Expansion of interferon-γ-secreting HIV-specific T cells during successful antiretroviral therapy.

OBJECTIVES: Antiretroviral therapy (ART) suppresses HIV viraemia, thereby reducing the antigenic drive for T cells to proliferate. Accordingly, selected HIV-specific T-cell responses have been described to contract within weeks of ART initiation. Here, we sought to investigate whether these findings apply to the entire repertoire of HIV-specific T cells. METHODS: Using interferon (IFN)-γ enzyme linked immuno spot (ELISpot), we performed retrospective 2-year proteome-wide monitoring of HIV-specific T cells in 17 individuals with undetectable viral loads during ART. The sample pool for each study subject consisted of one pre-ART time-point and at least two time-points after initiation of therapy. RESULTS: Peripheral pools of HIV-specific T cells decreased nonsignificantly within the first 2 years under ART in our cohort of patients, in terms of both breadth and magnitude. However, in most cases, the seeming decrease masked ongoing expansion of individual HIV-specific T-cell responses. We detected synchronous contraction and expansion of T-cell responses - with different peptide specificities - in 12 out of 17 study participants during follow-up. Importantly, the observed expansions and contractions of individual HIV-specific T-cell responses reached similar ranges, supporting the biological relevance of our findings. CONCLUSIONS: We conclude that successful ART enables both contraction and expansion of HIV-specific T-cell responses. Our results should prompt a renewed interest in HIV-specific T-cell dynamics under ART, in particular to elucidate the mechanisms that uncouple, to some extent, particular HIV-specific T-cell responses from variations in circulating antigen load and functionally characterize expanding/contracting T-cell populations beyond IFN-γ secretion. Assuming that expanding HIV-specific T-cell responses under ART are protective and functional, harnessing those mechanisms may provide novel opportunities for assisting viral control in chronically infected individuals.

Consistent cytotoxic-T-lymphocyte targeting of immunodominant regions in human immunodeficiency virus across multiple ethnicities.

Although there is increasing evidence that virus-specific cytotoxic-T-lymphocyte (CTL) responses play an important role in the control of human immunodeficiency virus (HIV) replication in vivo, only scarce CTL data are available for the ethnic populations currently most affected by the epidemic. In this study, we examined the CD8(+)-T-cell responses in African-American, Caucasian, Hispanic, and Caribbean populations in which clade B virus dominates and analyzed the potential factors influencing immune recognition. Total HIV-specific CD8(+)-T-cell responses were determined by enzyme-linked immunospot assays in 150 HIV-infected individuals by using a clade B consensus sequence peptide set spanning all HIV proteins. A total of 88% of the 410 tested peptides were recognized, and Nef- and Gag-specific responses dominated the total response for each ethnicity in terms of both breadth and magnitude. Three dominantly targeted regions within these proteins that were recognized by >90% of individuals in each ethnicity were identified. Overall, the total breadth and magnitude of CD8(+)-T-cell responses correlated with individuals' CD4 counts but not with viral loads. The frequency of recognition for each peptide was highly correlated with the relative conservation of the peptide sequence, the presence of predicted immunoproteasomal cleavage sites within the C-terminal half of the peptide, and a reduced frequency of amino acids that impair binding of optimal epitopes to the restricting class I molecules. The present study thus identifies factors that contribute to the immunogenicity of these highly targeted and relatively conserved sequences in HIV that may represent promising vaccine candidates for ethnically heterogeneous populations.

Persistent recognition of autologous virus by high-avidity CD8 T cells in chronic, progressive human immunodeficiency virus type 1 infection.

CD8 T-cell responses are thought to be crucial for control of viremia in human immunodeficiency virus (HIV) infection but ultimately fail to control viremia in most infected persons. Studies in acute infection have demonstrated strong CD8-mediated selection pressure and evolution of mutations conferring escape from recognition, but the ability of CD8 T-cell responses that persist in late-stage infection to recognize viruses present in vivo has not been determined. Therefore, we studied 24 subjects with advanced HIV disease (median viral load = 142,000 copies/ml; median CD4 count = 71/ micro l) and determined HIV-1-specific CD8 T-cell responses to all expressed viral proteins using overlapping peptides by gamma interferon Elispot assay. Chronic-stage virus was sequenced to evaluate autologous sequences within Gag epitopes, and functional avidity of detected responses was determined. In these subjects, the median number of epitopic regions targeted was 13 (range, 2 to 39) and the median cumulative magnitude of CD8 T-cell responses was 5,760 spot-forming cells/10(6) peripheral blood mononuclear cells (range, 185 to 24,700). On average six (range, one to 8) proteins were targeted. For 89% of evaluated CD8 T-cell responses, the autologous viral sequence was predicted to be well recognized by these responses and the majority of analyzed optimal epitopes were recognized with medium to high functional avidity by the contemporary CD8 T cells. Withdrawal of antigen by highly active antiretroviral therapy led to a significant decline both in breadth (P = 0.032) and magnitude (P = 0.0098) of these CD8 T-cell responses, providing further evidence that these responses had been driven by recognition of autologous virus. These results indicate that strong, broadly directed, and high-avidity gamma-interferon-positive CD8 T-cells directed at autologous virus persist in late disease stages, and the absence of mutations within viral epitopes indicates a lack of strong selection pressure mediated by these responses. These data imply functional impairment of CD8 T-cell responses in late-stage infection that may not be reflected by gamma interferon-based screening techniques.

