Deshratn Asthana

Mortality risk in selenium-deficient HIV-positive children

OBJECTIVE To determine the independent contribution of specific nutritional factors on disease progression and survival in HIV-1-infected children. POPULATION HIV-infected children (N = 24), who were perinatally exposed to the virus and symptomatic, were recruited between October and December of 1990 from the Jackson Memorial Pediatric Immunology Clinic, Miami, Florida, and observed for 5 years. METHODS Immune status was measured by CD4 cell count; nutritional status was determined using serum albumin and plasma trace elements including iron, zinc, and selenium. Cox proportional hazards regression models were used to evaluate the relationship of these parameters to survival. Use of antiretroviral treatment was considered in the statistical model, and age at death was considered a parameter of disease progression. RESULTS Over the course of the study, 12 children died of HIV-related causes. The final Cox multivariate analysis indicated that, of the variables evaluated, only CD4 cell count below 200 (risk ratio [RR] = 7.05; 95% confidence interval [CI], 1.87-26.5); p = .004], and low levels of plasma selenium (RR = 5.96; 95% CI, 1.32-26.81; p = .02) were significantly and independently related to mortality. Among the children who died, those with low selenium levels (< or =85 microg/L), died at a younger age, suggesting more rapid disease progression. CONCLUSIONS In pediatric HIV-infection, low plasma level of selenium is an independent predictor of mortality, and appears to be associated with faster disease progression.

Stressful events, pessimism, natural killer cell cytotoxicity, and cytotoxic/suppressor T cells in HIV+ black women at risk for cervical cancer.

Objective This study examines whether stressful negative life events and pessimism were associated with lower natural killer cell cytotoxicity (NKCC) and T cytotoxic/suppressor cell (CD8+CD3+) percentage in black women co-infected with human immunodeficiency virus Type 1 (HIV-1) and human papillomavirus (HPV), a viral initiator of cervical cancer. Method Psychosocial interviews, immunological evaluations, and cervical swabs for HPV detection and subtyping were conducted on 36 HIV+ African-American, Haitian, and Caribbean women. Results Greater pessimism was related to lower NKCC and cytotoxic/suppressor cells after controlling for presence/absence of HPV Types 16 or 18, behavioral/lifestyle factors, and subjective impact of negative life events. Conclusions A pessimistic attitude may be associated with immune decrements, and possibly poorer control over HPV infection and increased risk for future promotion of cervical dysplasia to invasive cervical cancer in HIV+ minority women co-infected with HPV.

Older age and plasma viral load in HIV-1 infection.

Background: The purpose of the study was to examine the relationship between age and plasma viral load in HIV-1-infected individuals. Design: The experimental method was to recruit older (> 50 years of age) and younger (18–39 years of age) HIV-1-infected individuals. The plasma viral load was measured using the Roche Molecular Systems UltraSensitive Roche HIV-1 Monitor test reflexively with the standard Amplicor HIV Monitor test to quantify viral load in the range of 50–750 000 copies of HIV-1 RNA/ml plasma. Subjects: A total of 135 HIV-1-seropositive individuals (at Centers for Disease Control and Prevention early symptomatic stage B or late symptomatic stage/AIDS C) were enrolled as part of a larger cohort also consisting of HIV-1-seronegative individuals. Results: A generalized linear models statistical analysis was conducted in order to evaluate age category as a predictor of plasma viral load. The result was a significant effect of age category, with older age associated with a lower plasma viral load. The association held controlling for antiretroviral therapy usage, disease stage, antiretroviral medication adherence, HIV-1 serostatus duration, alcohol and substance use, recent sexually transmitted disease, and sociodemographics (except income). Conclusion: Older age was associated with lower levels of HIV-1 replication in this sample, independent of antiretroviral therapy usage, regimen adherence, and disease stage. It is suggested that the effect may be caused by changes in viral evolution or immunological monitoring specific to older individuals with HIV-1 infection.

