Degeneracy and repertoire of the human HIV-1 Gag p17(77-85) CTL response

CD8+ CTL responses are important for the control of HIV-1 infection. The immunodominant HLA-A2-restricted Gag epitope, SLYNTVATL (SL9), is considered to be a poor immunogen because reactivity to it is rare in acute infection despite its paradoxical dominance in patients with chronic infection. We have previously reported SL9 to be a help-independent epitope in that it primes highly activated CTLs ex vivo from CD8+ T cells of seronegative healthy donors. These CTLs produce sufficient cytokines for extended autocrine proliferation but are sensitive to activation-induced cell death, which may cause them to be eliminated by a proinflammatory cytokine storm. Here we identified an agonist variant of the SL9 peptide, p41 (SLYNTVAAL), by screening a large synthetic combinatorial nonapeptide library with ex vivo-primed SL9-specific T cells. p41 invariably immunized SL9-cross-reactive CTLs from other donors ex vivo and H-2Db beta2m double knockout mice expressing a chimeric HLA-A*0201/H2-Db MHC class I molecule. Parallel human T cell cultures showed p41-specific CTLs to be less fastidious than SL9-CTLs in the level of costimulation required from APCs and the need for exogenous IL-2 to proliferate (help dependent). TCR sequencing revealed that the same clonotype can develop into either help-independent or help-dependent CTLs depending on the peptide used to activate the precursor CD8+ T cells. Although Ag-experienced SL9-T cells from two patients were also sensitive to IL-2-mediated cell death upon restimulation in vitro, the loss of SL9 T cells was minimized with p41. This study suggests that agonist sequences can replace aberrantly immunogenic native epitopes for the rational design of vaccines targeting HIV-1.     

Role of Salmonella and Yersinia in the pathogenesis of spondyloarthropathies and rheumatoid arthritis. II. Antibodies to Salmonella and Yersinia in sera and synovial fluids

IgA, IgG and IgM antibodies against Yersinia Yop proteins, Yersinia LPS and Salmonella LPS from different serogroups were determined by enzyme-linked immunosorbent assay (ELISA) in a 885 serum samples and 92 synovial fluids. The control group consisted of 200 healthy blood donors. Compared with control subjects, patients with arthritis showed significantly increased titres of antibodies against Yersinia Yop, Yersinia LPS and Salmonella LPS appropriately in 21.7%, 44.0% and 56.0% serum samples. The prevalence of positive antibody levels was highest in Yersinia serogroup O3 and Salmonella serogroup B and D antibodies. The IgA titres were found to be much higher in adults than in children and youngsters but IgM titres consequently decreased with age. Investigation of synovial fluids obtained from patients with arthritis showed that Yersinia and Salmonella antibodies in synovial fluid mirror those in serum by concentration, by specificity and by distribution in classes.     

Proteins reacting with anti-spectrin antibodies are present in Chlamydomonas cells.

It was found either in Western-blot analysis or in indirect immunofluorescence microscopy that cells of the alga Chlamydomonas reinhardtii contain polypeptides cross-reacting with antibodies directed against red blood cell spectrin. The protein could also be detected by immunoprecipitation with anti-spectrin antibodies. C. reinhardtii cells contain distinct polypeptide chains reacting with antibodies directed against either α- or β-spectrin subunits. This protein was extracted from the cells with low ionic strength solution but was not with nonionic detergent.     