Noncytolytic inhibition of X4 virus by bulk CD8(+) cells from human immunodeficiency virus type 1 (HIV-1)-infected persons and HIV-1-specific cytotoxic T lymphocytes is not mediated by beta-chemokines.

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) mediate immunologic selection pressure by both cytolytic and noncytolytic mechanisms. Non cytolytic mechanisms include the release of beta-chemokines blocking entry of R5 HIV-1 strains. In addition, CD8(+) cells inhibit X4 virus isolates via release of as yet poorly characterized soluble factors. To further characterize these factors, we performed detailed analysis of CTL as well as bulk CD8(+) T lymphocytes from six HIV-1-infected individuals and from six HIV-1-seronegative individuals. Kinetic studies revealed that secreted suppressive activities of HIV-1-specific CTL and bulk CD8(+) T lymphocytes from all HIV-1-infected persons are significantly higher than that of supernatants from seronegative controls. The suppressive activity could be blocked by monensin and brefeldin A, was heat labile, and appeared in a pattern different from that of secretion of chemokines (MDC, I-309, MIP-1alpha, MIP-1beta, and RANTES), cytokines (gamma interferon, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor), and interleukins (interleukin-13 and interleukin-16). This suppression activity was characterized by molecular size exclusion centrifugation and involves a suppressive activity of >50 kDa which could be bound to heparin and a nonbinding inhibitory activity of <50 kDa. Our data provide a functional link between CD8(+) cells and CTL in the noncytolytic inhibition of HIV-1 and suggest that suppression of X4 virus is mediated through proteins. The sizes of the proteins, their affinity for heparin, and the pattern of release indicate that these molecules are not chemokines.

Partial rescue of ethanol-induced neuronal apoptosis by growth factor activation of phosphoinositol-3-kinase.

BACKGROUND: Ethanol inhibition of insulin signaling pathways may contribute to impaired central nervous system (CNS) development in the fetal alcohol syndrome and brain atrophy associated with alcoholic neurodegeneration. Previous studies demonstrated ethanol inhibition of insulin-stimulated growth in PNET2 CNS-derived proliferative (immature) neuronal cells. We now provide evidence that the growth-inhibitory effect of ethanol in insulin-stimulated PNET2 cells is partly due to apoptosis. METHODS: Control and ethanol-treated PNET2 cells were stimulated with insulin and analyzed for viability, apoptosis, activation of pro-apoptosis and survival gene expression and signaling pathways, and evidence of caspase activation. RESULTS: Ethanol-treated PNET2 neuronal cells exhibited increased apoptosis mediated by increased levels of p53 and phospho-amino-terminal c-jun kinase (phospho-JNK), and reduced levels of Bcl-2, phosphoinositol 3-kinase (PI3 K), and intact (approximately 116 kD) poly (ADP ribose) polymerase (PARP), a deoxyribonucleic acid repair enzyme and important substrate for caspase 3. Partial rescue from ethanol-induced neuronal cell death was effected by culturing the cells in medium that contained 2% fetal calf serum instead of insulin, or insulin plus either insulin-like growth factor type 1 or nerve growth factor. The resulting enhanced viability was associated with reduced levels of p53 and phospho-JNK and increased levels of PI3 K and intact PARP. CONCLUSIONS: The findings suggest that ethanol-induced apoptosis of insulin-stimulated neuronal cells can be reduced by activating PI3 K and inhibiting pro-apoptosis gene expression and intracellular signaling through non-insulin-dependent pathways.

Differential effects of ethanol on insulin-signaling through the insulin receptor substrate-1.

