchi and Sasaki, which incorporates cross-reactivity. The inability of the immune system to 'catch up' with the continually escaping virus is an example of the 'Red Queen' (running to stay in place) evolutionary dynamics.
Frost SD, Wrin T, Smith DM, Kosakovsky Pond SL, Liu Y, Paxinos E, Chappey C, Galovich J, Beauchaine J, Petropoulos CJ, Little SJ, Richman DD. "Neutralizing antibody responses drive the evolution of human immunodeficiency virus type 1 envelope during recent HIV infection." Proc Natl Acad Sci USA. 2005 Dec 20;102(51):18514-9.

Autoantibodies may influence memory

2007-01-21T22:12:00.000-05:00

Most lupus patients have serum autoantibodies against double-stranded DNA (dsDNA) and some suffer from cognitive impairment and mood disturbance, symptoms of neuropsychiatric lupus (NPSLE). These researchers previously used phage display to find a pentapeptide that crossreacts with a pathogenic, monoclonal, dsDNA-specific autoantibody. They further found that the mouse and human receptors for the neurotransmitters NMDA (N-methyl-D-aspartate) and glutamate, NR2a and NR2b, contain this pentapeptide. They also showed that mice immunized with the pentapeptide developed antibodies that bound NR2 but did not cause neural injury unless the blood-brain-barrier (BBB) was disturbed. Similar antibodies can be detected in the cerebrospinal fluid of SLE patients but not healthy control subjects. Administering LPS to these immunized mice opened the BBB in the hippocampus, leading to antibody binding and cell loss in this area of the brain. The treated mice performed poorly in maze tests, suggesting memory impairment. Now, they show that administering epinephrine opened the BBB in the amygdala, exposing cells there to the toxic autoantibodies. The treated mice were less fearful. These may be seminal papers in understanding cognitive defects."
Huerta PT, Kowal C, Degiorgio LA, Volpe BT, Diamond B. "Immunity and behavior: Antibodies alter emotion." Proc Natl Acad Sci U S A. 2006 Jan 17;103(3):678-83.

TWEAK Regulates Cellular Immunity

2007-01-21T22:05:00.000-05:00

TNF-related weak inducer of apoptosis (TWEAK) is a member of the TNF superfamily, alias TNFSF12. The mouse gene was cloned by chance and the human gene was identified among EST clones. TWEAK is secreted by several cell types and binds, naturally, TWEAK-R, which is also known as FN14 because it is a 14 kDa protein that is inducible by fibroblast growth factor. FN14 is also distantly related to the TNFR family. Despite all this talk of families, TWEAK was an orphan when it came to functional relationships. Now, Maecker and colleagues report that TWEAK-knockout (TWEAK-KO) mice have altered immune systems. Specifically, the deficient mice have more natural killer cells and T helper type 1 (Th1) cells and their stimulated lymphocytes secrete more interferon-gamma and interleukin-12, suggesting that TWEAK normally acts to reduce these responses. TWEAK-KO mice succumb to shock caused by endotoxin, the bacterial cell wall component lipopolysaccharide (LPS), at lower doses than normal mice. On the other hand, TWEAK-KO mice resisted transplanted tumor cells better than normal mice. The authors also observed that TWEAK stimulates the rapid and prolonged association of NF-kappaB p65, a key transcription factor and intracellular signal, with the histone deacetylase HDAC-1, whereas TNF stimulates p65 association with the transciption coactivator p300. This intriguing observation is the likely molecular basis for the difference between TWEAK and TNF.
Maecker et al "TWEAK attenuates the transition from innate to adaptive immunity." Cell. 2005 123:931-44

What makes stem cells special?

2007-01-21T21:58:00.000-05:00

Stem cells are self-renewing and pluripotent, meaning that they can develop into any of the over 200 unique cell types in the body. Three transcription factors, Oct4, Sox2, and Nanog were previously identified as crucial for stem cell development. Oct4 binds Sox2 and together they regulate Nanog. Boyer and colleagues used chromatin immuno-precipitation (ChIP) to determine which genes these proteins bind (and presumably regulate). They found that they bind the promoters of many of the same genes and that many of these genes encode transcription factors that are important in development. Several genes encoding micro RNAs (miRNA) were also bound, which is particularly interesting since stem cells lacking miRNA processing cannot differentiate. Oct4 and Sox2 can activate or repress gene transcription. After correlating with expression profiles, the 3 factors bound 1303 active genes and 957 inactive genes. If the master switches and pathways are identified, it may be possible to convert any cell into a stem cell by activating the correct, hopefully few, genes.
Boyer et al Core transcriptional regulatory circuitry in human embryonic stem cells." Cell. 2005 Sep 23;122(6):947-56

ATP Generator Structure – Function

2007-01-21T21:49:00.000-05:00

Almost incredibly, a sedentary adult makes and uses 40 kg of ATP per day! ATP is made by the F0-F1 ATPase, a molecular motor with a rotating shaft and fixed "stator". One end of the shaft, F0, is buried in the mitochondrial inner membrane where the proton gradient causes it to rotate. A single gamma subunit connects the F0 to three alpha and beta subunits, together F1, which are responsible for synthesizing ATP from ADP and Pi (H2PO4-). As a catalytic motor and not just a catalyst, the F0-F1 ATPase is able to increase the rate of reaction away from the equilibrium (which strongly favors the reverse reaction, hydrolysis, because the concentrations of reactants and product in mitochondria are similar). The gamma subunit rotates too slowly, in the microsecond-to-millisecond range, for standard molecular dynamics simulation. To solve this, the authors applied "biasing forces" as the motor moved and assumed these forces would not change the mechanism. Positively charged amino acids on the gamma subunit attract negative amino acids on beta subunit, producing smooth and efficient ionic coupling. Rotation of the gamma subunit induces the opening of the beta subunits. The beta subunit closes spontaneously. Synthesis is not the reverse of hydrolysis, explaining why high concentrations of free ATP does not inhibit synthesis. Gao and colleagues propose a detailed model of how the motor harnesses the proton gradient to act against the equilibrium. Their quantitative model is based on the conceptual “binding change mechanism” model proposed by Boyer, where ATP synthesis proceeds by each beta subunit changing from “open”, weak nucleotide binding, to “tight”, high affinity ATP binding, to “loose”, with the release of ATP. The authors used this model to make accurate predictions about synthesis and hydrolysis kinetics and they invite others to test their detailed model.
Yi Qin Gao, Wei Yang and Martin Karplus, "A Structure-Based Model for the Synthesis and Hydrolysis of ATP by F1-ATPase" Cell Oct 21, 2005; 123(2):195-205

Biology of the 1918 Flu

2007-01-21T20:56:00.000-05:00

Gnome writes "The 1918 flu caused an unusually high death rate among healthy young adults (ages 15-34). Genomic RNA was recovered several years ago from autopsy material and victims buried in the Alaskan permafrost. The 1918 flu had unique hemagglutinin and neuraminidase proteins on its surface (H1N1). However, they did not cause its pathogenicity because later viruses with this combination are not particularly deadly (e.g., Texas/91). Now, Tumpey and colleagues have reconstructed the entire 1918 virus and tested it. Unlike current flu viruses, the 1918 virus does not require an exogenous protease to infect cells in culture. Moreover, all 13 mice infected through the nose by the 1918 virus developed severe lung inflammation and died within 7 days, while none infected with the Texas/91 strain died. Surprisingly, recombinants between 1918 flu and Texas/91 suggest that the viral polymerase acts in concert with the other 7 proteins to increase virulence. On the bright side, existing antiviral drugs and a novel siRNA therapy were previously shown to protect mice against certain 1918 recombinant viruses.
Tumpey et al., "Characterization of the Reconstructed 1918 Spanish Influenza Pandemic Virus" Science. 2005 Oct 7;310(5745):77-80.

