Breakpoint characterization of large deletions in EXT1 or EXT2 in 10 Multiple Osteochondromas families

<p>Abstract</p> <p>Background</p> <p>Osteochondromas (cartilage-capped bone tumors) are by far the most commonly treated of all primary benign bone tumors (50%). In 15% of cases, these tumors occur in the context of a hereditary syndrome called multiple osteochondromas (MO), an autosomal dominant skeletal disorder characterized by the formation of multiple cartilage-capped bone tumors at children's metaphyses. MO is caused by various mutations in <it>EXT1 </it>or <it>EXT2</it>, whereby large genomic deletions (single-or multi-exonic) are responsible for up to 8% of MO-cases.</p> <p>Methods</p> <p>Here we report on the first molecular characterization of ten large <it>EXT1</it>- and <it>EXT2</it>-deletions in MO-patients. Deletions were initially indentified using MLPA or FISH analysis and were subsequently characterized using an MO-specific tiling path array, allele-specific PCR-amplification and sequencing analysis.</p> <p>Results</p> <p>Within the set of ten large deletions, the deleted regions ranged from 2.7 to 260 kb. One <it>EXT2 </it>exon 8 deletion was found to be recurrent. All breakpoints were located outside the coding exons of <it>EXT1 </it>and <it>EXT2</it>. Non-allelic homologous recombination (NAHR) mediated by <it>Alu</it>-sequences, microhomology mediated replication dependent recombination (MMRDR) and non-homologous end-joining (NHEJ) were hypothesized as the causal mechanisms in different deletions.</p> <p>Conclusions</p> <p>Molecular characterization of <it>EXT1</it>- and <it>EXT2</it>-deletion breakpoints in MO-patients indicates that NAHR between <it>Alu-</it>sequences as well as NHEJ are causal and that the majority of these deletions are nonrecurring. These observations emphasize once more the huge genetic variability which is characteristic for MO. To our knowledge, this is the first study characterizing large genomic deletions in <it>EXT1 </it>and <it>EXT2</it>.</p

Low CD4+ T Cell Counts among African HIV-1 Infected Subjects with Group B KIR Haplotypes in the Absence of Specific Inhibitory KIR Ligands

Natural killer (NK) cells are regulated by interactions between polymorphic killer immunoglobulin-like receptors (KIR) and human leukocyte antigens (HLA). Genotypic combinations of KIR3DS1/L1 and HLA Bw4-80I were previously shown to influence HIV-1 disease progression, however other KIR genes have not been well studied. In this study, we analyzed the influence of all activating and inhibitory KIR, in association with the known HLA inhibitory KIR ligands, on markers of disease progression in a West African population of therapy-naïve HIV-1 infected subjects. We observed a significant association between carriage of a group B KIR haplotype and lower CD4+ T cell counts, with an additional effect for KIR3DS1 within the frame of this haplotype. In contrast, we found that individuals carrying genes for the inhibitory KIR ligands HLA-Bw4 as well as HLA-C1 showed significantly higher CD4+ T cell counts. These associations were independent from the viral load and from individual HIV-1 protective HLA alleles. Our data suggest that group B KIR haplotypes and lack of specific inhibitory KIR ligand genes, genotypes considered to favor NK cell activation, are predictive of HIV-1 disease progression

A Decline in CCL3-5 Chemokine Gene Expression during Primary Simian-Human Immunodeficiency Virus Infection

BACKGROUND: The CC-chemokines CCL3, CCL4 and CCL5 have been found to block the entry of CCR5-tropic HIV into host cells and to suppress the viral replication in vitro, but the in vivo role of endogenous CC-chemokines in HIV-1 infection is still incompletely understood. METHODOLOGY/PRINCIPLE FINDINGS: In this study, the primate host CCL3, CCL4 and CCL5 gene expression was evaluated in response to simian-human immunodeficiency virus (SHIV) infection in rhesus macaque model. Five rhesus macaques were inoculated with CCR5-tropic SHIV(SF162P4). The mRNA levels of CCL3, CCL4 and CCL5 were measured by real-time PCR at post inoculation day (PID) 0, 7, 14, 21, 35, 56 and 180 in peripheral blood. In addition, a selected subset of samples from CXCR4-tropic SHIV(Ku1)-infected macaques was included with objective to compare the differences in CC-chemokine down-regulation caused by the two SHIVs. Gut-associated lymphoid tissues (GALT) collected from SHIV(SF162P4)-infected animals were also tested by flow cytometry and confocal microscopy to corroborate the gene expression results. Predictably, higher viral loads and CD4+ T cell losses were observed at PID 14 in macaques infected with SHIV(Ku1) than with SHIV(SF162P4). A decline in CC-chemokine gene expression was also found during primary (PID 7-21), but not chronic (PID 180) stage of infection. CONCLUSIONS: It was determined that A) SHIV(SF162P4) down-regulated the CC-chemokine gene expression during acute stage of infection to a greater extent (p<0.05) than SHIV(Ku1), and B) such down-regulation was not paralleled with the CD4+ T cell depletion. Evaluation of CC-chemokine enhancing immunomodulators such as synthetic CpG-oligonucleotides could be explored in future HIV vaccine studies