Insulin stimulation increases cell proliferation and energy metabolism by activating the insulin receptor substrate I (IRS-1)-signaling pathways. This downstream signaling is mediated by interactions of specific tyrosyl phosphorylated (PY) IRS-1 motifs with SH2-containing molecules such as growth-factor receptor-bound protein 2 (Grb2) and Syp. Ethanol inhibits insulin-stimulated tyrosyl phosphorylation of IRS-1 and DNA synthesis. This study explores the roles of the Grb2- and Syp-binding motifs of IRS-1 in relation to the inhibitory effects of ethanol on insulin-stimulated DNA synthesis, proliferating cell nuclear antigen (PCNA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression, and activation of mitogen-activated protein kinase (MAPK), which is known to be essential for cell proliferation. NIH3T3 cells were stably transfected with wild-type IRS-1, or IRS-1 mutated at the Grb2 (IRS-1deltaGrb2), Syp (IRS-1deltaSyp), or Grb2 and Syp (IRS-1deltaGrb2deltaSyp)- binding sites. Cells transfected with IRS-1 had increased levels of DNA synthesis, PCNA, GAPDH, and activated MAPK. The IRS-1deltaGrb2 transfectants were highly responsive to insulin stimulation, achieving levels of GAPDH, PCNA, and activated MAPK that were higher than control. In contrast, the IRS-1deltaSyp and IRS-1deltaGrb2deltaSyp transfectants had reduced levels of DNA synthesis, PCNA, and activated MAPK. Ethanol exposure decreased insulin-stimulated DNA synthesis, PCNA, GAPDH, and activated MAPK levels in all clones, but the wild-type IRS-1 transfectants were relatively resistant, and the IRS-1deltaGrb2 transfectants were extraordinarily sensitive to these inhibitory effects of ethanol. The findings suggest that insulin-stimulated DNA synthesis and PCNA expression are mediated through the Syp-binding domain, whereas GAPDH expression and MAPK activation are modulated through both the Grb2 and Syp motifs of IRS-1. In addition, ethanol exposure may preferentially inhibit downstream signaling that requires interaction between Syp and PY-IRS-1.

Profiles of neuronal thread protein expression in Alzheimer's disease.

Neuronal thread proteins (NTPs) comprise a family of molecules expressed in brain and primitive neuroectodermal tumor cell lines. In Alzheimer's disease (AD), increased CNS levels of the 21 kD NTP species are correlated with dementia. The present study characterizes the nature and distribution of NTP expression using recently generated brain-derived polyclonal and monoclonal antibodies (MoAbs) to recombinant AD7c-NTP protein. In AD, high levels of NTP immunoreactivity were detected in neuronal perikarya, neuropil fibers, and white matter fibers (axons). In addition, 4 of the 23 AD7c-NTP MoAbs labeled degenerating neurons (with or without neurofibrillary tangles), axonal spheroids, dystrophic neurites, or irregular, wavy threadlike neuropil fibers in AD. Increased neuronal AD7c-NTP immunoreactivity in AD colocalized with perikaryal accumulations of tau-1, phosphorylated neurofilament, and the ganglioside, A2B5. In addition, AD7c-NTP immunoreactivity was detected in early neuritic plaques along with beta-amyloid-containing fibrils, but not in mature plaques, nor was it colocalized in beta A4-immunoreactive fibrils. This study demonstrates the profiles of NTP overexpression in relation to paired helical filament-associated neurodegenerative lesions in AD.

Effect of ethanol on p36 protein kinase substrate and insulin receptor substrate 1 expression and tyrosyl phosphorylation in human hepatocellular carcinoma cells.

Ethanol inhibits insulin (IN) and epidermal growth factor (EGF)-induced hepatocyte DNA synthesis. Growth factor receptor kinases, such as IN and EGF, phosphorylate insulin receptor substrate (IRS-1) and p36 protein kinase substrate, respectively, on tyrosine residues. IRS-1 and p36 are thought to be important intracellular signal transduction molecules involved in the regulation of cell growth. These investigations explored the effect of ethanol additions on the expression and tyrosyl phosphorylation (TP) of p36 and IRS-1 in a human hepatocellular carcinoma cell line (FOCUS) in relationship to cell proliferation induced by IN and serum growth factor stimulation. It was found that p36 was constitutively and highly expressed in serum-starved cells and protein, and mRNA levels did not change with cell proliferation induced by growth factors. However, exposure of FOCUS cells to ethanol additions substantially inhibited TP of p36. The early TP of IRS-1 induced by IN stimulation was also reduced by ethanol additions. Finally, there was a parallel decrease of FOCUS cell proliferation in ethanol-exposed cultures. These studies suggest that one possible mechanism of ethanol inhibitory effect on cell proliferation is through reduced TP of putative intracellular signal transduction molecules, such as p36 and IRS-1.