1 protein = 2 opposing weight-control hormones

2007-01-21T20:49:00.000-05:00

Obesity, a body mass index over 30 kg/m^2, is increasing in America and many western societies. Obesity is correlated with poor health, including diabetes and cardiovascular disease. Body weight is regulated by peptide hormones secreted from the gut and brain. Zhang and colleagues noted that ghrelin, a 28 amino acid, gut-derived peptide that stimulates appetite, is post-translationally trimmed from a larger, 117 amino acid protein. They identified in this larger protein not only a signal sequence but also a whole new, 23 amino acid peptide downstream of a potential convertase cleavage site. They named this second protein obestatin, for obesity-suppressing. They found obestatin in rat gut tissue. Whereas fasting rats had more ghrelin in their blood and fed rats had less, obestatin was stable between the groups. Injecting mice with synthetic obestatin blocked ghrelin-induced weight gains. Finally, they identified the hitherto orphan GPR39 as the G protein-coupled receptor. One gene encodes one protein that yields two antagonist peptides! This story promises to be an interesting and important example of post-translationally regulated protein expression.
Zhang et al., "Obestatin, a Peptide Encoded by the Ghrelin Gene, Opposes Ghrelin's Effects on Food Intake" Science 11 November 2005.

Interleukin-1 in Systemic Juvenile arthritis

2007-01-21T20:36:00.000-05:00

Juvenile idiopathic arthritis (JIA), joint inflammation with unknown cause in children, affects an estimated 250,000 children in the US alone. About 10% of these cases begin with fevers and/or rash that precede joint symptoms by months or years, a subset called systemic onset JIA (SoJIA). These are hard to diagnose and most do not respond to TNF blockade, a therapy that helps many adult rheumatoid arthritis patients. Here, Pascual and colleagues show that the disease is instead driven by the other major proinflammatory cytokine, interleukin 1 (IL-1). They found that activate peripheral blood leukocytes from SoJIA patients make more IL-1 than do those from healthy people. Sera from SoJIA patients increased the expression of immune-related genes by leukocytes from healthy people. Most strikingly, an IL-1 receptor antagonist (IL-1Ra, Anakinra) reduced fever, arthritis, and other symptoms or markers of disease in all 9 SoJIA patients treated.
Pascual et al., J Exp. Med. 201(9) 1479-1486.

Publishing your mistakes?

2007-01-21T20:11:00.000-05:00

Most scientists suspect that erroneous results occasionally make their way into publication. Now, Ioannidis confirms these suspicions, and worse, with statistical simulations. In a remarkably readable paper, he shows that "most published research findings are false"! Not surprisingly, bias increases the chances that the finding is false and large effects are more likely to be true than are small effects. Less expected is the conclusion that "a research finding is less likely to be true... when more teams are involved in a scientific field". In other words, "hot" fields have more mistakes published. The analysis is straightforward and may suggest reasonable ways to reduce the errors in publications.
Ioannidis, JPA. August 2005 PLoS Medicine 2(8):696-701

Insulin-Specific T Cells in Long-Term Diabetics

2007-01-21T19:57:00.000-05:00

Type 1 diabetes is unusual among autoimmune diseases because diagnosis is greatly aided by laboratory tests. Levels of blood sugar and glycosylated hemoglobin are excellent measures of disease progression. The symptoms of disease are treated with insulin, which would normally be produced by the pancreatic beta cells that are destroyed in the autoimmune process. In common with other autoimmune diseases, the cause of type 1 diabetes is not known, though insulin itself has been a leading candidate autoantigen. Here, Kent and colleagues show they could find insulin-specific T lymphocytes in the lymph nodes draining the pancreas in 2 long-term diabetics, but not in 3 non-diabetic individuals or 1 recently-diagnosed diabetic. They argue against the T cells having been stimulated by therapeutic insulin because none were found in the spleen of one diabetic. They convincingly demonstrate that the T cell response is specific for the insulin peptide (residues 1-15 of the A chain) bound by host histocompatibility molecules (HLA-DR). The insulin-specific T cells were helper (CD4+) cells and, curiously, secreted a suspected tolerogenic helper type 2 cytokine (interleukin-13) and not a proinflamatory helper type 1 cytokine (interferon-gamma) in vitro. These observations strengthen the argument that long-term diabetes correlates with increased immune reactivity against
insulin.
Kent et al., Nature, May 12, 2005. 435(7039):224-8.

Alternate splicing may generate a fly immune receptor

2007-01-21T19:48:00.000-05:00

Fruit flies have a large protein called Dscam (Down syndrome cell adhesion molecule) with 24 exons. Four of these exons are alternatively spliced from 12, 48, 33, and 2 variants, potentially generating over 18,000 slightly different Dscam proteins. The protein is expressed by brain cells and cells involved with protection against infectious agents: called fat bodies and hemocytes. Dscam is found on the surface of these cells and the extracellular portion of the protein, clearved from the cytoplasmic portion, is found in the insect equivalent of blood, called the hemolymph. The function of Dscam was not known, but Schmucker and colleagues suspected a role in immune protection. They found that many of the gene variants are expressed in the fat bodies and hemocytes, though even more variants are expressed in the brain. Most strikingly, reducing expression of Dscam with RNA inhibition or using null mutants reduces the phagocytosis of bacteria by hemocytes. Two Dscam variants, but not a third, bind one strain of bacteria, supporting the notion that these variants might protect against different pathogens. Moreover, the alternative splicing of Dscam is conserved in other insects. Although the human homologue is not alternatively spliced, might there be other Dscam-like receptors, analogous to Toll-like-receptors (TLRs) in the mammalian genome?"
Watson et al., Science. 2005 Sep 16 309(5742):1874

Immune Recognition of Vascular Endothelial Cells

2007-01-21T17:44:00.000-05:00

Immunologists think about central or peripheral tolerance, depending on whether the potential antigen is found within the lymphoid tissues. The central immune system includes the bloodstream, lymph, and primary lymphoid organs such as the thymus and lymph nodes. From an immunologist's perspective, the "periphery" is all the body's non-lymphoid organs and tissues. This distinction overlooks the interface between these tissues - the endothelium - the single cell layer that lines the blood and lymph vessels. Given the pervasiveness of blood vessels, it is not surprising that endothelial cells (EC) have been implicated in many immune processes, most notably atherosclerosis and transplant rejection. These investigators directly tested the ability of the immune system to recognize a new protein expressed by EC using two strains of transgenic mice that express beta-galactosidase only in their EC because their genomes have the beta-gal gene, lacZ , under control of promoters specific for endothelial cells. One transgenic line, VWF-lacZ, expresses beta-gal only in heart and brain endothelium. A second line, TIE2-lacZ, expresses beta-gal in practically all endothelial cells. Upon immunization with beta-gal protein, these mice responded normally, generating strong antibodies and T cell responses. They also responded normally after immunization with a DNA vector driving expression of beta-gal. After immunization, vascular EC in the transgenic mice continued to express beta-gal and the vessels showed no signs of inflammation when analyzed microscopically. This suggested the EC were simply ignored. However, TIE2-lacZ skin (which contains beta-gal+ EC) stimulated
beta-gal specific antibodies when it was transplanted onto non-transgenic littermates. Moreover, no antibodies were stimulated upon transplantation onto VWF-lacZ mice, suggesting that the immune system in these mice were tolerant, not ignorant, of the beta-gal expressed by EC. They conclude that the immune system subtly tolerates and does not ignore EC proteins.
Annette L Rothermel, Yinong Wang, Jeffrey Schechner, Barry Mook-Kanamori, William C Aird, Jordan S Pober, George Tellides and David R Johnson. Endothelial cells present antigens in vivo BMC Immunology March 2004, 5:5

