Robert Asaad

Poor CD4 T cell restoration after suppression of HIV-1 replication may reflect lower thymic function.

ObjectiveTo characterize immune phenotype and thymic function in HIV-1-infected adults with excellent virologic and poor immunologic responses to highly active antiretroviral therapy (HAART). MethodsCross-sectional study of patients with CD4 T cell rises of ⩾ 200 × 106 cells/l (CD4 responders; n = 10) or < 100 × 106 cells/l (poor responders; n = 12) in the first year of therapy. ResultsPoor responders were older than CD4 responders (46 versus 38 years;P < 0.01) and, before HAART, had higher CD4 cell counts (170 versus 35 × 106 cells/l;P = 0.11) and CD8 cell counts (780 versus 536 × 106 cells/l ; P = 0.02). After a median of 160 weeks of therapy, CD4 responders had more circulating naive phenotype (CD45+CD62L+) CD4 cells (227 versus 44 × 106 cells/l ; P = 0.001) and naive phenotype CD8 cells (487 versus 174 × 106 cells/l ; P = 0.004) than did poor responders (after 130 weeks). Computed tomographic scans showed minimal thymic tissue in 11/12 poor responders and abundant tissue in 7/10 responders (P = 0.006). Poor responders had fewer CD4 cells containing T cell receptor excision circles (TREC) compared with CD4 responders (2.12 versus 27.5 × 106 cells/l ; P = 0.004) and had shorter telomeres in CD4 cells (3.8 versus 5.3 kb ; P = 0.05). Metabolic labeling studies with deuterated glucose indicated that the lower frequency of TREC-containing lymphocytes in poor responders was not caused by accelerated proliferation kinetics. ConclusionPoor CD4 T cell increases observed in some patients with good virologic response to HAART may be caused by failure of thymic T cell production.

Immunologic Failure Despite Suppressive Antiretroviral Therapy Is Related to Activation and Turnover of Memory CD4 Cells

BACKGROUND Failure to normalize CD4(+) T-cell numbers despite effective antiretroviral therapy is an important problem in human immunodeficiency virus (HIV) infection. METHODS To evaluate potential determinants of immune failure in this setting, we performed a comprehensive immunophenotypic characterization of patients with immune failure despite HIV suppression, persons who experienced CD4(+) T-cell restoration with therapy, and healthy controls. RESULTS Profound depletion of all CD4(+) T-cell maturation subsets and depletion of naive CD8(+) T cells was found in immune failure, implying failure of T-cell production/expansion. In immune failure, both CD4(+) and CD8(+) cells were activated but only memory CD4(+) cells were cycling at increased frequency. This may be the consequence of inflammation induced by in vivo exposure to microbial products, as soluble levels of the endotoxin receptor CD14(+) and interleukin 6 were elevated in immune failure. In multivariate analyses, naive T-cell depletion, phenotypic activation (CD38(+) and HLA-DR expression), cycling of memory CD4(+) T cells, and levels of soluble CD14 (sCD14) distinguished immune failure from immune success, even when adjusted for CD4(+) T-cell nadir, age at treatment initiation, and other clinical indices. CONCLUSIONS Immune activation that appears related to exposure to microbial elements distinguishes immune failure from immune success in treated HIV infection.

Increased tissue factor expression on circulating monocytes in chronic HIV infection: relationship to in vivo coagulation and immune activation.

HIV infection is associated with an increased risk of thrombosis; and as antiretroviral therapy has increased the lifespan of HIV-infected patients, their risk for cardiovascular events is expected to increase. A large clinical study found recently that all-cause mortality for HIV(+) patients was related to plasma levels of interleukin-6 and to D-dimer products of fibrinolysis. We provide evidence that this elevated risk for coagulation may be related to increased proportions of monocytes expressing cell surface tissue factor (TF, thromboplastin) in persons with HIV infection. Monocyte TF expression could be induced in vitro by lipopolysaccharide and flagellin, but not by interleukin-6. Monocyte expression of TF was correlated with HIV levels in plasma, with indices of immune activation, and with plasma levels of soluble CD14, a marker of in vivo lipopolysaccharide exposure. TF levels also correlated with plasma levels of D-dimers, reflective of in vivo clot formation and fibrinolysis. Thus, drivers of immune activation in HIV disease, such as HIV replication, and potentially, microbial translocation, may activate clotting cascades and contribute to thrombus formation and cardiovascular morbidities in HIV infection.

Nadir CD4+ T-cell count and numbers of CD28+ CD4+ T-cells predict functional responses to immunizations in chronic HIV-1 infection.

