Rare coding variant analysis in a large cohort of Ashkenazi Jewish families with inflammatory bowel disease.

Rare variants are thought to contribute to the genetics of inflammatory bowel disease (IBD), which is more common amongst the Ashkenazi Jewish (AJ) population. A family-based approach using exome sequencing of AJ individuals with IBD was employed with a view to identify novel rare genetic variants for this disease. Exome sequencing was performed on 960 Jewish individuals including 513 from 199 multiplex families with up to eight cases. Rare, damaging variants in loci prioritized by linkage analysis and those shared by multiple affected individuals within the same family were identified. Independent evidence of association of each variant with disease was assessed. A number of candidate variants were identified, including in genes involved in the immune system. The ability to achieve statistical significance in independent case/control replication data was limited by power and was only achieved for variants in the well-established Crohn's disease gene, NOD2. This work demonstrates the challenges of identifying disease-associated rare damaging variants from exome data, even amongst a favorable cohort of familial cases from a genetic isolate. Further research of the prioritized rare candidate variants is required to confirm their association with the disease.

Regulation by Per-Arnt-Sim (PAS) kinase of pancreatic duodenal homeobox-1 nuclear import in pancreatic beta-cells.

The transcription factor PDX-1 (pancreatic duodenal homeobox-1) is required for normal pancreatic development and for the function of insulin-producing islet beta-cells in mammals. We have shown previously that glucose regulates insulin gene expression in part through the activation and translocation of PDX-1 from the nuclear periphery to the nucleoplasm. We have also found that PASK [PAS (Per-Arnt-Sim) kinase], a member of the nutrient-regulated family of protein kinases, is activated in response to glucose challenge in beta-cells and is involved in the regulation of expression of PDX-1. Purified PASK efficiently phosphorylated recombinant PDX-1 in vitro on a single site (Thr-152). To determine the impact of phosphorylation at this site, we generated wild-type and mutant (T152A, T152D and T152E) forms of PDX-1 and examined the distribution of each of these in clonal MIN6 beta-cells by immunocytochemical analysis. Unexpectedly, only the T152D mutation significantly affected subcellular distribution, increasing the ratio of nuclear/cytosolic labelling at low and high glucose concentrations, suggesting that phosphorylation at Thr-152 inhibits nuclear uptake in response to glucose. Based on these results, experiments to examine the contribution of Thr-152 to the overall phosphorylation of PDX-1 in intact cells will be undertaken.

Serum imbalance of cytokines in melanoma patients.

The cytokines interleukin (IL)6 and IL10 appear to be involved in the progression of melanoma because they are secreted by malignant cells and their serum levels are associated with poor survival and with advanced stages of the disease. Antitumour immunity is considered to be a T-cell response, mediated mainly by type 1 cytokines such as IL12 and interferon-gamma (IFNgamma). We evaluated the serum levels of cytokines involved in the host response against tumour (IL12, IFNgamma) and/or the progression of melanoma (IL6, IL10) in 45 melanoma patients with localized and metastatic disease and in 45 controls, using commercially available enzyme-linked immunosorbent assay (ELISA) kits. In the controls, IL6 and IL12 were nearly undetectable, whereas the IL10 and IFNgamma ranges were 0.5-9 pg/ml and 2-4.8 pg/ml, respectively. In the melanoma patients, pathologically high values were found in 44.4% for IL6, in 24.4% for IL10, and in 60% for IL12. Significantly higher values were found for IL6 and IL12, and lower values for IFNgamma. This study highlights a significant difference in serum cytokine profiles between controls and melanoma patients, which is mainly due to the high levels of IL6 and IL12 and the low levels of IFNgamma.

Human immunodeficiency virus (HIV)-specific IgA and HIV neutralizing activity in the serum of exposed seronegative partners of HIV-seropositive persons.

The presence and activity of human immunodeficiency virus (HIV)-specific antibodies were analyzed in the sera of 15 sexually exposed seronegative persons who had systemic HIV-specific cell-mediated immunity and IgA-mediated mucosal immunity and in their HIV-infected partners. The HIV-positive subjects had HIV-specific serum IgG and IgA; the seronegative persons had HIV-specific serum IgA in the absence of IgG. Testing of the seronegative persons 1 year after the interruption of at-risk sex showed that no IgG seroconversion had occurred and that HIV-specific IgA serum concentrations had declined. Serum from the HIV-exposed seronegative persons was analyzed for the ability to neutralize primary HIV-1 isolates. Neutralizing activity was detected in 5 of 15 sera and in 2 cases was retained by serum-purified IgA. Thus, the immunologic picture for resistance to HIV infection should include HIV-specific cell-mediated immunity as well as HIV-specific IgA-mediated mucosal and systemic immunity.