Mitochondrial tRNA mutant causes metabolic defects

2007-01-21T11:45:00.000-05:00

Mitochondria are cellular organelles that use oxidative phosphorylation to convert the energy of reduced dietary carbon into ATP, which provides energy for practically all processes. Human mitochondria possess a mostly vestigial 16 kb genome, which encodes ribosomal and 22 transfer RNAs along with 13 proteins. There are more mitochondria in cells with high energy needs such as heart muscle cells, where mitochondria and myofibrils make up 85% of cell volume. Cardiovascular disease is associated with high blood pressure and high serum cholesterol. These investigators analyzed the genetics of a syndrome that includes hypertension (blood pressure >140/90 mm Hg), hypercholesterolemia (serum cholesterol >200 mg/dl), and hypomagnesemia (serum magnesium below 1.8 mg/dl) in 4 generations of a large family. A genome-wide linkage
analysis yielded no candidates. They found that the 32 family members of both sexes with hypomagnesemia all descended from one female, suggesting a mitochondrial defect. Upon sequencing and single-strand conformational polymorphism analysis, they found 14 variants, 13 of which were previously described, and one new variant at nucleotide 4291 of the mitochondrial genome, within an isoleucine tRNA. This tRNA mutation is found only on this maternal lineage, not among the thousands of previously sequenced mitochondrial genomes.
Wilson et al. Science 306: 1190, November 2004

Common infection might set stage for lupus

2007-01-21T11:00:00.000-05:00

Infection is known to trigger certain autoimmune diseases, e.g., rheumatic fever and ankylosing spondylitis. The initial immune response against the pathogen is thought to also recognize "self" proteins called autoantigens and may later spread to recognize additional autoantigens. These investigators wished to test their suspicion that Epstein Barr virus (EBV) triggers systemic lupus erythematosus (SLE). They previously showed that the first SLE autoantigen is the RNA-binding protein Ro. Here, they analyzed blood samples from a repository drawn from more than 5 million people. They found 29 people who later developed lupus and who had donated a blood sample before they developed Ro-specific autoantibodies. Nine of these early sera contained autoantibodies that recognized only a single small portion of Ro. All of these early, Ro-specific antibodies cross-reacted with a completely different small fragment of the EBV nuclear protein EBNA-1, suggesting that the crossreaction could be an early event in the disease. To test this, they immunized rabbits with peptides matching Ro or EBNA-1. Strikingly, both groups of rabbits developed lupus-like symptoms, including reduced numbers of white blood cells (leukopenia), clotting factors (thrombocytopenia) and kidney function. This paper connects a common infection with lupus. It remains to be determined why lupus develops in relatively few of the many EBV infected adults.
McClain et al. Nat.Med. 11, 85 - 89 (2004) "Early events in lupus humoral autoimmunity suggest initiation through molecular mimicry"

Defective Sweet Taste Receptor in Cats

2007-01-07T11:24:00.000-05:00

Most mammals can taste bitter, sweet, salty, sour, and amino acids, such as glutamate (umami). The receptors for sweet and umami are paired G proteins, called T1Rs, that are expressed exclusively in the taste buds. T1R1 and T1R3 together respond to the umami taste. Similarly, T1R2 and T1R3 together recognize diverse natural and synthetic sweeteners. T1R2 and T1R3 are encoded by the genes Tas1r2 and Tas1r3. Cats, unlike puppies, do not prefer sweet foods. Previous work had demonstrated that alleles of Tas1r3 predict sugar intake. These investigators tested a simple hypothesis: cats are indifferent to sugar because their sweet taste genes are defective. Here, they show that the cat Tas1r2 gene has a large deletion in the coding region and no messenger RNA can be detected. They found similar deletions in the genes of tiger, cheetah and domestic cats and conclude that this is important in the evolution of the cat as carnivore.
Li et al. PLoS Genetics Volume 1 | Issue 1 | JULY 2005"

Vitamin D and Autoimmunity

2007-01-03T23:56:00.000-05:00

Vitamin D deficiency has been implicated in several autoimmune disease, including systemic lupus erythematosus (SLE), type 1 diabetes, rheumatoid arthritis, inflammatory bowel disease and multiple sclerosis (MS). Munger and colleges tested the vitamin D levels in the serum samples collected in the US Army repository. They found that "high circulating levels of vitamin D are associated with a lower risk of multiple sclerosis". When they investigated cause-and-effect, they found:

“Average 25- hydroxyvitamin D levels among individuals who developed MS were stable during the years preceding symptom onset (P=.42 for trend) but significantly decreased after onset of symptoms (P=.002). Mean 25-hydroxyvitamin D levels were 71.8 nmol/L more than 6 years before symptom onset (51 cases),…”

Surprisingly, they continue:

“These results argue against the possibility that the low preclinical 25-hydroxyvitamin D levels among individuals with MS are a consequence rather than a cause of the disease,..."

This finding seems to suggest that MS causes the decrease in vitamin D levels, not vice versa. Also, the average levels preceding the development of symptoms (71.8 nmol/L) is actually protective (figure). This seems inconsistent with a conclusion that low vitamin D contributes to MS (a conclusion they do not draw explicitly, instead stating they have established an “association”).

Toll-free immune responses

2006-12-26T20:30:00.000-05:00

Toll receptors mediate invertebrate protection against bacteria and yeast. Homologous mammalian molecules, the “Toll-like receptors” (TLR), were identified almost a decade ago and were immediately implicated in immunity and autoimmunity. TLRs were thought to provide crucial adjuvant stimulation to trigger the specific adaptive immune response. So you can imagine their surprise when Nemazee and colleagues found that mice mice without functional TLRs nevertheless made strong antibody responses against antigens in four typical adjuvants, including Freund’s Complete Adjuvant (FCA) (Figure). These mice were genetically deficient in both MyD88 and TRIF, which mediate all known TLR signaling. Mice were immunized with the antigen trinitrophenol - hemocyanin (TNP-Hy) in Freund's complete adjuvant. Similar results were obtained with another common experimental antigen, TNP-KLH (keyhole limpet hemocyanin). The deficient mice also responded normally following TNP-Hy immunization with the adjuvant alum, which is used in humans. Moreover, the TLR deficient mice also respond strongly against adjuvants containing TLR ligands. The authors modestly state that these findings “may have important implications in the use and development of vaccine adjuvants”.