Presence of hepatitis B and C viral genomes in US blood donors as detected by polymerase chain reaction amplification.

Hepatitis C virus (HCV) represents a major cause of posttransfusion hepatitis worldwide. Posttransfusion hepatitis associated with hepatitis B virus (HBV) continues to occur. HBsAg-negative donor sera from the Rhode Island Blood Center between 1987 and 1988 were screened using more sensitive techniques to assess the prevalence of low level HBV infection. Group I consists of 866 healthy blood donors without HBV serologic markers, group II consists of 377 donors with ALT elevations (> 45 IU/L), group II consists of 148 donors positive for anti-HBc, and group IV consists of eight donors positive for both surrogate markers. A sensitive monoclonal immunoradiometric assay (M-IRMA) was employed for detection of HBsAg-associated epitopes (detection limit of 20 pg/ml) in serum. A subset of sera were analyzed for the presence of HBV DNA using the method of anti-HBs capture of HBV related virions in serum followed by polymerase chain reaction (PCR) amplification. Using these techniques, 0.8% and 1.7% of donors were positive for HBsAg and HBV DNA respectively in group I. In contrast, 0.9% and 9.5% in group II and 0.7% and 18.1% in group III were positive, respectively. There were eight donors with both ALT elevation and anti-HBc; and four (50%) of these were positive for HBV DNA. In the group with anti-HBc, the majority (80%) of donors with HBV DNA had either no or low (signal to noise ratio < 10) anti-HBs titer. Using anti-HCV testing and reverse transcription-PCR for detection of HCV genomes, we detected evidence of HCV infection in nine of the 49 donors with low level HBV DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of ethanol and extracellular matrix on induction of p36 protein kinase substrate expression in rat hepatocytes.

p36 plays a direct role in DNA synthesis and is overexpressed in transformed cells. It is also an important component linking the cell membrane to intracellular cytoskeletal components. Experiments were performed in primary rat hepatocyte culture stimulated with epidermal growth factor (EGF) to determine if p36 expression was related to DNA synthesis or to the effect of the extracellular matrix on hepatocyte differentiation; ethanol was employed as an agent to inhibit hormone stimulated hepatocyte DNA synthesis. It was found that hepatocyte p36 expression was highly dependent on the type of extracellular matrix and the time in culture. There was no correlation of p36 expression with DNA synthesis and, therefore, p36 levels appeared more closely related to the differentiated phenotype, induced by the extracellular matrix interactions rather than cellular proliferation.

Hepatitis B virus infection in patients with idiopathic liver disease.

We studied 67 HBsAg-negative Israeli patients (36 negative for all HBV serological markers as group 1 and 31 positive for antibodies to HBs and HBc as group 2) with chronic liver disease and cirrhosis of unknown origin using a rapid, sensitive and specific assay for the detection of low levels of hepatitis B virus in serum. This technique uses a high-affinity monoclonal antibody to HBs against an a domain epitope of HBsAg to capture the virion, followed by hepatitis B virus DNA amplification with the polymerase chain reaction. In addition, 55 subjects without liver disease served as controls: Group 3 (n = 32) was negative for all hepatitis B virus markers; group 4 (n = 23) was positive for antibodies to HBs and HBc. We found 11 individuals in group 1 (31%) and 10 in group 2 (29%) harboring low levels of hepatitis B virus DNA in serum. In contrast, no one in group 3 or group 4 was positive by this technique (p less than 0.0001). Using polymerase chain reaction primers spanning other regions of the hepatitis B virus genome and a method of restriction-fragment analysis of polymerase chain reaction-amplified sequences, we detected significant DNA sequence heterogeneity, suggesting infection with distinct hepatitis B virus strains. DNA extracted from paraffin-embedded liver biopsy specimens of 42 patients from groups 1 and 2 was shown to contain hepatitis B virus DNA by polymerase chain reaction in 11 of 12 patients with circulating virion DNA. More important, 18 additional patients whose sera were negative by HBs-antibody capture/polymerase chain reaction amplification had hepatitis B virus DNA sequences in their livers.(ABSTRACT TRUNCATED AT 250 WORDS)

Geographic classification of dengue-2 virus strains by antigen signature analysis.