A role for mucosal immunity in resistance to HIV infection.

In a recent, thought-provoking novel by Elizabeth McCracken (The Giant's House. Avon Books, New York, 1997), two characters discuss love and its impossibilities. One brashly claims to be "immune to love", explaining the concept to his perplexed interlocutor, "...people become immune to love like they become immune to any disease. Either they had it bad early in life, like chicken pox and that's that; or they keep getting exposed to it in little doses and build up an immunity; or somehow they just don't catch it, something in'em is born resistant. I'm the last type. I'm immune to love and poison ivy". (p. 275) (E. McCracken, The Giant's House. Avon Books, New York, 1997). Substitute the words 'HIV infection' for 'love' and this intriguing metaphor summarizes the state of the art working hypotheses for the phenomenon of resistance to HIV infection in HIV-exposed individuals who, against all odds, do not seroconvert. These hypotheses will be discussed hereafter and particular emphasis will be placed upon a possible role for mucosal immunity in this phenomenon.

Protein kinase CK2alpha' is induced by serum as a delayed early gene and cooperates with Ha-ras in fibroblast transformation.

Protein kinase CK2 is an ubiquitous and pleiotropic Ser/Thr protein kinase composed of two catalytic (alpha and/or alpha') and two noncatalytic (beta) subunits forming a heterotetrameric holoenzyme involved in cell growth and differentiation. Here we report the identification, cloning, and oncogenic activity of the murine CK2alpha' subunit. Serum treatment of quiescent mouse fibroblasts induces CK2alpha' mRNA expression, which peaks at 4 h. The kinetics of CK2alpha' expression correlate with increased kinase activity toward a specific CK2 holoenzyme peptide substrate. The ectopic expression of CK2alpha' (or CK2alpha) cooperates with Ha-ras in foci formation of rat primary embryo fibroblasts. Moreover, we observed that BALB/c 3T3 fibroblasts transformed with Ha-ras and CK2alpha' show a faster growth rate than cells transformed with Ha-ras alone. In these cells the higher growth rate correlates with an increase in calmodulin phosphorylation, a protein substrate specifically affected by isolated CK2 catalytic subunits but not by CK2 holoenzyme, suggesting that unbalanced expression of a CK2 catalytic subunit synergizes with Ha-ras in cell transformation.

Chlamydia pneumoniae antibody response in patients with acute myocardial infarction and their follow-up.

STUDY POPULATIONS: This study concerned the possible relations between seroreactivity to Chlamydia pneumoniae and myocardial infarction. A group of 29 patients with acute myocardial infarction (AMI), 74 members of a healthy control group, and a subgroup of 24 members of a healthy control group matched for age, sex, and coronary risk factors (HCM) were included in the study. In addition, we evaluated the AMI group in a 1-year patients' follow-up study. We used two different tests to detect anti-C. pneumoniae antibodies: recombinant enzyme immunoassay antilipopolysaccharide antibodies and a reference microimmunofluorescence test. RESULTS: High titers of C. pneumoniae microimmunofluorescence antibodies were found in 89.65% of the AMI group and in 25% of the HCM group (p = 0.0000065). Immunoglobulin A-microimmunofluorescence was 51.72% in the AMI group and 20.83% in the HCM group (p = 0.0042). Immunoglobulin G and immunoglobulin A antilipopolysoccharide titers were 65.51% and 62.60% in the AMI group and 20.83% in the HCM group, respectively (p = 0.006). High concentrations of interleukin-6 were found in 86.20% of our AMI group (p value = 54.38 pg/ml) when compared with the control group. A good correlation between interleukin-6 levels and immunoglobulin A-lipopolysaccharide titers (r = 0.658) was found. CONCLUSION: The presence of a high prevalence rate and high titers of immunoglobulin G and immunoglobulin A-specific anti-C. pneumoniae antibodies in AMI at admission demonstrated the presence of a specific anti-C. pneumoniae immunization in the AMI population.

HIV-specific mucosal and cellular immunity in HIV-seronegative partners of HIV-seropositive individuals.