Physiology

2006-12-21T12:49:00.000-05:00

The NIH has a treasure of video recordings of biomedical lectures available for viewing. One recent lecture by Dr. James Bassingthwaite (U Washington) was an enjoyable tour of physiology, the thinking man's biology, and introduction to the 'Physiome' Projects. Though physiology is rarely mentioned these days, replaced by newer terms such as "systems" or "integrative" biology, he reminds us that physiology has always focused on connecting different branches of biology, answering the how and why in biology. He makes a strong case for sharing models and working together to improve them. The Physiome projects provide the JSim software and many models that run on JSim. The lecture's scope is broad, ranging from modeling to personalized medicine, always with a foot or two solidly planted on the ground.

CNS and centrality of the cytoskeleon

2006-12-12T13:00:00.000-05:00

That cytoskeletal proteins provide a fundamental organising principle for a cell has been appreciated for a long time. However, the centrality of these proteins in disease progression and aging has been realised more recently. Ikeda and coworkers have reported that mutations in bIII spectrin, a cytoskeletal protein expressed abundantly in Purkinje cells, result in spinocerebellar ataxia type 5 (SCA5). Interestingly, the pedigree studied was from President Lincoln's family. The mutations reported result in destabilization of the glutamate transporter EAAT4 resulting in impaired glutamate signalling. While mutations in subunits of the EAAT4 have been shown to affect their assembly and therefore their function, this report draws attention to the importance of the cytoskeleton in proper assembly and stability of subcellular organelles and domains.

Modeling terror: botulism in milk

2006-11-25T23:43:00.000-05:00

Gnome writes "What if the milk from a single processing plant were poisoned with botulism? Poisoning cold drinks with botulinum toxin is one of the 3 “greatest threats to humans” (along with smallpox and airborne anthrax attacks) according to some experts. Authors Wein and Liu analyzed this question mathematically using the supply-chain values of the California dairy industry (which produces >20% of the milk in the US). With “enough” toxin and without “timely” diagnosis, they predict a catastrophe. For example, <1 g of toxin could cause 100,000 poisonings. Ten grams could kill over a half million people. Particularly chilling is that “due to children's higher consumption rate and greater toxin sensitivity”, they estimate 61% of the dead would be children (1 g). They recommend testing and perhaps altered heat pasteurization to inactivate the toxin at a total cost of less than 1 cent per gallon. The publication of this paper was controversial. Ironically, the full editorial on the balance between openness and secrecy, which discusses the decision to publish this paper, is not freely available.
Wein and Liu PNAS July 12, 2005 vol. 102:9984 Analyzing a bioterror attack on the food supply: The case of botulinum toxin in milk"

Genes induced by SARS infection

2006-11-25T23:33:00.000-05:00

drj writes "SARS (severe acute respiratory syndrome) is a form of pneumonia with a mortality rate of ~10%. No new cases have been diagnosed since mid-2004, quelling fears of a pandemic. It remains unclear why this coronavirus was is so infectious and deadly. Here, Reghunathan and colleagues analyzed the genes induced in peripheral blood mononuclear cells (PBMC) of 10 SARS patients. They report that 186 genes are expressed at different levels in the PBMC of SARS-infected people compared to PBMC from uninfected people. Several genes are strongly induced, including those encoding lactotransferrin (LTF), a marker of neutrophil degranulation, the calcium binding protein S100A9, which regulates monocyte and neutrophil migration, and the pro-apoptotic protein lipocalin 2 (Lcn2). Surprisingly, genes encoding inflammatory cytokines (such as the interferons) and other immune-related genes (such as HLA class I) are not induced. This unusual response to viral infection may contribute to the pathogenesis of SARS. Reghunathan et al. BMC Immunology 2005, 6:2 Expression profile of immune response genes in patients with Severe Acute Respiratory Syndrome"

Neutrophil DNA traps pathogens

2006-11-25T23:20:00.000-05:00

drj writes "Most white blood cells are neutrophils, which are small, very short lived (hours-days) cells that are crucial in immune defense. Once triggered, they leave the bloodstream and enter inflamed tissues where they engulf and kill bacteria and fungi with hydrogen peroxide and protein toxins. Now, Brinkmann and colleagues show that stimulated neutrophils also produce DNA-containing fibers that can trap and kill bacteria. They suggest these fibers, which they named neutrophil extracellular traps (NETs), are key mediators of anti-bacterial activity. Dead and dying neutrophils are major constituents of pus, from which DNA (‘nuclein’) was discovered in 1869 by Friedreich Meisher.
Brinkmann et al. Science. 2004 Mar 5;303(5663):1532-5. Neutrophil extracellular traps kill bacteria"

Bird song’s genetic key

2006-11-25T23:05:00.000-05:00

drj writes "Canary (Serinus canaria) songs consist of arranged phrases, themselves composed of repeated, shorter tonal whistles called syllables. Syllables are learned and then arranged into sequences that are unique to each species. This structure is found even in birds raised in isolation, though their repertoire of syllables is smaller. If tutored, isolated birds can instead learn songs without such structure. Gardner et al. now show that imitated songs are remixed into the innate canary song structure as tutored male birds develop into adults. Testosterone accelerated the song restructuring and sexual maturity. The authors provide this maxim: freedom in youth, rules in adulthood . The songs of the juveniles, adults, tutor, and tutored birds are in the on-line supplementary materials.
Gardner et al. Science 308:1046, 13 May 2005 "Freedom and Rules: The Acquisition and Reprogramming of a Bird's Learned Song"

Chemokine levels influence virus infection

2006-11-25T22:39:00.000-05:00

drj writes "HIV-1 infects cells by binding to CCR5, a cell surface receptor for the immune-signaling protein (chemokine) CCL3L1 (MIP-1alpha). One allele (version) of the CCR5 gene has a 32-bp deletion (CCR5delta32). People with two copies of CCR5delta32 are relatively resistant to HIV-1 infection and even one copy delays progression to AIDS. A relatively high frequency of the CCR5delta32 allele in people of European origin (10%) might be due to selection by plague or hemorrhagic fever. Now, Gonzalez and colleagues show that variation in the copies of the gene encoding CCL3L1 is also important. People with more copies of the CCL3L1 gene are more resistant to HIV and AIDS. (This correlation may not hold in some populations.) The simple explanation is that CCL3L1 competes with HIV-1 for binding to CCR5. It's surprising that a receptor can be occupied enough to make a difference. CCR5 is already a target for therapy with small molecule inhibitors. Unfortunately, HIV-1 has demonstrated its ability to develop resistance to some small molecule inhibitors of CCR5.
Gonzalez et al. Science. 2005 307(5714):1434-40. The influence of CCL3L1 gene-containing segmental duplications on HIV-1/AIDS susceptibility.

