Maria C. Villacres

Vitamin D deficiency in HIV-infected and HIV-uninfected women in the United States

Background:Vitamin D deficiency is of increasing concern in HIV-infected persons because of its reported association with a number of negative health outcomes that are common in HIV. We undertook this study to determine the prevalence and predictors of vitamin D deficiency among a nationally representative cohort of middle-aged, ethnically diverse, HIV-infected and HIV-uninfected women enrolled in the Womens Interagency HIV Study (WIHS). Methods:Vitamin D testing was performed by Quest Diagnostics on frozen sera using the liquid chromatography/mass spectroscopy method. Vitamin D deficiency was defined as 25(OH)D ≤20 ng/mL. Comparisons of continuous and categorical characteristics among HIV-infected and HIV-uninfected women were made by Wilcoxon tests and Pearson χ2 tests, respectively. Results:One thousand seven hundred seventy-eight women (1268 HIV positive) were studied. Sixty-three percent had vitamin D deficiency (60% HIV positive vs. 72% HIV negative; P < 0.001). Multivariable predictors of vitamin D deficiency were being African American (adjusted odds ratio 3.02), Hispanic (adjusted odds ratio 1.40), body mass index (adjusted odds ratio 1.43), age (adjusted odds ratio 0.84), HIV positive (adjusted odds ratio 0.76), glomerular filtration rate <90·mL−1·min−1 (adjusted odds ratio 0.94), and WIHS sites Los Angeles (adjusted odds ratio 0.66) and Chicago (adjusted odds ratio 0.63). In the HIV-positive women, multivariate predictors were undetectable HIV RNA (adjusted odds ratio 0.69), CD4 50-200 cells per cubic millimeter (adjusted odds ratio 1.60), CD4 <50 cells per cubic millimeter (adjusted odds ratio 1.94), and recent protease inhibitor use (adjusted odds ratio 0.67). Conclusions:In this study of more than 1700 women in the United States, most women with or without HIV infection had low vitamin D levels and African American women had the highest rates of vitamin D deficiency. An understanding of the role that vitamin D deficiency plays in non-AIDS-related morbidities is planned for investigation in WIHS.

Association of the IFNL4-ΔG Allele With Impaired Spontaneous Clearance of Hepatitis C Virus

Interferon lambda 4 protein can be generated in IFNL4-ΔG carriers but not IFNL4-TT homozygotes. We studied 890 anti-hepatitis C virus (HCV)-positive participants in the Womens Interagency HIV Study. Among blacks (n = 555), HCV was more often cleared for those with genotype IFNL4-TT/TT (32.6%; odds ratio [OR], 3.59; P = 3.3 × 10(-5)) than IFNL4-TT/ΔG (11.3%; OR, 0.95; P = .86) or IFNL4-ΔG/ΔG (11.9%; referent). Pooling these data with published results in blacks (n = 1678), ORs were 3.84 (P = 8.6 × 10(-14)) for IFNL4-TT/TT and 1.44 (P = .03) IFNL4-TT/ΔG, and the area under the curve was 0.64 for IFNL4-ΔG genotype and 0.61 for rs12979860 (IL28B). IFNL4-ΔG is strongly associated with impaired spontaneous HCV clearance.

