Humoral immunity in tuberculin skin test anergy and its role in high-risk persons exposed to active tuberculosis.

The most common test to identify latent tuberculosis is the tuberculin skin test that detects T cell responses of delayed type hypersensitivity type IV. Since it produces false negative reactions in active tuberculosis or in high-risk persons exposed to tuberculosis patients as shown in this report, we studied antibody profiles to explain the anergy of such responses in high-risk individuals without active infection. Our results showed that humoral immunity against tuberculin, regardless of the result of the tuberculin skin test is important for protection from active tuberculosis and that the presence of high antibody titers is a more reliable indicator of infection latency suggesting that latency can be based on the levels of antibodies together with in vitro proliferation of peripheral blood mononuclear cells in the presence of the purified protein derivative. Importantly, anti-tuberculin IgG antibody levels mediate the anergy described herein, which could also prevent reactivation of disease in high-risk individuals with high antibody titers. Such anti-tuberculin IgG antibodies were also found associated with blocking and/or stimulation of in vitro cultures of PBMC with tuberculin. In this regard, future studies need to establish if immune responses to Mycobacterium tuberculosis can generate a broad spectrum of reactions either toward Th1 responses favoring stimulation by cytokines or by antibodies and those toward diminished responses by Th2 cytokines or blocking by antibodies; possibly involving mechanisms of antibody dependent protection from Mtb by different subclasses of IgG.

Kinase requirements in human cells: III. Altered kinase requirements in VHL-/- cancer cells detected in a pilot synthetic lethal screen.

Clear cell renal carcinomas are the most common form of kidney cancer and frequently are linked to biallelic inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. The VHL gene product, pVHL, has multiple functions including directing the polyubiquitylation of the HIF transcription factor. We screened 100 shRNA vectors, directed against 88 kinases, for their ability to inhibit the viability of VHL-/- renal carcinoma cells preferentially compared with isogenic cells in which pVHL function was restored. shRNAs for "hits" identified in the primary screen were interrogated in secondary screens that included shRNA titration studies. Multiple shRNAs against CDK6, MET, and MAP2K1 (also known as MEK1) preferentially inhibited the viability of 786-O and RCC4 VHL-/- cells compared with their wild-type pVHL-reconstituted counterparts. The sensitivity of pVHL-proficient cells to these shRNAs was not restored upon HIF activation, suggesting that loss of an hypoxia-inducible factor (HIF)-independent pVHL function formed the basis for selectivity. A small-molecule Cdk4/6 inhibitor displayed enhanced activity against VHL-/- renal carcinoma cells, suggesting that in some cases hits from shRNA screens such as described here might translate into therapeutic targets.

Genetic fixity in the human major histocompatibility complex and block size diversity in the class I region including HLA-E.

BACKGROUND: The definition of human MHC class I haplotypes through association of HLA-A, HLA-Cw and HLA-B has been used to analyze ethnicity, population migrations and disease association. RESULTS: Here, we present HLA-E allele haplotype association and population linkage disequilibrium (LD) analysis within the ~1.3 Mb bounded by HLA-B/Cw and HLA-A to increase the resolution of identified class I haplotypes. Through local breakdown of LD, we inferred ancestral recombination points both upstream and downstream of HLA-E contributing to alternative block structures within previously identified haplotypes. Through single nucleotide polymorphism (SNP) analysis of the MHC region, we also confirmed the essential genetic fixity, previously inferred by MHC allele analysis, of three conserved extended haplotypes (CEHs), and we demonstrated that commercially-available SNP analysis can be used in the MHC to help define CEHs and CEH fragments. CONCLUSION: We conclude that to generate high-resolution maps for relating MHC haplotypes to disease susceptibility, both SNP and MHC allele analysis must be conducted as complementary techniques.

Allorecognition of an HLA-A*01 aberrant allele by an HLA identical family member carrying the HLA-A*0101 allele.