Multi-pathway cancer inhibition

2006-11-25T22:26:00.000-05:00

Gnome writes "Non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancers, the leading cause of death due to cancer. A growth factor (TGF-alpha) is thought to promote NSCLC proliferation through binding to the epidermal growth factor receptor (EGFR), which is found at high levels on the tumor cell surface. A competitive inhibitor of EGFR, gefitinib (trade name “Iressa”), failed in clinical trials but physicians reported dramatic improvement in ~10% of patients. Surprisingly, further investigation revealed that these patients’ tumors nearly all carried mutations around the ATP-binding site of EGFR, precisely the target of the drug. Several different mutations were detected. [This contrasts with a similar drug imatinib (STI-571, Gleevec), where mutations or amplifications of the target kinase reduce drug efficacy against leukemias (CML)]. Unfortunately, the benefit of gefitinib is transient (average 7 months) because additional EGFR mutations arise. Here, Kwak and colleagues show that gefitinib-resistant mutants are sensitive to non-competitive (irreversible) EGFR inhibitors named HKI-272, HKI-357, and EKB-569. These inhibitors alter the movement of EGFR in the cell. Although these studies must be extended to animals, targeting several pathways is well established, successful strategy in anti-viral treatment.
Kwak et al. PNAS 102:7665 (2005) Irreversible inhibitors of the EGF receptor may circumvent acquired resistance to gefitinib"

Chemotherapy for Poxvirus

2006-11-22T19:00:00.000-05:00

Anonymous Coward writes "Antiviral medications typically target enzymes encoded by the virus, such as reverse transcriptase or proteases. Now, viral dependence on host cell functions is being exploited. Poxviruses, the largest known animal viruses (~200 genes), encode growth factors that bind cellular receptors and increase virulence. For example, the smallpox growth factor tightly binds ErbB-1 (0.14 nM), the cell's epidermal growth factor receptor that also binds smallpox itself. Yang et al. now report the identification of a small molecule inhibitor of the ErbB-1 protein tyrosine kinase. The inhibitor is a synthetic molecule, called CI-1033, that blocks the ATP-binding site of ErbB-1 (IC50 = 0.8 nM). Although CI-1033 did not inhibit replication of a related poxvirus, vaccinia virus, it reduced ErbB-1 activity and intercellular spreading. The most convincing proof of principle came from mice treated with CI-1033 before exposure. They were protected from an otherwise fatal pneumonia following infection with vaccinia. The window of protection is narrow and protection required continuous treatment.
Most human tumors possess ErbB abnormalities, a fact that has promoted the development of orally-administered, small molecule inhibitors for cancer therapy (Gleevec, Iressa). This paper suggests a potential dual use for these medications. Fauci and Challberg provide a comprehensive introduction. See also the thorough review of poxviruses growth and antivirals from Harrison et al.
Yang et al. J. Clin. Invest. 115:379-387 "Antiviral chemotherapy facilitates control of poxvirus infections through inhibition of cellular signal transduction".

"Down Syndrome Critical Region" isn't?

2006-11-22T18:52:00.000-05:00

Gnome writes "Down syndrome (DS) results from the most common viable human chromosomal abnormality, occurring in 1 out of 700 live births. In addition to an entire extra chromosome 21 (free trisomy), DS is also caused by translocated or duplicated portions of chromosome 21. In 1987, the "Down Syndrome Critical Region" (DSCR) was mapped to the short arm of chromosome 21 using quantitative Southern blots of genomes of DS patients and rare patients with ostensibly normal karyotypes. The DSCR was later refined as a 5.4 megabase region around 21q22.3. Known genes in the DSCR are highly conserved between humans and mice (on chromosome 16). Chimeric mice containing a human chromosome 21 exhibit learning deficits and cardiac defects reminiscent of Down syndrome. Even the fruit fly homolog of the DSCR regulates learning. Now, investigators generated transgenic mice with precise DSCR trisomy or monosomy by clever use of asymmetric Cre-Lox sites. They report that mice with three copies of the DSCR do not show the same facial skeletal abnormalities found in mice with larger duplicated regions. Surprisingly, trisomic DSCR mice are larger and monosomic DSCR are smaller than normal, though mice trisomic through a ~3-fold larger, overlapping region are smaller than normal. The authors state that these results refute the predictions of the nearly 3 decade old hypothesis that DS is caused by dose effects of genes within the DSCR."
Olson et al. Science 2004 Oct 22;306(5696):687-90
A Chromosome 21 Critical Region Does Not Cause Specific Down Syndrome Phenotype

Brain wiring patterns

2006-11-22T18:32:00.000-05:00

Gnome writes "Complex electronic circuits designed by humans are composed of many repeated, simpler modules such as amplifiers or logic gates. Does the brain have a similar organization? Reigl and colleagues looked for such patterns in C. elegans, a worm whose nervous system wiring of its 302 neurons could be analyzed from electron micrographs of serial sections. Here, they analyzed the frequency of connection patterns up to 5 neurons. [They concede that their analysis would miss singular circuits, such as a crucial “rectifier in a power supply” (returning to the electronic analogy).] Nevertheless, they found that certain connection patterns between 2, 3, and 4 but not 5 neurons occurred more often than chance. For example, a triplet where 1 neuron stimulates 2 neurons, one of which stimulates the other, occurs much more often than expected. They conclude that these motifs could perform stereotypical functions in the worm nervous system. Many useful circuits need many more connections.
Reigl et al., BMC Biology 2004, 2:25
Search for computational modules in the C. elegans brain

Mitochondria and Cardiovascular disease

2006-11-22T18:26:00.000-05:00

Gnome writes "Aerobic capacity (fitness) correlates with less cardiovascular disease. Is aerobic capacity inherited? Here, rats from a genetically “heterogeneous” stock were selected for endurance running. Low and high capacity lines were bred. Investigators measured a 3.5-fold difference in aerobic capacity after 11 generations. Capacity of both lines changed through the generations, from the starting average 355 m to exhaustion to a low of 191 m and a high of 853 m. Poor runners had higher blood pressure, higher serum triglycerides, and lower glucose tolerance than good runners. Training didn't help poor runners much. Several mitochondrial proteins are much lower in poor runners and the authors attribute heritable decreases in fitness to decreased mitochondia function. Since mitochondria are maternally inherited, it would be interesting to compare the offspring of high capacity mothers with low capacity fathers and vice versa.
Wisloff et al. Science 2005 Jan 21;307(5708):418-20
Cardiovascular risk factors emerge after artificial selection for low aerobic capacity.

Mouse Lupus prevented with FcgRIIB

2006-11-20T17:39:00.000-05:00

drj writes "Mice from some lupus-prone strains make less of an inhibitory antibody receptor (FcgRIIB). Here, mice from these strains were given bone marrow cells that express more FcgRIIB. They lived longer, did not develop as many autoantibodies, and did not develop kidney inflammation (glomerularnephritis). In these experiments, the bone marrow cells from mice representing 3 different lupus models (NZM, BXSB, and B6 FcgRIIB-knockout) were infected (transduced) with retroviruses encoding FcgRIIB. Only about 40% of the B cells made FcgRIIB and the actual increase in expression was "modest" (~43% on B cells; on knockout B cells the increase was presumably qualitative). FcgRIIB may prevent lupus by limiting the development of autoantibodies. Human variations (polymorphisms) that predispose to lupus have been identified in FcgRIIA and FcgRIIIA (review), suggesting a role in human disease. It remains to be seen whether in addition to preventing the onset, retroviral transduction could cure lupus (gene therapy)."
McGaha et al., Science 307:590-593, Jan. 2005 "Restoration of Tolerance in Lupus by Targeted Inhibitory Receptor Expression".