Microbiology and Cytokine Levels Around Healthy Dental Implants and Teeth

BACKGROUND Elicitation of the relationship of periodontopathogens and pro-inflammatory cytokines to bone resorption and formation is significant to a growing body of research known as osteoimmunology. It is essential that clinically healthy peri-implant and periodontal sites are studied to contribute comparison data for investigations that are addressing diseased sites. PURPOSE The purpose of this study was to describe levels of selected pro-inflammatory cytokines in clinically healthy peri-implant and periodontal sites, and to examine whether cytokine levels may be related to specific bacterial/viral pathogens. MATERIALS AND METHODS Eleven subjects (mean age 56.2 +/- 10) participated in the study. Subgingival microbial samples were cultured for periodontopathic bacteria. Gingival crevicular fluid samples were analyzed by nested polymerase chain reaction for Cytomegalovirus (HCMV) and were tested for the quantification of Interleukin (IL)-8, IL-1beta, IL-6, IL-10, Tumor Necrosis Factor (TNF)-alpha, and IL-12p70 using flow cytometry (FACS). Findings for microbiota composition and cytokine levels were compared between implants and teeth (chi square, Kruskall-Wallis, Mann-Whitney; p < or = .05). RESULTS Both the frequency (%) and levels (%) of periodontopathic bacteria were higher around teeth than implants. The concentration (picogram per milliliter) of cytokines was more prominent around implants than teeth, reaching nearly twofold differences in some instances. Cytokine levels were higher when the sites analyzed were positive for any bacteria tested. HCMV was not detected. CONCLUSIONS Pro-inflammatory cytokine production was unrelated to heavy bacterial challenge. Nevertheless, when periodontopathic bacteria were detected by culture, cytokine levels were increased around both implants and teeth. Studies are needed to investigate the pro-inflammatory cytokines (especially IL-1beta and TNF-alpha) produced in spite of minimal bacterial accumulation.

Assessing mortality in women with hepatitis C virus and HIV using indirect markers of fibrosis.

Objective:Co-infection with hepatitis C virus (HCV) is a major cause of morbidity and mortality in HIV-infected individuals. However, predictors of mortality are poorly defined and most studies have focused predominantly on co-infection in men. We evaluated whether two indirect markers of hepatic fibrosis, aspartate aminotransferase-to-platelet ratio index (APRI) and FIB-4 scores, were predictive of mortality in a well defined longitudinal cohort of HCV/HIV-co-infected women on HAART. Methods:HCV/HIV-co-infected women on antiretroviral therapy enrolled in Womens Interagency HIV Study (WIHS), a National Institutes of Health-funded prospective, multicenter, cohort study of women with and at risk for HIV infection were included. Using Cox regression analysis, associations between APRI and FIB-4 with all-cause mortality were assessed. Results:Four hundred and fifty HCV/HIV-co-infected women, of whom 191 women died, had a median follow-up of 6.6 years and 5739 WIHS visits. Compared with women with low APRI or FIB-4 levels, severe fibrosis was significantly associated with an increased risk of all-cause mortality {APRI: hazard ratio 2.78 [95% confidence interval (CI) 1.87, 4.12]; FIB-4: hazard ratio 2.58 (95% CI 1.68, 3.95)}. Crude death rates per 1000 patient-years increased with increasing liver fibrosis: 34.8 for mild, 51.3 for moderate and 167.9 for severe fibrosis as measured by FIB-4. Importantly, both APRI and FIB-4 increased during the 5 years prior to death for all women: the slope of increase was greater for women dying a liver-related death compared with nonliver-related death. Conclusion:Both APRI and FIB-4 are independently associated with all-cause mortality in HCV/HIV-co-infected women and may have clinical prognostic utility among women with HIV and HCV.

Factors associated with hepatitis C viremia in a large cohort of HIV-infected and - uninfected women

BACKGROUND Co-infection with hepatitis C virus (HCV) is common among HIV-infected women. OBJECTIVE To further our understanding of the risk factors for HCV viremia and the predictors of HCV viral load among women. STUDY DESIGN We investigated sociodemographic, immunologic, and virologic factors associated with presence and level of HCV viremia among 1049 HCV-seropositive women, 882 of whom were HIV-infected and 167 HIV-uninfected at their entry into the Womens Interagency HIV Study. RESULTS Plasma HCV RNA was detected in 852 (81%) of these 1049 women (range: 1.2-7.8 log(10)copies/ml). HCV-viremic women were more likely to have an HIV RNA level >100,000 copies/ml (P=0.0004), to have reported smoking (P=0.01), or to be Black (P=0.005). They were less likely to have current or resolved hepatitis B infection. HCV RNA levels were higher in women who were >35 years old, or HIV-infected. Current smoking and history of drug use (crack/freebase cocaine, marijuana, amphetamines, or heroin) were each associated with both presence and level of viremia. CONCLUSIONS Substance abuse counseling aimed at eliminating ongoing use of illicit drugs and tobacco may reduce clinical progression, improve response to treatment, and decrease HCV transmission by lowering levels of HCV viremia in women.