Objective: To ascertain whether delaying the initiation of highly active antiretroviral therapy (HAART) compromises functional immune reconstitution in HIV-1 infection in persons who regain ‘normal’ CD4 T-cell counts after suppressive antiretroviral therapies. Design: Prospective open-label study carried out at two University-affiliated HIV-outpatient clinics in the USA. Subjects and methods: Response to immunization was used as a model for in vivo functional immune competence in 29 HIV-1 infected patients with CD4 T-cell counts > 450 × 106cells/l and HIV-RNA < 400 copies/ml for > 12 months after HAART and nine HIV-1 seronegative controls. After immunization with tetanus toxoid, diphtheria-toxoid, and keyhole limpet hemocyanin, immune response scores (IRS) were calculated using postimmunization antibody concentrations, lymphocyte proliferation, and delayed-type hypersensitivity responses to vaccine antigens. Results: Despite normal numbers of circulating CD4 T-cells, the CD4 T-cell nadir before HAART initiation predicted the immune response to immunization (ρ = 0.5; P < 0.005) while current CD4 T-cell count did not. Likewise, CD4 T-lymphocyte expression of the co-stimulatory molecule CD28 was also an independent predictor of response to immunization (ρ = 0.5; P < 0.005). Conclusions: Even among persons who controlled HIV replication and normalized CD4 T-cell counts with HAART, pretreatment CD4 T-cell count and numbers of circulating CD4+CD28+ T-cells at immunization, but not current CD4 T-cell count, predict the ability to respond to vaccination. Delaying the initiation of HAART in chronic HIV-1 infection results in impaired functional immune restoration despite normalization of circulating CD4 T-cell numbers.

Selective Impairments in Dendritic Cell-Associated Function Distinguish Hepatitis C Virus and HIV Infection

Impaired APC functions may play important roles in chronicity of hepatitis C virus (HCV) and HIV infections. To investigate the separate and combined effects of HCV and HIV infection on immature dendritic cells (DCs), we evaluated myeloid-derived DC (MDC) and plasmacytoid-derived DC (PDC) frequencies and functions, measured by Toll-like receptor ligand-induced IFN-α and IL-12, in healthy controls and subjects with chronic HCV, HIV, and HCV-HIV infection. To evaluate the relation between innate and adaptive immunity, we measured HCV-specific IFN-γ-producing T cell frequency. MDC frequencies tended to be reduced in HIV infection (1.8-fold), while PDC frequencies were minimally reduced in HCV infection (1.4-fold). In contrast, a striking reduction in non-PDC-associated IFN-α production was observed in HIV-infected subjects (17-fold), while PDC-associated IFN-α production was markedly reduced in HCV-infected subjects (20-fold). Both non-PDC and PDC functions were impaired in HCV-HIV coinfection. MDC-associated IL-12 production was markedly reduced in both HCV and HIV-infected subjects (over 10-fold). Functional defects were attenuated with slowly progressive HIV infection. The proportion of subjects with HCV-specific T cell responses, and the number of Ags recognized were reduced in HCV-HIV subjects as compared with HCV singly infected subjects. A positive association was observed between MDC-associated IL-12 production and HCV-specific T cell frequency in HCV-infected subjects. These results indicate that immature DC function is dysregulated in HIV and HCV infections, but differentially, and that these defects are attenuated in slowly progressive HIV infection. These selectively different impairments may contribute to the reduced adaptive immune response to HCV in HCV-HIV coinfection.

Changing Spectrum of Mortality Due to Human Immunodeficiency Virus: Analysis of 260 Deaths during 1995–1999

We analyzed the deaths in an outpatient human immunodeficiency virus (HIV) care clinic at University Hospitals in Cleveland from January 1995 through December 1999. The number of annual deaths decreased progressively, from 112 in 1995 to 32 in 1999. The median final CD4(+) cell count before death increased progressively from 10 cells/microL in 1995 to 90 cells/microL in 1999 (P<.01); 20%--25% of patients who died from 1997 through 1999 had plasma HIV RNA levels below detection limits. From 1995 through 1998, deaths due to infection, to end-stage acquired immune deficiency syndrome, and to malignancies decreased, whereas the proportion of deaths due to end-organ failures and of uncertain relationship to HIV infection increased. The spectrum of mortality in HIV disease has changed recently; although opportunistic infections cause death less frequently, deaths are occurring in people who have control of HIV replication and with some preservation of immune function. These observations underscore the need to monitor the etiologies of HIV-associated mortality and to better our understanding of the relationships among immune defenses, treatment-related toxicities, and end-organ failure in patients with HIV disease.

Direct Visualization of Cytokine-Producing Recall Antigen-Specific CD4 Memory T Cells in Healthy Individuals and HIV Patients

We have used computer-assisted cytokine ELISA spot analysis to measure the frequencies, the type of cytokine, and the amount of cytokine produced by individual recall Ag-specific CD4 memory cells in freshly isolated blood. We studied the memory cells specific for tetanus toxoid and purified protein derivative in 18 healthy individuals and in 22 HIV-infected patients on highly active antiretroviral therapy (HAART). In healthy individuals, the frequency, cytokine signature, and cytokine production per cell of these memory cells were stable over time. Although it is presently unclear whether the maintenance of the memory T cell pool depends upon Ag persistence, cross-reactive Ag stimulation, or cytokine-driven bystander stimulations and expansions, our data strongly argue for a stable memory cell pool in healthy individuals. In HIV patients, however, the frequency of these memory cells was a function of the viral load. The decreased numbers of functional memory cells in patients with high viral loads might provide one mechanism behind the immunodeficient state. Although the cytokine output per cell was unaffected in most patients (20 of 24), in some patients (4 of 24) it was >100-fold reduced, which might provide an additional mechanism to account for the reduced immunocompetence of these patients. The ability to visualize directly and quantify the cytokine produced by the low frequency memory cells in freshly isolated blood that have been physiologically stimulated by Ag should aid comprehensive studies of the Ag-specific memory cell pool in vivo, in health and disease.