TRAIL-R attenuates innate immunity

2006-11-20T17:11:00.000-05:00

drj writes: "TRAIL, officially known as TNFSF10, induces apoptosis of virus-infected or transformed cells but not normal cells, suggesting a potentially "magic bullet" therapeutic. TRAIL expression is inducible on immune effector cells: Natural Killers (NK) and T cells, macrophages, and dendritic cells. TRAIL knockout mice are more susceptible to tumor metastasis and chemical carcinogenesis. TRAIL-R (receptor) expression is apparently induced by transformation or infection.
Here, Diehl and collegues describe the generation and characterization of TRAIL-R knockout (TRAIL-R-KO) mice. (Mice have only one signaling receptor; humans have two: TNFRSF10A, B.) TRAIL-R-KO mice have normal lymphocyte populations but produce more of the cytokines IL-12, IFN-alpha, and IFN-gamma and clear cytomegalovirus more rapidly. TRAIL-R-KO macrophages stimulated with pathogen-derived molecules (e.g., LPS) though the Toll-like receptors show increased TRAIL expression and enhanced cytokine production. Although early signaling is normal, activation of the transciption factor NF-kB and degradation of its inhibitor IkB-a is prolonged (12 h).
What is the point of inhibiting the innate response? Unlike the adaptive response, there are no expanded populations of cells to inactivate and remove." Diehl et al., Immunity 21:877-889, December, 2004
TRAIL-R as a Negative Regulator of Innate Immune Cell Responses

Interleukin-7, T cells, and Rheumatoid Arthritis

2006-11-20T17:03:00.000-05:00

drj writes "Transient lymphocyte depletion is an effective therapy for some autoimmune diseases, including
juvenile rheumatoid arthritis and multiple sclerosis (MS). Lymphocytes are usually replenished by treatment with growth factors after depletion or with stem cells after ablation. However, CD4 T cells remain depleted for extended periods following therapy of rheumatoid arthritis (RA). Ponchel and colleagues now demonstrate significantly reduced levels of circulating interleukin-7 (IL-7), a T cell growth factor , in the sera of RA patients. Serum IL-7 increased transiently in cancer patients but not RA patients following lymphocyte depletion and autologous stem cell reconstitution.
It would be relatively easy to supplement RA treatment with recombinant IL-7. As the authors acknowledge, however, this would require careful monitoring since IL-7 has been implicated in MS, lymphoma and other cancers."
Ponchel et al., Arthr. Res. & Ther. 7, 2005
"Interleukin-7 deficiency in rheumatoid arthritis: consequences for therapy-induced lymphopenia"

Gut check time?

2006-11-07T12:00:00.000-05:00

drj writes "Dieters complain about growing fatter despite eating less and exercising more. Backhed and colleagues now provide surprising support for half this claim. Germ-free mice “conventionalized” with microorganisms from the guts of normal mice increase their body fat 60% in just 2 weeks despite eating less. Activity levels were not reported. The bacteria were transmitted by spreading the contents of the distal intestine from normal mice onto the fur of germ-free mice. The microorganisms apparently act by increasing the uptake of sugars from the gut into the bloodstream. Adaptive immunity is not involved in this response because conventionalized Rag1-knockout animals behave similarly. The authors propose that Western diets may predispose to obesity by promoting certain gut microorganisms.
Will obesity, like ulcers caused by Helicobacter pylori, now be understood as having a key microbiological component? Is there a market for bugs from skinny people?"
"The gut microbiota as an environmental factor that regulates fat storage" Fredrik Backhed, Hao Ding, Ting Wang, Lora V. Hooper, Gou Young Koh, Andras Nagy, Clay F. Semenkovich, and Jeffrey I. Gordon. Proc Natl Acad Sci U S A. 2004 Nov 2;101(44):15718-23. PMID: 15505215

Deamidation & Deimination in Disease

2006-11-07T11:40:00.000-05:00

Julian Banerji writes "Deamidation of ASN residues in Bcl-xl may regulate apoptosis (Zhao). Deimination of histones can regulate their activity (Cuthbert and Wang.) Deiminated ARG in Fibronectin is the target of some autoantibodies in RA (Hida). The involvement of Peptidylarginine deimidase (PAD) in disease is explored in Vossenaar. The location of PAD is characterized in synovial tissue by Chang. A Japanese group has found polymorphisms in PAD linked to RA (Suzuki). The stories speak for themselves. Something else that's pretty cool: large scale unfolding of chromatin by estrogen receptor (Carpenter) "

Pecking Ordered

2006-11-07T11:31:00.000-05:00

drj writes "Darwin found on the Galapagos Islands 14 species of finches with different beak shapes. He proposed that their different beaks allowed them to exploit different foods – short and wide for cracking seeds vs. long and narrow for reaching into flowers. Abzhanov et al. now show that expression of the bone morphogenic protein 4 gene (Bmp4) is higher in the upper beak epithelium of embryonic chicks growing shorter, stronger beaks, suggesting that BMP4 shapes beaks. Expression of Bmp2 and Bmp4 message, in contrast, correlated with the size of the beak. Surprisingly, expression of Bmp4 in the lower beak epithelium does not vary despite shape differences that are comparable to those in the upper beak. Infection with a virus expressing BMP4 protein produced shorter, wider beaks whereas infection with a virus expressing Noggin, which antagonizes BMP2/4/7 activity, produced longer, narrower beaks. The authors are also “tempted to speculate” that Sonic hedgehog (shh) and Fibroblast growth factor 8 (fgf8) are the nuclear factors driving expression of bmp4. The accompanying article by Wu et al. also concludes that BMP4 shapes beaks. Who regulates the regulators (ssh, fgf8)?
Bmp4 and Morphological Variation of Beaks in Darwin’s Finches Arhat Abzhanov, Meredith Protas, B. Rosemary Grant, Peter R. Grant, Clifford J. Tabin. Science 305:1462, September 3, 2004

J Exp Med Classics online

2006-11-07T11:25:00.000-05:00

drj writes "The Journal of Experimental Medicine has produced pdf (Acrobat) versions of all papers they've published since 1896! Now you can peruse the classic 1944 Avery, MacLeod, and McCarty paper that first showed “desoxyribonucleic acid” (DNA) was the “transforming substance”. Or, one of their follow-up papers, for example an "Improved Method for the Isolation of the Transforming Substance”, very early in a very long string of DNA methods papers. Or here’s one of my favorites, the genetic regulation of the immune response. For a glance backward at less memorable papers, try the I-J era. Three cheers and much appreciation to the publisher, the
Rockefeller University Press, with support from The Medical Library Center of New York. This stuff is too old for PubMed (which covers back to the mid-1960s) and even for OLDMEDLINE, which currently goes back to 1951. You’ve got to seek and find it on your own. Search here for your favorite classics (old and new).
What's your favorite J Exp Med paper?"

Synthetic prions

2006-11-06T21:43:00.000-05:00

carbonbasedunit writes "In the 20 years since Prusiner popularized Griffith’s suggestion that infectious protein causes Creutzfeld-Jacob disease, the "protein-only" hypothesis has matured from radical to conventional. The hardy doubters not cowed by the 1997 Nobel prize still argued that the infectious agent in brain preparations had not been proven to be protein but could instead be contaminating nucleic acids (review).
Now Legname et al. show that when polymerized into amyloid fibrils and injected into the brains of mice, recombinant mouse prion protein produced in bacteria induces neurologic disease. They previously showed that disease was accelerated in disease-prone mice treated with a synthetic 55 amino acid peptide matching a fragment of a mutant (P101L) human prion that causes spontaneous disease in younger adults. Here, they produced fibrils from a recombinant, truncated mouse prion in mice that do not develop the disease spontaneously. Rather than having fortuitously refolded in vitro a natural prion form, the authors instead suggest based on histology that a novel strain of prion was created. Is there a potential Pandora’s box of prions?
Synthetic mammalian prions. Legname G, Baskakov IV, Nguyen HO, Riesner D, Cohen FE, DeArmond SJ, Prusiner SB. Science. 2004 Jul 30;305(5684):673-6.