The Role of Cytokines which Signal through the Common γ Chain Cytokine Receptor in the Reversal of HIV Specific CD4(+) and CD8(+) T Cell Anergy

BACKGROUND: HIV specific T cells are putatively anergic in vivo. IL-2, a member of a class of cytokines that binds to receptors containing the common gamma chain (γc) has been shown to reverse anergy. We examined the role of γc cytokines in reversing HIV specific T cell anergy. METHODS: PBMC from untreated HIV-infected individuals were briefly exposed to a panel of γc cytokines, and frequencies of gag specific T cells were enumerated by intracellular IFN-γ flow cytometry. RESULTS: Of the γc cytokines, brief exposure to IL-2, IL-15, or combined IL-15/IL-7 significantly enhanced (range 2–7 fold) the CD4(+) and CD8(+) T cell IFN-γ responses to HIV gag, with IL-15 giving the greatest enhancement. The effects of cytokines were not due to enhanced proliferation of pre-existing antigen specific cells, but were due to a combination of enhanced cytokine production from antigen specific T cells plus activation of non-epitope specific T cells. CONCLUSIONS: These observations support the notion that a significant number of HIV specific T cells are circulating in an anergic state. IL-2, IL-7 and particularly IL-15 as an immune modulator to reverse HIV-1 specific T cell anergy should be investigated, with the caveat that non-specific activation of T cells may also be induced

Killer immunoglobulin-like receptor and human leukocyte antigen-C genotypes in rheumatoid arthritis primary responders and non-responders to anti-TNF-α therapy

The identification of patients who will respond to anti-tumor necrosis factor alpha (anti-TNF-α) therapy will improve the efficacy, safety, and economic impact of these agents. We investigated whether killer cell immunoglobulin-like receptor (KIR) genes are related to response to anti-TNF-α therapy in patients with rheumatoid arthritis (RA). Sixty-four RA patients and 100 healthy controls were genotyped for 16 KIR genes and human leukocyte antigen-C (HLA-C) group 1/2 using polymerase chain reaction sequence-specific oligonucleotide probes (PCR-SSOP). Each patient received anti-TNF-α therapy (adalimumab, etanercept, or infliximab), and clinical responses were evaluated after 3 months using the disease activity score in 28 joints (DAS28). We investigated the correlations between the carriership of KIR genes, HLA-C group 1/2 genes, and clinical data with response to therapy. Patients responding to therapy showed a significantly higher frequency of KIR2DS2/KIR2DL2 (67.7% R vs. 33.3% NR; P = 0.012). A positive clinical outcome was associated with an activating KIR–HLA genotype; KIR2DS2(+)HLA-C group 1/2 homozygous. Inversely, non-response was associated with the relatively inhibitory KIR2DS2(–)HLA-C group 1/2 heterozygous genotype. The KIR and HLA-C genotype of an RA patient may provide predictive information for response to anti-TNF-α therapy

Innate partnership of HLA-B and KIR3DL1 subtypes against HIV-1

Allotypes of the natural killer (NK) cell receptor KIR3DL1 vary in both NK cell expression patterns and inhibitory capacity upon binding to their ligands, HLA-B Bw4 molecules, present on target cells. Using a sample size of over 1,500 human immunodeficiency virus (HIV)+ individuals, we show that various distinct allelic combinations of the KIR3DL1 and HLA-B loci significantly and strongly influence both AIDS progression and plasma HIV RNA abundance in a consistent manner. These genetic data correlate very well with previously defined functional differences that distinguish KIR3DL1 allotypes. The various epistatic effects observed here for common, distinct KIR3DL1 and HLA-B Bw4 combinations are unprecedented with regard to any pair of genetic loci in human disease, and indicate that NK cells may have a critical role in the natural history of HIV infection

Reduction of Natural Killer but Not Effector CD8 T Lymphoyctes in Three Consecutive Cases of Severe/Lethal H1N1/09 Influenza A Virus Infection

Background: The cause of severe disease in some patients infected with pandemic influenza A virus is unclear. Methodology/Principal Findings: We present the cellular immunology profile in the blood, and detailed clinical (and postmortem) findings of three patients with rapidly progressive infection, including a pregnant patient who died. The striking finding is of reduction in natural killer (NK) cells but preservation of activated effector CD8 T lymphocytes; with viraemia in the patient who had no NK cells. Comparison with control groups suggests that the reduction of NK cells is unique to these severely ill patients. Conclusion/Significance: Our report shows markedly reduced NK cells in the three patients that we sampled and raises the hypothesis that NK may have a more significant role than T lymphocytes in controlling viral burden when the host is confronted with a new influenza A virus subtype