Tobacco Smoking Increases Immune Activation and Impairs T-Cell Function in HIV Infected Patients on Antiretrovirals: A Cross-Sectional Pilot Study

Background The influence of tobacco smoking on the immune system of HIV infected individuals is largely unknown. We investigated the impact of tobacco smoking on immune activation, microbial translocation, immune exhaustion and T-cell function in HIV infected individuals. Method HIV infected smokers and non-smokers (n = 25 each) with documented viral suppression on combination antiretroviral therapy and HIV uninfected smokers and non-smokers (n = 15 each) were enrolled. Markers of immune activation (CD38 and HLA-DR) and immune exhaustion (PD1, Tim3 and CTLA4) were analyzed in peripheral blood mononuclear cells (PBMCs) by flow cytometry. Plasma markers of microbial translocation (soluble-CD14 - sCD14 and lipopolysaccharide - LPS) were measured. Antigen specific functions of CD4+ and CD8+ T-cells were measured, by flow cytometry, in PBMCs after 6 hours stimulation with Cytomegalovirus, Epstein-Barr virus and Influenza Virus (CEF) peptide pool. Results Compared to non-smokers, smokers of HIV infected and uninfected groups showed significantly higher CD4+ and CD8+ T-cell activation with increased frequencies of CD38+HLA-DR+ cells with a higher magnitude in HIV infected smokers. Expressions of immune exhaustion markers (PD1, Tim3 and CTLA4) either alone or in combinations were significantly higher in smokers, especially on CD4+ T-cells. Compared to HIV uninfected non-smokers, microbial translocation (sCD14 and LPS) was higher in smokers of both groups and directly correlated with CD4+ and CD8+ T-cell activation. Antigen specific T-cell function showed significantly lower cytokine response of CD4+ and CD8+ T-cells to CEF peptide-pool stimulation in smokers of both HIV infected and uninfected groups. Conclusions Our results suggest that smoking and HIV infection independently influence T-cell immune activation and function and together they present the worst immune profile. Since smoking is widespread among HIV infected individuals, studies are warranted to further evaluate the cumulative effect of smoking on impairment of the immune system and accelerated disease progression.

Impaired Restoration of Plasmacytoid Dendritic Cells in HIV-1-Infected Patients with Poor CD4 T Cell Reconstitution Is Associated with Decrease in Capacity to Produce IFN-α but Not Proinflammatory Cytokines

We analyzed reconstitution characteristics of plasmacytoid dendritic cells (PDCs) and myeloid DCs-1 in 38 HIV-1-infected patients with impaired restoration of CD4 T cell counts despite prolonged suppression of plasma viremia (discordant) and compared them with 42 patients showing good immunological and virological responses following highly active antiretroviral therapy (HAART). While myeloid DCs showed spontaneous recovery following HAART in both the groups, the discordant patients demonstrated poor peripheral reconstitution of PDCs as compared with concordant patients. The ability of PDCs to produce IFN-α following stimulation with TLR7 ligand imiquimod and TLR9 ligand CpG ODN-2216 was also impaired in discordant patients even after 2 years following initiation of HAART. Lower IFN-α expression in the PDCs following TLR stimulation was further associated with lower expression of transcription factor, IFN regulatory factor-7. In contrast, production of TNF-α and IL-6 following TLR stimulation was comparable in both groups of patients, indicating that impaired reconstitution characteristics do not affect the capacity of PDCs to produce proinflammatory cytokines. The discordant patients had significantly lower baseline CD4 T cell counts and higher baseline viral load at the initiation of HAART implying that lower baseline CD4 T cell counts and higher plasma viral load are associated with impaired restoration of CD4 T cells and PDCs, thus, increasing the susceptibility of discordant patients toward opportunistic infections despite virological control.

Beyond the Brain The Role of Brain-Derived Neurotrophic Factor in Viroimmune Responses to Antiretroviral Therapy among People Living with HIV with and without Alcohol Use

Objective: Given the emerging data suggesting the key role of brain-derived neurotrophic factor (BDNF) in the immune system, we assessed longitudinally whether BDNF depletions induced by hazardous alcohol use (HAU) would impact a response to antiretroviral therapy (ART). Methods: In a prospective single-site cohort, virological and immunological responses to ART in 200 hazardous and 200 nonhazardous users were obtained, along with plasma BDNF levels. Results: Hazardous drinkers were more likely to have BDNF levels <4000 pg/mL (odds ratio [OR] = 1.6, P = .01). Participants with BDNF <4000 pg/mL were less likely to have CD4 counts of more than 500 cells/mm3 (P = .02) and to achieve viral suppression over the follow-up period (OR = 1.5, P = .03). Multivariate analysis confirmed the significant role of HAU and low BDNF in predicting viroimmune responses. Conclusion: Hazardous alcohol use was associated with BDNF alterations, which in turn were linked to a limited response to ART in terms of viral suppression and CD4 count improvements.