Innate + Adaptive = Immunity

2006-11-06T21:37:00.000-05:00

drj writes "Innate immunity is initiated by host cells recognizing stable molecular patterns associated with pathogenic microbes and viruses. These molecular patterns are thought to be reliable triggers because they are required for the life of the pathogen. Here, French et al. show that viruses can change these patterns and evade innate immunity in mice deficient in adaptive immune responses. Early infection by murine cytomegalovirus is controlled by natural killer (NK) cells, which use one form of Ly49H to recognize a viral protein, m157, that is expressed on infected cells. Mice possessing a form of Ly49H that is not activated by this virus die in 6 days. Recombinase-deficient mice without adaptive immunity efficiently suppress viral replication for only about 3 weeks, when they succumb. Viruses recovered from these mice do not trigger Ly49H+ NK cells and nearly all possess mutations in m157, probably allowing them to resist innate immunity. This paper demonstrates an essential role for adaptive immunity in clearing virus, thereby reducing the risk of a mutant evading the innate immune system. Even for this double-stranded DNA virus, mutations occur frequently enough to present a serious health risk. The authors suggest that cytomegalovirus disease, which develops more than a month after transplant in some immune suppressed recipients, may be due to a similar selection of mutant viruses escaping the innate immune response.
Escape of Mutant Double-Stranded DNA Virus from Innate Immune Control Anthony R. French, Jeanette T. Pingel, Markus Wagner, Ivan Bubic, Liping Yang, Sungjin Kim,Ulrich Koszinowski, Stipan Jonjic, and Wayne M. Yokoyama. Immunity, Vol. 20, 747–756, June, 2004

Asthma – the Chitin hypothesis

2006-11-06T21:27:00.000-05:00

drj writes "Chitin is a polysaccharide that forms fungal cell walls and the exoskeleton of insects and crustacea. Some 'lower' organisms use enzymes that degrade chitin, chitinases, for defense. Chitinases are also found in mammals but their function is unknown. Here, Zhu et al. show that acidic mammalian chitinase (AMCase) accumulates in the lung epithelium of human asthmatics and in mice treated to induce asthma. In mice, the accumulation depended on "type 2" (allergic) helper T lymphocytes and their characteristic cytokines, IL-4 and IL-13. Most promising was the reduction in inflammation caused by blocking AMCase with specific antibodies, suggesting it is a good target for therapy. The authors discuss the potential links between chitin-containing insects, hygiene, and asthma."
Acidic Mammalian Chitinase in Asthmatic Th2 Inflammation and IL-13 Pathway Activation Zhou Zhu, Tao Zheng, Robert J. Homer, Yoon-Keun Kim, Ning Yuan Chen, Lauren Cohn, Qutayba Hamid, Jack A. EliasScience304: 1678-82 Zhu et al., Science 304:1678-82, April 2004

Splice and die!

2006-11-06T21:15:00.000-05:00

drj writes "Cytotoxic T lymphocytes (CTL) are triggered by short peptides (8-10 amino acids) held by MHC class I molecules on the target cell. Peptides often derive from cleaved and trimmed full-length proteins but an
earlier report described a spliced peptide that flanks a 40 amino acid deletion within the protein fibroblast growth factor-5 (FGF-5). Vigneron et al. now report the second example of a spliced peptide, a 4 amino acid deletion within a melanocyte glycoprotein 100 (gp100) peptide. They also found that “highly purified” proteasomes are capable of peptide splicing and suggested that closely spaced threonines within the proteasome catalyze protein cleavage and peptide ligation. They estimate that only 1 spliced peptide is produced from 10,000 gp100 molecules but even this low efficiency is good enough to trigger CTL. This phenomenon expands enormously the universe of potential peptides (and will greatly complicate the prediction of MHC binding peptides).
An Antigenic Peptide Produced by Peptide Splicing in the Proteasome Vigneron N, Stroobant V, Chapiro J, Ooms A, Degiovanni G, Morel S, van der Bruggen P, Boon T, Van den Eynde BJ.
Science 304:587-23 APRIL 2004"

Autoimmunity and Ubiquitin ligases

2006-10-10T20:43:00.000-05:00

drj writes "Mutations in the AIRE (autoimmune regulator) gene cause ‘autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy’ (APECED, OMIM 240300), a rare autoimmune disease marked by lymphocyte infiltration and antibody deposition in multiple organs (review). Known human AIRE mutant alleles are recessive and were identified by positional cloning at 21q22.3 in 1997. Mice deficient in AIRE also develop autoimmune disease. AIRE protein is mostly nuclear and can activate transcription when linked to a DNA binding domain. AIRE increases the ectopic (out of place) expression of a ‘substantial’ number of genes in the thymus, which may promote T cell tolerance (self shadow). Failure to encounter these proteins in the thymus may permit autoimmune T cells to survive and mature, leading to autoimmunity. Here, disease-causing mutations in AIRE are shown to abolish ubiquitin (Ub) ligase activity without reducing transcription activation, strongly suggesting that Ub ligation is AIRE’s primary activity. The Cbl family of Ub ligases are known to deactivate the T cell receptor by targeting the associated tyrosine kinases. Is it too simplistic to ask which is (are) AIRE’s mechanism(s)?
Uchida et al., J. Exp. Med. 199(2) 167-172, 2004"
AIRE Functions As an E3 Ubiquitin Ligase. Daisuke Uchida, Shigetsugu Hatakeyama, Akemi Matsushima, Hongwei Han, Satoshi Ishido, Hak Hotta, Jun Kudoh, Nobuyoshi Shimizu, Vassilis Doucas, Keiichi I. Nakayama, Noriyuki Kuroda, and Mitsuru Matsumoto J. Exp. Med. Volume 199, Number 2, January 19, 2004 167–172

Clinical trial ethics reconsidered?

2006-10-10T20:38:00.000-05:00

drj writes "Ethical design of randomized controlled clinical trials has required that there be substantial uncertainty about which treatment is most likely to benefit the patient, a principle known as 'equipoise'. Fries and Krishnan examined all 45 trials reported at a professional rheumatology meeting and found that all trials gave results favorable to the industry sponsor, demonstrating a significant deviation from equipoise because the results were predictable. (Nearly all patients would be concerned with even a 70:30 outcome distribution.) The authors suggest that extensive preliminary findings produced a beneficial 'design bias'. This is good, they believe, because true equipoise is inefficient, exposes more patients to risk, and conflicts with other major ethical principles. They propose a principle of "positive expected outcomes". Is this a refreshing acknowledgement of reality and useful guide or just a rewording of accepted clinical practice where treatment group size already varies based on expected outcomes?"
Fries & Krishnan, Arthr. Res. & Ther. 6(3), 2004. James F Fries and Eswar Krishnan "Equipoise, design bias, and randomized controlled trials: the elusive ethics of new drug development" Arthritis Research & Therapy Vol 6 No 3