Defective response to Toll-like receptor 3 and 4 ligands by activated monocytes in chronic hepatitis C virus infection

Summary.  Toll‐like receptors (TLR) have a critical role in innate immunity against pathogens. We investigated the cytokine response to TLR stimulation in peripheral blood cells of subjects infected with hepatitis C virus (HCV) and / or human immunodeficiency virus (HIV) in the Women Interagency HIV Study (WIHS) cohort. Interleukin (IL)‐6 in response to TLR3 and TLR4 ligands such as polyinosinic‐polycytidylic acid and lipopolysaccharide was significantly compromised in HCV‐infected women. High spontaneous secretion of IL‐6 suggested pre‐existing cell activation as a factor mediating reduced responses to TLR3 and TLR4 stimulation. To a lesser extent, tumour necrosis factor‐α and IL‐1β responses to TLR stimulation were also compromised. Monocytes, but not B cells or NK cells, were identified as the cell population spontaneously secreting cytokines and also as the cells responding to TLR stimulation. These results highlight a functional defect in antigen‐presenting cells of women with HCV infection or co‐infection. In women with existing HIV co‐infection, decreased cytokine function of antigen‐presenting cells suggests another mechanism contributing to immune dysfunction in addition to the HIV‐associated CD4 defect.

Asymptomatic HLA-A*02:01–Restricted Epitopes from Herpes Simplex Virus Glycoprotein B Preferentially Recall Polyfunctional CD8+ T Cells from Seropositive Asymptomatic Individuals and Protect HLA Transgenic Mice against Ocular Herpes

Evidence from C57BL/6 mice suggests that CD8+ T cells, specific to the immunodominant HSV-1 glycoprotein B (gB) H-2b–restricted epitope (gB498–505), protect against ocular herpes infection and disease. However, the possible role of CD8+ T cells, specific to HLA-restricted gB epitopes, in protective immunity seen in HSV-1–seropositive asymptomatic (ASYMP) healthy individuals (who have never had clinical herpes) remains to be determined. In this study, we used multiple prediction algorithms to identify 10 potential HLA-A*02:01–restricted CD8+ T cell epitopes from the HSV-1 gB amino acid sequence. Six of these epitopes exhibited high-affinity binding to HLA-A*02:01 molecules. In 10 sequentially studied HLA-A*02:01–positive, HSV-1–seropositive ASYMP individuals, the most frequent, robust, and polyfunctional CD8+ T cell responses, as assessed by a combination of tetramer, IFN-γ-ELISPOT, CFSE proliferation, CD107a/b cytotoxic degranulation, and multiplex cytokine assays, were directed mainly against epitopes gB342–350 and gB561–569. In contrast, in 10 HLA-A*02:01–positive, HSV-1–seropositive symptomatic (SYMP) individuals (with a history of numerous episodes of recurrent clinical herpes disease) frequent, but less robust, CD8+ T cell responses were directed mainly against nonoverlapping epitopes (gB183–191 and gB441–449). ASYMP individuals had a significantly higher proportion of HSV-gB–specific CD8+ T cells expressing CD107a/b degranulation marker and producing effector cytokines IL-2, IFN-γ, and TNF-α than did SYMP individuals. Moreover, immunization of a novel herpes-susceptible HLA-A*02:01 transgenic mouse model with ASYMP epitopes, but not with SYMP epitopes, induced strong CD8+ T cell–dependent protective immunity against ocular herpes infection and disease. These findings should guide the development of a safe and effective T cell–based herpes vaccine.