Dengue-2 virus strains from different locations were compared by T1-RNAse-resistant oligonucleotide fingerprinting and antigen signature analysis. The latter technique involved construction of radioimmunoassays using monoclonal antibodies that recognize nine distinct dengue-2 type-specific and flavivirus cross-reactive epitopes over a range of antigen concentrations. A statistical method was used to align unknown dengue antigen concentrations in different strain preparations, allowing comparison of binding profiles. Twenty-six dengue-2 virus strains were separated into five distinct groups (topotypes) on the basis of unique RNA fingerprints. Two of these were represented by New Guinea C, the prototype virus isolated in 1944, and a Philippine strain; others were segregated on the basis of greater than or equal to 80% shared oligonucleotides into similarity groups representing Burma/Thailand (8 strains), Puerto Rico (12 strains), and Jamaica (4 strains). Signature analysis of the prototype and four geographic topotype strains revealed striking antigenic differences. In contrast, a high degree of antigenic similarity was found among strains from the same geographic region. Variation between antigenically distinct strains occurred at both type-specific and group-reactive epitopes, but the widest differences appeared at group-reactive determinants. Signature analysis provides a more rapid and simpler means than RNA fingerprinting of monitoring changes or new introductions of dengue virus populations in a geographic region.

Sensitive and specific monoclonal immunoassay for detecting yellow fever virus in laboratory and clinical specimens.

A solid-phase radioimmunoassay (RIA) was developed for the detection of yellow fever (YF) virus in infected cell culture supernatant fluid and clinical samples. The test employed a flavivirus group-reactive monoclonal antibody attached to a polystyrene bead support and a radiolabeled type-specific antibody probe in a simultaneous sandwich RIA format. Optimal assay conditions specified a 16-h incubation at high temperature (45 degrees C). Monoclonal antibody to tetanus toxoid was added to the radiolabeled probe to inhibit nonspecific binding. The sensitivity of the assay for cell culture-propagated virus was 2.0 log10 50% mosquito infectious doses per 100 microliters or 100 pg of gradient-purified virion protein per 100 microliters. Specificity, assessed with human sera from 512 patients with liver diseases other than YF, including acute viral hepatitis, showed a false-positive rate of 0.0 to 0.6%. Sera from experimentally infected rhesus macaques containing greater than 3.0 log10 units/100 microliter of YF virus were positive by RIA. Sera and liver tissue from human patients were found to be positive.

S1 nuclease mapping of viral RNAs from a temperature-sensitive transformation mutant of murine sarcoma virus.

The structures of murine sarcoma virus (MuSV) ts110 viral RNA and intracellular RNA present in MuSV ts110-infected cells (6m2 cells) have been examined by S1 nuclease analysis. A previous study involving heteroduplex analysis of MuSV ts110 viral RNAs hybridized to wild-type DNA revealed the presence of two MuSV ts110 RNAs, 4.0 and 3.5 kilobases (kb) in length, containing overlapping central deletions relative to wild-type MuSV 124 viral RNA (Junghans et al., J. Mol. Biol. 161:229-255, 1982). Here we show that the deletion (termed delta 1) in the 4.0-kb RNA has a 5' border located at about nucleotide 2409 (using the numbering system of Van Beveren et al., Cell 27:97-108, 1981), a position 63 bases upstream of the junction of the p30 and p10 coding sequences. The 3' border of the delta 1 deletion is found 1,473 bases downstream at approximately nucleotide 3883, 10 nucleotides downstream of the first mos gene initiation codon. In the 3.5-kb MuSV ts110 RNA, the 5' border of the deleted central region (termed delta 2) is located in a splice consensus donor site at approximately nucleotide 2017, 330 bases downstream from the junction of the p12 and p30 coding sequences, and extends about 1,915 bases in the downstream direction to nucleotide 3935, found in a splice consensus acceptor site about 55 nucleotides downstream of the first mos gene initiation codon and 30 bases upstream of the second initiation codon. No alteration of polyadenylate addition sites was observed in either MuSV ts110 RNA species, as compared with MuSV 349 RNA. The observation that the 5' and 3' borders of the deletion in the 3.5-kb RNA are within in-frame splice donor and acceptor sites suggests strongly that the 3.5-kb RNA is derived from the 4.0-kb RNA by a temperature-sensitive splice mechanism. Data presented here show unequivocally that formation of the 3.5-kb MuSV ts110 RNA from which the P85gag-mos polypeptide is translated is temperature sensitive. At 33 degrees C, with S1 analysis, the 3.5-kb RNA is found readily in 6m2 cells. Within 4 h of a shift to 39 degrees C, however, only trace amounts of this RNA can be found. Moreover, reshifting 6m2 cells to 33 degrees C permits the reappearance of the 3.5-kb RNA at its original level.