The HIV-1 HLA-A2-SLYNTVATL is a help-independent CTL epitope

The CTL response to the HLA-A*0201-restricted, HIV-1 p17 Gag(77-85) epitope (SLYNTVATL; SL9) has been extensively studied in patients. Although this reactivity is exceptionally prominent in chronically infected patients and inversely correlated to viral load, SL9-specific CTLs (SL9-CTLs) are rarely detected in acute infection. To explore the cellular basis for this unusual manifestation, SL9-CTLs primed ex vivo from naive circulating CD8(+) T cells of healthy, seronegative donors were generated and characterized. SL9 appeared to differ from other well-studied A*0201-restricted epitopes in several significant respects. In contrast to published reports for influenza and melanoma peptides and the HIV gag IV9 epitope studied here in parallel, SL9-CTLs were primed by immature but not mature autologous dendritic cells. Highly activated SL9-CTLs produce sufficient autocrine mediators to sustain clonal expansion and CTL differentiation for months without CD4(+) T cells or exogenous IL-2. Moreover, SL9-CTLs were sensitive to paracrine IL-2-induced apoptosis. IL-2 independence and sensitivity to paracrine IL-2 were also characteristic of SL9-CTLs immunized by dendritic cells transduced by a nonreplicating lentiviral vector encoding full-length Gag. In vitro-primed SL9-CTLs resembled those derived from patients in degeneracy of recognition and functional avidities for both SL9 and its natural mutations. Together, these data show that SL9 is a highly immunogenic, help-independent HIV epitope. The scarcity of SL9-CTLs in acute infection may result from cytokine-induced apoptosis with the intense activation of the innate immunity. In contrast, SL9-CTLs that constitutively produce autocrine help would predominate during CD4-diminished chronic infection.     

The role of Salmonella and Yersinia in the pathogenesis of spondyloarthropathies and rheumatoid arthritis. I. Detection of bacteria, bacterial DNA, lipopolysaccharide and ECA antigen in synovial fluid or blood

To investigate the role of Salmonella and Yersinia in the pathogenesis of spondyloarthropathies and rheumatoid arthritis synovial specimens from 92 patients were analysed for the presence of bacterial DNA with the use of polymerase chain reaction and for the presence of lipopolysaccharide and enterobacterial common antigen (ECA) with the use of Dot-ELISA. In addition, peripheral blood samples were available for PCR analysis from 68 patients. Salmonella and Yersinia chromosomal DNA was not found in any of the synovial specimens and blood samples from the patients. All of the synovial fluids were also culture-negative. Salmonella LPS antigens were observed in 8 (8.6%), Yersinia in 20 (21.7%) and ECA antigens in 32 (34.9%) synovial specimens. Our findings revealed the presence of bacterial degradation products, but not bacteria from the genus Salmonella and Yersinia or their DNA in the synovial fluid or blood of patients with spondyloarthropathies and rheumatoid arthritis.     

Value of Perfusion-Weighted MR Imaging in the Assessment of Early Cerebral Alterations in Neurologically Asymptomatic HIV-1-Positive and HCV-Positive Patients

Background and Purpose

Asymptomatic central nervous system (CNS) involvement occurs in the early stage of the human immunodeficiency virus (HIV) infection. It has been documented that the hepatitis C virus (HCV) can replicate in the CNS. The aim of the study was to evaluate early disturbances in cerebral microcirculation using magnetic resonance (MR) perfusion-weighted imaging (PWI) in asymptomatic HIV-1-positive and HCV-positive patients, as well as to assess the correlation between PWI measurements and the clinical data.

Materials and Methods

Fifty-six patients: 17 HIV-1-positive non-treated, 18 HIV-1-positive treated with combination antiretroviral therapy (cART), 7 HIV-1/HCV-positive non-treated, 14 HCV-positive before antiviral therapy and 18 control subjects were enrolled in the study. PWI was performed with a 1.5T MR unit using dynamic susceptibility contrast (DSC) method. Cerebral blood volume (CBV) measurements relative to cerebellum (rCBV) were evaluated in the posterior cingulated region (PCG), basal ganglia (BG), temporoparietal (TPC) and frontal cortices (FC), as well as in white matter of frontoparietal areas. Correlations of rCBV values with immunologic data and liver histology activity index (HAI) were analyzed.