Making dendritic cells from the inside out: Lentiviral vector-mediated gene delivery of granulocyte-macrophage colony-stimulating factor and interleukin 4 into CD14+ monocytes generates dendritic cells in vitro

We have evaluated a one-hit lentiviral transduction approach to genetically modifying monocytes in order to promote autocrine and paracrine production of factors required for their differentiation into immature dendritic cells (DCs). High-titer third-generation self-inactivating lentiviral vectors expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) efficiently achieved simultaneous and persistent codelivery of the transgenes into purified human CD14+ monocytes. Coexpression of GM-CSF and IL-4 in CD14+ cells was sufficient to induce their differentiation into a DC-like phenotype, as evidenced by their morphology, immature immunophenotypic profile (CD14-, CD1a+, CD80+, CD86+, MHC-I+, MHC-II+), and their ability to further develop into a mature phenotype (CD83+) on further treatment with soluble CD40 ligand. Mixed lymphocyte reactions showed that the T cell-stimulating activity of lentivirus-modified DCs was superior to that of DCs grown by conventional methods. Lentivirus-modified DCs displayed efficient antigen-specific, MHC class I-restricted stimulation of autologous CD8+ T cells, as shown by IFN-gamma production and CTL assays. DCs coexpressing GM-CSF and IL-4 could be kept metabolically active and viable in culture for 14 days in the absence of exogenously added growth factors, unlike conventionally produced DCs. Coexpression of FLT3 ligand did not improve the viability, expansion, or immunologic performance of lentivirus-modified DCs. This article demonstrates the proof-of-concept to genetically convert monocytes to DC-type antigen-presenting cells with lentiviral vectors.

Vitamin D insufficiency may impair CD4 recovery among Women's Interagency HIV Study participants with advanced disease on HAART

Background:Recent studies in HIV-infected men report an association between low vitamin D (25OH-D) and CD4 recovery on HAART. We sought to test this relationship in the Womens Interagency HIV Study (WIHS). Methods:We examined 204 HIV-infected women with advanced disease, who started HAART after enrollment in the WIHS. We measured vitamin D (25OH-D) levels about 6 months prior to HAART initiation. The relationship between CD4 recovery (defined as increases of ≥50, 100, and 200 cells at 6, 12, and 24 months) and exposure variables was examined using logistic regression models at 6, 12 and 24 months post-HAART initiation in unadjusted and adjusted analyses, and using multivariable longitudinal Generalized Estimating Equations (GEE). Vitamin D insufficiency was defined as 25OH-D levels at least 30 ng/ml. Results:The majority were non-Hispanic black (60%) and had insufficient vitamin D levels (89%). In adjusted analyses, at 24 months after HAART, insufficient vitamin D level (OR 0.20, 95% CI 0.05–0.83) was associated with decreased odds of CD4 recovery. The undetectable viral load (OR 11.38, 95% CI 4.31–30.05) was associated with CD4 recovery. The multivariable GEE model found that average immune reconstitution attenuated significantly (P < 0.01) over time among those with insufficient vitamin D levels compared with those with sufficient vitamin D levels. Conclusion:Vitamin D insufficiency is associated with diminished late CD4 recovery after HAART initiation among US women living with advanced HIV. The mechanism of this association on late CD4 recovery may be late vitamin D-associated production of naive CD4 cells during immune reconstitution.

The Profile of Inflammatory Cytokines in Gingival Crevicular Fluid around Healthy Osseointegrated Implants

OBJECTIVE Regardless of gingival health and subgingival microbiology, production of cytokines within peri-implant tissues may be different from that of teeth. The objective of this study was to describe the peri-implant levels of pro-inflammatory cytokines and subgingival microbiology in clinically healthy sites. MATERIALS AND METHODS Subgingival plaque and gingival crevicular fluid (GCF) were obtained from 28 clinically healthy implants and 26 teeth selected from 24 individuals. Microbial composition was determined by selective anaerobic culture techniques. Pro-inflammatory cytokines were quantified by flow cytometry analysis of GCF. The concentration of cytokines between implants and teeth were compared with the independent t-test. RESULTS The concentration of cytokines was higher in GCF from healthy implants than in teeth. The profile of cytokines was characteristic of an innate immune response. A more frequent detection of periodontopathic bacteria was observed in teeth than implants. Cultivable levels of periodontopathic bacteria were similar between implants and teeth. CONCLUSIONS Despite gingival tissue health and scarce plaque accumulation, the profile of inflammatory cytokines in implant crevicular fluid was distinctive of an innate immune response and in higher concentration than in teeth. Other than bacterial stimulus, intrinsic factors related to implants may account for more cytokine production than teeth.