Effects of Ageing on the Immune System: Infants to Elderly

Physiological ageing is accompanied by decline in immune system function and immune alteration during ageing increases susceptibility to infections. We retrospectively analysed the data for complete blood count (CBC) and lympho‐cyte subsets from infant to elderly age groups to determine changes during ageing. Data from dual‐platform flow cytometry and CBC were analysed to determine the percentage (%) and absolute cell counts (Abs) of peripheral blood lymphocyte subsets (CD3, CD4, CD8, CD19 and CD56+16+ cells) in infants (1 month to 1 year), children (1 year to 6 years), adolescents (12 years to 18 years), adults (21 years to 50) and elderly (70 years to 92 years). Differences in plasma cytokine levels in adults and elderly were also analysed using Randox system. Comparisons among age groups from infants through adults revealed progressive declines in the percentage of total lymphocytes and absolute numbers of T and B cells. The NK cells declined from infancy to adulthood but increased in elderly participants. The percentages of T cells increased with age from infant to adulthood and then declined. Pro‐inflammatory cytokines, TNF‐α and IL‐6, were higher in elderly people compared to adults. The elderly group had significantly higher levels of monocyte chemoattractant protein‐1 (MCP‐1) and lower levels of epidermal growth factor (EGF) compared to adults. Our findings confirm and extend earlier reports on age‐related changes in lymphocyte subpopulations and data generated from this study is useful for clinicians and researchers, patient management in various age groups for the interpretation of disease‐related changes, as well as therapy‐dependent alterations.

Intracellular Substrates for the Primer-Unblocking Reaction by Human Immunodeficiency Virus Type 1 Reverse Transcriptase: Detection and Quantitation in Extracts from Quiescent- and Activated-Lymphocyte Subpopulations

ABSTRACT Treatment of human immunodeficiency virus type 1 (HIV-1)-infected patients with 3′-azido-3′-deoxythymidine (AZT) selects for mutant forms of viral reverse transcriptase (RT) with increased ability to remove chain-terminating nucleotides from blocked DNA chains. We tested various cell extracts for the presence of endogenous acceptor substrates for this reaction. Cell extracts incubated with HIV-1 RT and [32P]ddAMP-terminated DNA primer/template gave rise to 32P-labeled adenosine 2′,3′-dideoxyadenosine 5′,5′′′−P1,P4-tetraphosphate (Ap4ddA), ddATP, Gp4ddA, and Ap3ddA, corresponding to the transfer of [32P]ddAMP to ATP, PPi, GTP, and ADP, respectively. Incubation with [32P]AZT monophosphate (AZTMP)-terminated primer/template gave rise to the analogous 32P-labeled AZT derivatives. Based on the rates of formation of the specific excision products, ATP and PPi levels were determined: ATP was present at 1.3 to 2.2 mM in H9 cells, macrophages, and unstimulated CD4+ or CD8+ T cells, while PPi was present at 7 to 15 μM. Under these conditions, the ATP-dependent reaction predominated, and excision by the AZT-resistant mutant RT was more efficient than wild type RT. Activated CD4+ or CD8+ T cells contained 1.4 to 2.7 mM ATP and 55 to 79 μM PPi. These cellular PPi concentrations are lower than previously reported; nonetheless, the PPi-dependent reaction predominated in extracts from activated T cells, and excision by mutant and wild-type RT occurred with similar efficiency. While PPi-dependent excision may contribute to AZT resistance in vivo, it is likely that selection of AZT-resistant mutants occurs primarily in an environment where the ATP-dependent reaction predominates.

Reference ranges of lymphocyte subsets in healthy adults and adolescents with special mention of T cell maturation subsets in adults of South Florida.