Results

Significantly lower rCBV values were found in the right TPC and left FC as well as in PCG in HIV-1-positive naïve (p = 0.009; p = 0.020; p = 0.012), HIV-1 cART treated (p = 0.007; p = 0.009; p = 0.033), HIV-1/HCV-positive (p = 0.007; p = 0.027; p = 0.045) and HCV-positive patients (p = 0.010; p = 0.005; p = 0.045) compared to controls. HIV-1-positive cART treated and HIV-1/HCV-positive patients demonstrated lower rCBV values in the right FC (p = 0.009; p = 0.032, respectively) and the left TPC (p = 0.036; p = 0.005, respectively), while HCV-positive subjects revealed lower rCBV values in the left TPC region (p = 0.003). We found significantly elevated rCBV values in BG in HCV-positive patients (p = 0.0002; p<0.0001) compared to controls as well as to all HIV-1-positive subjects. There were no significant correlations of rCBV values and CD4 T cell count or HAI score.

Conclusions

PWI examination enables the assessment of HIV-related as well as HCV-related early cerebral dysfunction in asymptomatic subjects. HCV-infected patients seem to reveal the most pronounced perfusion changes.     

Nerve growth factor regulates the expression of the cholinergic locus and the high-affinity choline transporter via the Akt/PKB signaling pathway

Nerve growth factor (NGF) is a trophic and survival factor for cholinergic neurons, and it induces the expression of several genes that are essential for synthesis and storage of acetylcholine (ACh), specifically choline acetyltransferase, vesicular ACh transporter (VAChT), and choline transporter. We have found previously that the phosphatidylinositol 3'-kinase pathway, but not the MEK/MAPK pathway, is the mediator of NGF-induced cholinergic differentiation. Here we demonstrate, in the rat pheochromocytoma cell line PC12 and in primary mouse neuronal cultures, that NGF-evoked up-regulation of these three cholinergic-specific genes is mediated by the anti-apoptotic signaling molecule Akt/protein kinase B. Inhibition of Akt activation by the pharmacological inhibitor 1L-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO), or by a peptide fragment derived from the proto-oncogene TLC1, eliminated NGF-stimulated increases in cholinergic gene expression, as demonstrated by RT-PCR and reporter gene assays. Moreover, treatment with HIMO reversed NGF-evoked increases in choline acetyltransferase activity and ACh production. In co-transfection assays with the reporter construct, a dominant-negative Akt plasmid and Akt1-specific small interfering RNA also attenuated NGF-induced cholinergic promoter activity. Our data indicate that, in addition to its well-described role in promoting neuronal survival, Akt can also mediate signals necessary for neurochemical differentiation.     

Peripheral Arterial Disease and Ankle-Brachial Index Abnormalites in Young and Middle-Aged HIV-Positive Patients in Lower Silesia,Poland

Background

Peripheral arterial disease (PAD) is a clinical manifestation of atherosclerosis and mainly refers to elderly patients, having a negative impact on their functionality and quality of life. The findings of previous studies in HIV-infected patients have shown that cardiovascular risk is higher and PAD occurs more frequently than in the general population. There are also contradictory observations. Much less is known about the ankle-brachial index (ABI) value in asymptomatic HIV-infected patients. The aim of this study was to evaluate the prevalence of PAD and ankle-brachial index abnormalities as well as to determine risk factors related to the disease in a group of Polish HIV–positive patients.

Methods and Findings

One hundred and eleven young to middle aged HIV–positive subjects and 40 noninfected subjects were enrolled into the study. Resting ABI measurements were performed and cardiovascular risk was analysed as well. Subgroups were created according to the ABI values: low (PAD), borderline, normal, high and altered ABI. Symptomatic PAD was observed in 2 HIV–positive patients, asymptomatic PAD was not diagnosed. The ABI value is lower and more varied, in 22.5% of the study group altered ABI values were found. Six subjects demonstrated borderline ABI, and 15 high ABI, including >1.4. In the control group no low or very high values were reported. A relation between low ABI and cardiovascular family history and between altered ABI and high–density–lipoprotein cholesterol (HDL–C) level was demonstrated.

Conclusions

In young and middle–aged HIV–positive patients, symptomatic PAD prevalence is comparable to that observed in the overall population. Among asymptomatic patients PAD is not reported. The ABI value in HIV–positive patients is more varied compared to the HIV–negative subjects; the altered ABI shows a strong relation with low HDL–C levels and metabolic syndrome.