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

OBJECTIVE: To examine the safety and the immunologic and virologic consequences of corticosteroid use in HIV-1 infection. METHODS: A 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 x 10(6) 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. RESULTS: No 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 alpha levels and CD38+ CD8+ cells decreased significantly in those taking prednisone. CONCLUSION: Short-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.

Preferential S phase entry and apoptosis of CD4(+) T lymphocytes of HIV-1-infected patients after in vitro cultivation.

We have studied the relationship between spontaneous apoptosis and cell cycle perturbations in circulating peripheral blood lymphocytes of HIV-1-infected patients and healthy controls. PBMC obtained from HIV-1-infected patients and healthy controls were incubated in culture medium for 48 h. Cells were separated into CD4(+) and CD8(+) populations using immunomagnetic beads. Apoptosis and cell cycle phases were measured by propidium iodide staining and bromodeoxyuridine (BrdU) incorporation followed by flow cytometric analyses. In experiments using cells obtained from HIV-1-infected patients, spontaneous apoptosis was more frequent in CD4(+) T lymphocytes than in CD8(+) T lymphocytes (17.6% vs 9.5%, P < 0.005). Among healthy controls, spontaneous apoptosis in CD4(+) and CD8(+) T lymphocytes was comparable (4.5% vs 5.1%). Lymphocytes obtained from patients were more frequently in S phase than healthy controls' cells (2.2 +/- 0.9% vs 0.5 +/- 0.2%, P < 0.002) and patients' CD4(+) cells tended to enter S phase more frequently than controls' CD4(+) cells (4.2% +/- 3.5% vs 1.8% +/- 0.5% P < 0.04), whereas the frequency of S phase CD8(+) T cells was not different among patients (2.8% +/- 2.9%) and controls (1.8% +/- 0.5%) (P > 0.4). Kinetic analyses using BrdU and PI staining revealed that S phase cells were more likely to become apoptotic than resting (G(0)-G(1)) cells (28.4% +/- 10.3% vs 11.3% +/- 9.9% in patients, P < 0.04, and 15.3% +/- 2.8% vs 1.8% +/- 0.5% in controls, P < 0.003). Lymphocytes obtained from HIV-1-infected persons are activated in vivo to enter S phase and to undergo spontaneous apoptosis after brief in vitro cultivation. The present studies indicate that most apoptotic cells in this system are CD4(+) and kinetic analyses reveal that S phase cells are more likely to undergo spontaneous apoptosis than G(0)-G(1) cells. Accelerated cell death in HIV-1 disease may contribute to the failure of lymphocyte responsiveness to appropriate T cell receptor stimulation.

Lymphocyte proliferation assays may underestimate antigen responsiveness in human immunodeficiency virus infection.

The objective of this study was to examine the relationships among lymphocyte proliferation, interferon-gamma (IFN-gamma) production, and apoptosis in peripheral blood mononuclear cells (PBMC) of HIV-1-infected patients and controls. PBMC were prepared from 19 HIV-1-infected patients and 16 healthy controls. Using tetanus toxoid (TT) as a recall antigen, we assessed lymphocyte proliferation using [(3)H]thymidine incorporation after 2, 4, 6, and 7 days' culture and IFN-gamma production in 48-h culture supernatants by ELISA. Apoptosis was measured using TdT-mediated dUTP nick-end labeling. Median stimulation indices (SIs) in HIV-1-infected patients were 2.8 and 3.7 as opposed to 24.9 and 25.1 in healthy controls after 6 and 7 days' culture, respectively (P < 0. 001). Among the controls, peak proliferation was seen after 7 days in culture whereas in patients, SIs peaked at 4 days and fell progressively by days 6 and 7. At 2 and 4 days of stimulation with tetanus, patients' T cells showed increased apoptosis (19 and 25%) vs 12 and 15% apoptosis seen in controls' cells, P < 0.05. Interferon-gamma in 48-h supernatants of TT-stimulated PBMC was comparable among patients and controls. Whereas in our system, 6 and 7 day assays of lymphocyte proliferation provide increasing responses to TT among healthy controls, these durations of culture may underestimate antigen responsiveness in HIV-1 infection. Cell death due to apoptosis may account for this phenomenon. Whether shorter term or longer term assays of lymphocyte responsiveness more accurately reflect in vivo immune competence is unknown. Nonetheless, shorter duration assays may provide a more realistic estimate of the frequency of antigen-reactive cells in persons with HIV-1 infection.

