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