Inflammatory mediator sustains SLE T cells

2006-10-10T20:25:00.000-05:00

drj writes "Self-reactive antibodies are a hallmark of systemic lupus erythematosus (SLE, lupus), a life-threatening autoimmune disease that disproportionately affects women in their child bearing years. Production of these antibodies is driven by autoreactive T cells that are normally eliminated but persist in SLE patients. Xu and colleagues compared the activities of thousands of genes in the T cells of SLE patients and normal controls. They found that stimulatory conditions that caused normal T cells to be eliminated instead caused SLE T cells to increase their expression of cyclooxygenase-2 (COX-2) and survive. Moreover, inhibiting COX-2 activity reversed the abnormal resistance of SLE T cells. The increased COX-2 was specific to SLE T cells and did not occur in T cells from other autoimmune diseases or cancer cells. Certain COX-2 inhibitors but not others were able to restore normal responses to SLE T cells, providing a rational basis for the use of these drugs for therapy and suggesting approaches to designing more potent and specific therapeutic drugs for use in SLE.
Xu et al, Nat. Med. February 29, 2004. L, Zhang L, Yi Y, Kang HK, Datta SK. "Human lupus T cells resist inactivation and escape death by upregulating COX-2". Nature Medicine February 29, 2004"

Arthritis suppression by TNF alpha regulators

2006-10-10T20:15:00.000-05:00

drj writes "Many rheumatoid arthritis patients are helped by treatments that block the inflammatory cytokine TNF alpha. TNF alpha production is reduced by two proteins that bind AU-rich regions of mRNA: TIA-1 inhibits protein translation and TTP promotes mRNA degradation. Mice deficient in either TIA-1 or TTP (gene “knockouts”) develop arthritis. Not surprisingly, Phillips and colleagues show that TIA-1/TTP double knockout mice develop worse arthritis. Single knockout macrophages contain more TNF alpha protein than normal macrophages and double knockout macrophages contain much more, most markedly the membrane bound form (mTNF alpha). Surprisingly, much less TNF alpha protein is secreted by double knockout macrophages than either single knockout, suggesting TIA-1 and TTP influence secretion or mTNF alpha processing. The investigation turned to the less usual suspects, neutrophils, which also make TNF alpha and are extraordinarily abundant in the bone marrow of double knockout mice. Indeed, activated bone marrow cells from double knockout mice express much more TNF alpha than single TTP knockout macrophages. However, purified neutrophils from TTP knockout mice show only a modest increase and double knockout show no further increase, leaving the origin of the increased TNF obscure. Although failing to establish a complete mechanism, this investigation demonstrates several important interactions and represents the kind of analysis that is necessary to dissect complex disease genetics.
Phillips et al. PNAS 101:2011-2016. February 17, 2004. Arthritis suppressor genes TIA-1 and TTP dampen the expression of tumor necrosis factor alpha, cyclooxygenase 2, and inflammatory arthritis Kristine Phillips, Nancy Kedersha, Lily Shen, Perry J. Blackshear, and Paul Anderson
Additional information: Macrophages and neutrophils were activated with bacterial endotoxin, LPS. Double knockout mice are born runts and remain about half the weight of single knockout or normal mice.

Innate intracellular resistance to HIV-1

2006-09-07T21:41:00.000-05:00

drj writes "Human immunodeficiency virus type 1 (HIV-1), the cause of acquired immunodeficiency syndrome (AIDS) in humans, efficiently enters Old World monkey cells but it is not reverse transcribed. This block probably protects monkeys from productive HIV-1 infection and AIDS. Stremlau and colleagues searched for an Old World monkey (rhesus) gene blocking HIV-1 infection by transferring an expression library into a susceptible human cell line (HeLa) and testing for HIV-1 resistance. They found TRIM5a, a 497 amino acid protein located in cytoplasmic bodies and named only for its structural "tripartite motif". Inhibition might result from the ubiquitin ligase activities of TRIM5a, which marks a protein for degradation but here might simply interfere with capsid disassembly. Notably, the rhesus TRIM5a does not block simian immunodeficiency virus (SIV) infection. Human TRIM5a differs in several key residues that are presumably involved in recognizing HIV-1 capsid.
Stremlau et al. Nature 427:858. February 26, 2004.
'The cytoplasmic body component TRIM5a restricts HIV-1 infection in Old World monkeys' Matthew Stremlau, Christopher M. Owens, Michel J. Perron, Michael Kiessling, Patrick Autissier & Joseph Sodroski"

mRNA quality control

2006-09-07T21:32:00.000-05:00

drj writes "Messenger RNA (mRNA) with a premature termination codon (PTC) undergoes nonsense mediated decay (NMD). NMD is thought to protect against amino-terminal protein fragments that could be disruptive, for example, by acting as dominant negative mutants. Muscular dystrophy, Marfan syndrome, and other human diseases are caused by premature termination. Hillman and colleagues earlier determined that over one-third of alternatively-spliced human mRNAs have a PTC. Since NMD mechanisms were only recently discovered, they suspected many mRNAs in the databases might contain previously unrecognized PTCs and would have undergone NMD in vivo. Here, they aligned protein sequences from SWISS-PROT with gene sequences from Genbank to identify ~8% of entries that contain a PTC. Important biological effects could be attributed to NMD. For example, an intronic polymorphism linked to type II diabetes reduces 4 of 8 calpain-10 mRNA isoforms. These same 4 isoforms contain a PTC, suggesting NMD underlies reduced expression, causing disease. Similar analysis of CDC-like kinases and a receptor containing a death domain suggests previously unsuspected roles for NMD.
Hillman et al. Genome Biology 5:R8. January 2, 2004
Background: In mammals, "premature" is a stop codon >50 nucleotides upstream of the last intron. mRNAs with PTC are flagged for degradation by the persistent splice junction protein complexes, which are normally cleared by the ribosome.
An unappreciated role for RNA surveillance R Tyler Hillman, Richard E Green and Steven E Brenner. Genome Biology 2004, 5:R8. online at genomebiology"

Lymphocyte migration triggered by sphingosine-1-phosphate

2006-09-06T21:04:00.000-05:00

drj writes "Why did the lymphocyte leave the lymphoid organ? A chemical gradient was long suspected and now some details have been identified. Matloubian et al. found very few lymphocytes in the blood of mice with lymphocytes that are deficient in one particular receptor for sphingosine-1-phosphate (S1P), called S1P1. They show that developing S1P1-deficient thymocytes remain in the thymus and B cells remain in peripheral lymphoid organs, though there is only a mild alteration in the the subpopulations within these organs. Normal thymocytes but not deficient thymocytes move in response to a S1P gradient. Antigen stimulated T cells transiently reduce S1P1 expression, which is mostly restored within days, suggesting a molecular basis for transient retention in lymphoid organs. An immunosuppressant drug currently in clinical trials, FTY720, binds S1P1, leading to receptor downregulation and reduced migration. The data strongly support a compelling model, albeit one with many details yet to be determined."
M. Matloubian et al. Nature 427:355-360. January 22, 2004

Enzymology - The Next Generation

2006-09-06T16:20:00.000-05:00

drj writes "This thought-provoking overview of current enzymology in The Scientist [free registration required] briefly introduces advances in structural studies, mechanistic theories, ribozymes, and potential commercial applications. My favorite was conceiving of the cell as a network of enzymes operating at rates determined by their (intrinsic) Km and the concentration of substrates and cofactors. Not a new idea, perhaps, but well described and evocative. The network is dramatized by these mapping of metabolic pathways and cellular and molecular processes (through ExPASy, courtesy of Roche). (originally posted on MedDot.org)

"Enzymology's New Frontiers" M. Greener, The Scientist. 2004;Vol. 18 1:16-20 January 19, 2004"