2007 Flu vaccine: Pain, and gain?

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

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

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

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

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

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”

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

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

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

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?

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

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

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

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

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

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

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

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

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

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?

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

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?

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

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

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

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