HIV-specific mucosal and cellular immunity was analyzed in heterosexual couples discordant for HIV status in serum and in HIV-unexposed controls. HIV-specific IgA but not IgG was present in urine and vaginal wash samples from HIV-exposed seronegative individuals (ESN), whereas both IgA and IgG were observed in their HIV-seropositive partners; antibodies were not detected in low-risk controls. Envelope protein (Env) peptide-stimulated interleukin-2 (IL-2) production by peripheral blood mononuclear cells (PBMCs) was detected in 9 out of 16 ESNs, 5 out of 16 HIV-infected patients and 1 out of 50 controls. Env peptide-stimulated PBMCs of ESNs produced more IL-2 and less IL-10 compared with those of HIV-infected individuals; no differences were observed in chemokine production or in CCR5 expression. These data demonstrate that a compartmentalized immune response to pathogens is possible in humans and raise the possibility of protective roles for cell-mediated immunity and mucosal IgA in HIV-seronegative individuals exposed to HIV.

[Anti-Chlamydia pneumoniae antibodies and production of interleukin 6 in acute myocardial infarct]. / Anticorpi anti Chlamydia pneumoniae e produzione di interleuchina 6 nell'infarto miocardico acuto.

UNLABELLED: Chlamydia pneumoniae (C.p.) has been correlated with acute myocardial infarction (AMI). High levels of anti-C.p. antibodies and circulating immune complexes containing C.p. lypopolyaaccharide (LPS) antigens have been demonstrated in AMI. LPS antigen and especially Chlamydial LPS is one of the best antigen and it is also a very good Interleukin inductor. Moreover, interleukin 6 (IL-6) has been observed in AMI patients. The aim of our study was to assess the possible relationships between anti-C.p. immune response and IL-6 production in AMI patients. We studied 17 consecutive patients with myocardial infarction (12 males and 5 females; mean age 62; range 46-72). Blood samples were obtained immediately after hospital admission. There were 17 control subjects (HCM) (mean age 62; range 45-72) who were matched for the main coronary risk factors (gender, age, diabetes, hypertension, hypercolesterolemia, smoking, family history of ischemic heart disease). In addition, we evaluated the AMI patients in a one-year follow-up study (FU). RESULTS: High levels of C.p. IgG MIF were found in 82.3% of our AMI patients and in 29.4% of HCM subjects (p = 0.0000065). IgA-MIF were 70.5% in AMI patients and 29.4% in HCM (p = 0.0042). High levels of C.p. IgG and IgA anti-LPS were found, with a very high prevalence rate of 76.4% and 64.7% in AMI patients, and both rates were 47.0% (p = 0.158; p = 0.489) in HCM. Very high levels of IL-6 were found (m = 54.38 pg/ml) in 100% of the AMI patients (normal values in our population: 0-10.86 pg/ml) and only detectable levels in 5.8% of HCM. A good linear correlation was demonstrated between IL-6 and IgA levels in the first sample (r = 0.655). The high levels of anti-C.p. IgG, IgA and IL-6, with a good correlation between IL-6 and IgA levels, may confirm the presence of an active infection and probably of a reinfection.

Biological removal of inorganic Hg(II) as gaseous elemental Hg(0) by continuous culture of a Hg-resistant Pseudomonas putida strain FB-1.

A strain of broad-spectrum, mercury-resistant Pseudomonas putida FB1 was used to remove mercury as the gaseous element (Hg(0)) from a continuous axenic culture, fed with a synthetic medium containing 1 mg Hg l(-1) as HgCl2. Mercury determinations were performed in steady-state cultures using various culture fractions [whole culture, filtered supernatant, bacterial cells (dry wt), recovery trap liquid] in order to determine the removal efficiency at different dilution rates (from 0.1 to 3.0 day(-1)). The removal efficiency ranged from 99.2% to 99.8%, and the residual Hg was maintained below 5 µ l(-1) (the maximum allowable concentration of Hg in liquid wastes according to Italian law) at a dilution rate of 1.0 day(-1), corresponding to a Hg flux of 40 µg l(-1) h(-1). Hg accumulation by cell biomass was negligible for dilution rates under 1.0 day(-1). A progressive accumulation of Hg, both in the liquid phase and in cells, occurred at a higher dilution rate (3.0 day(-1); close to washout), corresponding to a Hg concentration of 25 µg g(-1) (dry wt). The estimated Km and Vmax for Hg reduction were 0.241 mg l(-1) and 9.5 mg g(-1) h(-1), respectively. In batch experiments maximum Hg removal occurred at the optimum growth temperature (28°C) of P. putida. The maximum recovery of Hg in the liquid trap was 78%.