Granzyme B ELISPOT assay for ex vivo measurements of T cell immunity

A major goal in immunodiagnostics has been the development of assay systems that can measure CD8(+) T cell immunity in humans, directly ex vivo, at high resolution, and with high throughput. We established granzyme B (grB) enzyme-linked immunospot assay (ELISPOT) in conjunction with image analysis to this end. Using grB transfected and untransfected Chinese hamster ovary (CHO) cells and T cell lines, we show that the antibody pair utilized was grB-specific and that only activated T cells secrete grB. GrB release began within 4 h after antigen stimulation and stopped within 40 h. Side-by-side comparison showed grB ELISPOT assays to have a higher resolution than classic chromium-release assays in terms of signal-to-noise ratio. The linearity of the relation of the number of CD8(+) effector T cells plated to grB spots detected suggests that grB ELISPOT assays measure the frequencies of grB-secreting cells directly. Reactivity to HIV peptides was seen in grB ELISPOT assays of freshly isolated PBMC from HIV patients, consistent with the detection of peptide-specific memory cells. The higher resolution and lower labor and material investment should make grB ELISPOT assays an attractive alternative to chromium-release assays in monitoring the clonal sizes of specific CD8 memory cells in vivo.

Interferon-α is the primary plasma type-I IFN in HIV-1 infection and correlates with immune activation and disease markers.

Type-I interferon (IFN-I) has been increasingly implicated in HIV-1 pathogenesis. Various studies have shown elevated IFN-I and an IFN-I-induced gene and protein expression signature in HIV-1 infection, yet the elevated IFN-I species has not been conclusively identified, its source remains obscure and its role in driving HIV-1 pathogenesis is controversial. We assessed IFN-I species in plasma by ELISAs and bioassay, and we investigated potential sources of IFN-I in blood and lymph node tissue by qRT-PCR. Furthermore, we measured the effect of therapeutic administration of IFNα in HCV-infected subjects to model the effect of IFNα on chronic immune activation. IFN-I bioactivity was significantly increased in plasma of untreated HIV-1-infected subjects relative to uninfected subjects (p = 0.012), and IFNα was the predominant IFN-I subtype correlating with IFN-I bioactivity (r = 0.658, p<0.001). IFNα was not detectable in plasma of subjects receiving anti-retroviral therapy. Elevated expression of IFNα mRNA was limited to lymph node tissue cells, suggesting that peripheral blood leukocytes are not a major source of IFNα in untreated chronic HIV-1 infection. Plasma IFN-I levels correlated inversely with CD4 T cell count (p = 0.003) and positively with levels of plasma HIV-1 RNA and CD38 expression on CD8 T cells (p = 0.009). In hepatitis C virus-infected subjects, treatment with IFN-I and ribavirin increased expression of CD38 on CD8 T cells (p = 0.003). These studies identify IFNα derived from lymph nodes, rather than blood leukocytes, as a possible source of the IFN-I signature that contributes to immune activation in HIV-1 infection.

Placebo-controlled trial of prednisone in advanced HIV-1 infection.

ObjectiveTo examine the safety and the immunologic and virologic consequences of corticosteroid use in HIV-1 infection. MethodsA randomized, double-blinded, placebo-controlled trial of corticosteroid administration in 41 patients with advanced HIV-1 infection. Patients had a baseline median CD4 cell count of 131 × 106 cells/l at enrollment and 85% had a history of opportunistic infection. All but one of the patients had been taking stable antiretroviral regimen, including a protease inhibitor in 36, for a median duration of 158 days. Patients were randomized to 8 weeks of prednisone 0.5 mg/kg daily or placebo. ResultsNo AIDS-defining events occurred; two patients in each group developed oral candidiasis, and two patients on prednisone developed mild herpes simplex flares. None who developed oral candidiasis or herpes simplex was receiving prophylaxis and each responded promptly to therapy. In the prednisone group, two patients developed hyperglycemia and one diabetic increased insulin requirements. CD4 cell counts and plasma HIV-1 RNA levels did not change, but plasma tumor necrosis factor α levels and CD38+CD8+ cells decreased significantly in those taking prednisone. ConclusionShort-term prednisone administration is well tolerated and reasonably safe in advanced HIV-1 disease and decreases immune activation without effects on HIV-1 RNA levels or CD4 cell counts. These results suggest that, in stable HIV-1 disease, these immune activation markers are more likely consequences of but not inducers of HIV-1 replication.