We identified and characterized an HLA-A1 aberrant allele (A*0118N) resulting from a novel molecular mechanism; this allele was present in an unusually informative family with a near identical parental HLA haplotype (c d) differing only by one nucleotide substitution in one HLA-A allele, A*0118N, of the maternal HLA haplotype (c) and not of the paternal HLA haplotype (a). Although serologic HLA typing showed a "blank," DNA molecular HLA typing detected a HLA-A*0118N allele. Sequence based typing identified the substitution of guanine by cytosine at the nucleotide position 215, which resulted in the replacement of arginine by proline at position 48 of the HLA-A1 H chain. The loss of surface protein expression was also found by FACS analysis. Isoelectric-focusing analysis detected a HLA-A H chain with a unique isoelectric-focusing pattern, which does not associate with the L chain (beta(2)-microglobulin). These results suggest that the residue 48-containing interaction site on the alpha(1) domain plays a critical role in the association between HLA class I H chain and beta(2)-microglobulin. Functional studies showed that the T cells of the propositus (HLA haplotypes c d) carrying this null allele recognized its wild-type counterpart, HLA-A*010101, in her HLA-identical son that carries the HLA-A*0101 heterodimer. This is the first example of the generation of cytotoxic T cells in the absence of proliferation of CD4(+) T cells (mixed lymphocyte culture) and the description of an aberrant allele, A*0118N, that may behave as a minor histocompatibility Ag, with implications in allorecognition by cytolytic T cells in solid organ and stem cell transplantation.

Interaction of KIR genes and G1M immunoglobulin allotypes confer susceptibility to type 2 diabetes in Puerto Rican Americans.

The susceptibility to type 2 diabetes (T2D) involves genetic factors. We studied the distribution of KIR and MHC class I ligands phenotype and genotype frequencies, as well as immunoglobulin KM and GM allotype frequencies in a group of patients (N = 95) with T2D and ethnically matched healthy controls (N = 74) with Puerto Rican ethnic background. We found a slight increase of the 2DL3/2DL3 homozygous genotype in T2D. Moreover, the association between 2DL3/2DL3 genotype was significant in the presence of 2DS4 (pC = 0.01). Also, we observed an epistatic effect of the interaction of 2DL3/2DL3, 2DS4 with allele z of G1M in T2D (pC = 0.004, OR = 3.60, 95% CI, 1.62-8.10). This genetic interaction between KIR and G1M allotypes, associated with T2D, was also significant by multiple logistic regression analysis (p < 0.0001, OR = 4.90, 95% CI, 2.12-11.3). We did not detect population stratification using unlinked short tandem repeat (STR) markers, demonstrating that the patients and controls were ethnically matched. Hence, we have demonstrated in this study an epistatic interaction between KIR genes and the G1M allotype that influences the susceptibility to T2D in Puerto Rican Americans. Our findings are important for understanding the autoimmune or innate immune inflammatory-mediated mechanisms involved in the pathogenesis of T2D.

Increased FasL expression correlates with apoptotic changes in granulocytes cultured with oxidized clozapine.

Clozapine has been associated with a 1% incidence of agranulocytosis. The formation of an oxidized intermediate clozapine metabolite has been implicated in direct polymorphonuclear (PMN) toxicity. We utilized two separate systems to analyze the role of oxidized clozapine in inducing apoptosis in treated cells. Human PMN cells incubated with clozapine (0-10 microM) in the presence of 0.1 mM H2O2 demonstrated a progressive decrease of surface CD16 expression along with increased apoptosis. RT-PCR analysis showed decreased CD16 but increased FasL gene expression in clozapine-treated PMN cells. No change in constitutive Fas expression was observed in treated cells. In HL-60 cells induced to differentiate with retinoic acid (RA), a similar increase in FasL expression, but no associated changes in CD16 gene expression, was observed following clozapine treatments. Our results demonstrate increased FasL gene expression in oxidized clozapine-induced apoptotic neutrophils suggesting that apoptosis in granulocytes treated with clozapine involves Fas/FasL interaction that initiates a cascade of events leading to clozapine-induced agranulocytosis.