BACKGROUND Analysis of peripheral blood lymphocyte subsets has become an essential tool in the evaluation of outcome of diagnostic and research related questions in immunological and pathological conditions. Periodic evaluation and establishment of normal lymphocyte reference ranges are required in clinical and research settings of various immunodeficiency disorders for evaluation of the significance of observations. It is also important that age and gender specific lymphocyte subset reference ranges should be locally established for meaningful comparison and accurate result interpretation as age plays a significant role in the development of immune system. METHODS We performed dual platform flow cytometry to determine reference ranges for lymphocyte subsets (CD3, CD4, CD8, CD19 [B cells] and CD16+CD56+ [Natural Killer - NK cells]) in 50 adolescents (age range: 12-18) and 100 adults (age range: 21-67) along with T cell maturation, activation and co-stimulatory molecules in healthy multiracial adult population of South Florida. RESULTS The lymphocyte reference ranges percentages [absolute counts - Abs, cells/μl] unadjusted for gender differences for adolescents are: CD3: 49-83 [939-2959]; CD4: 27-53 [467-1563]; CD8: 16-40 [259-1262]; CD19+ B cells: 8-31 [169-1297] and CD16+CD56+ NK cells: 3-30 [59-1178] and for adults are: CD3: 65-88 [983-3572]; CD4: 26-62 [491-2000]; CD8: 14-44 [314-2,087]; CD19+ B cells: 2-27 [64-800] and CD16+CD56+ NK cells: 2-27 [27-693]. The ranges for CD4:CD8 ratio for adolescents and adults are 0.7-2.6 and 0.6-4.4, respectively. Gender based analysis of relative percentages of lymphocyte subsets showed no significant differences between adult and adolescent males and females. The mean CD4:CD8 ratio was significantly higher in adult females than males (P=0.04) and in adolescents this difference was not significant between genders. The mean CD3 and CD4 T cell percentages were higher and CD19 cell percentages were lower in adults compared to adolescents (P<0.0001). Absolute lymphocyte counts showed a positive correlation with the absolute counts of CD3+, CD4+, CD8+, CD19+, CD16+CD56+, CD45RO+ and CD45RA+ cells (all correlations with P<0.0001 except CD45RO [P=0.01] and CD45RA [P=0.03]). CONCLUSION The reference values of peripheral blood lymphocyte subsets were analyzed in healthy adolescent and adult population of South Florida. This study indicates the need for periodic evaluation and establishment of lymphocyte reference ranges for patient population served based on gender and age since these could influence immune status and treatment outcome.

Biochip array-based analysis of plasma cytokines in HIV patients with immunological and virological discordance.

Assessment of cytokines in body fluids or cells provides important information in understanding the disease process and designing treatment strategies. Recent introduction of antibody‐based protein arrays have provided investigators simultaneous and specific detection of multiple analytes in a single sample using minimum volumes. In this study, we used a biochip array system capable of measuring 12 cytokines and growth factors (IL‐2, IL‐4, IL‐6, IL‐8, IL‐10, IL‐1α, IL‐1β, IFN‐γ, TNF‐α, monocyte chemoattractant protein‐1 (MCP‐1), vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF)) in HIV patients with immunological and virological discordance (discordant) to find out differences if any, in their plasma cytokine profiles when compared with concordant HIV‐infected individuals. A sandwich chemiluminescent assay was performed with plasma specimens of 110 HIV patients (55 discordant, 55 concordant) and 22 normal healthy individuals followed by enzyme‐linked immunosorbent assay (ELISA) to the confirm levels of cytokines and growth factors that showed significant differences in the two groups. The discordant HIV patients showed significantly higher levels of plasma VEGF (P = 0.001) and EGF (P = 0.034) levels when compared with concordant patients. Overall, the patients showed significantly higher levels of TNF‐α, MCP‐1 and VEGF when compared with the normal healthy controls (P < 0.05). ELISA for VEGF (P < 0.001) and EGF (P = 0.004) confirmed the comparison obtained with biochip array, between the discordant and concordant patients. The results of cytokine quantitation by biochip array and ELISA confirmed that this technology is not only comparable but also has a good potential in the future applications involving measurement of multiple cytokines with limiting specimens.