HIV infection perturbs DNA content of lymphoid cells: partial correction after 'suppression' of virus replication.

OBJECTIVE: To examine the DNA content of circulating lymphocytes obtained from HIV-1-infected persons and to explore the effects of antiretroviral therapy on these indices. DESIGN: Cross-sectional analysis and 48-week open label treatment trial (AIDS Clinical Trials Group Protocol 315) of zidovudine, lamivudine and ritonavir. METHODS: Peripheral blood lymphocytes were obtained from HIV-1-infected patients and healthy controls and after 48 h of in vitro cultivation were stained with propidium iodide and analyzed for DNA content by flow cytometry. RESULTS: HIV-1-infected patients had more hypodiploid cells (19%), fewer G0-G1 phase cells (70%) and more S phase cells (10%) than did healthy controls (8%, 85% and 5% respectively; P = 0.002). Patients with sustained suppression of plasma HIV-1 RNA levels after antiretroviral therapy had only modest improvements in these indices. In contrast, patients who failed to suppress plasma HIV-1 RNA levels had decreases in G0-G1 cells to 54% (P = 0.032) and increases in S phase cells to 24% (P = 0.055). Plasma HIV-1 RNA levels and the percentage of S phase cells were correlated (r, 0.23; P = 0.047). In patients failing antiretroviral therapy, there was an inverse correlation between the percentage of G0-G1 cells and expression of the activation antigens CD38 and HLA-DR on CD4 cells (r, -0.409; P = 0.016) and CD8 cells (r, -0.363; P = 0.035). CONCLUSIONS: Lymphocytes obtained from HIV-1-infected patients display perturbations in DNA content after brief cultivation in vitro reflective of immune activation in vivo. The marginal improvement in these indices after 'successful' suppression of HIV-1 replication suggests that even low levels of HIV-1 replication are sufficient to induce immune activation and perturbations in DNA content.

CD4+-T-cell counts, spontaneous apoptosis, and Fas expression in peripheral blood mononuclear cells obtained from human immunodeficiency virus type 1-infected subjects.

We examined the relationships among CD4+-T-cell counts, spontaneous apoptosis, and Fas expression among peripheral blood mononuclear cells obtained from human immunodeficiency virus type 1 (HIV-1)-infected patients. After 2 days of incubation, propidium iodide DNA staining and flow cytometry revealed that peripheral blood mononuclear cells from subjects with the lowest CD4+-cell numbers (0 to 99/microl; n = 20) showed the highest frequency of apoptosis: 22.4% +/- 2.7% (mean +/- standard error) versus 13.8% +/- 1.2% and 12.7% +/- 1.4% among peripheral blood mononuclear cells obtained from patients with 100 to 499 CD4+ cells/microl (n = 19) and >500 CD4+ cells/microl (n = 17), respectively. Each of these means differed significantly from the mean frequency of apoptosis (6.3% +/- 0.7%) of peripheral blood mononuclear cells obtained from HIV-1-seronegative controls (P < 0.001, Student's t test). After incubation, the percentage of peripheral blood mononuclear cells expressing Fas antigen was increased for the HIV-1-infected subjects, and this was most evident for patients with more advanced disease. Among patients with fewer than 100 CD4+ cells/microl, 64.4% +/- 5.4% of peripheral blood mononuclear cells were Fas+, as opposed to 25.8% +/- 3.0% and 14.5% +/- 1.7% Fas+ cells among patients with more than 100 CD4+ cells/microl and healthy controls, respectively (P < 0.05 for each group comparison). Interestingly, in all populations, most apoptotic cells did not express Fas. Thus, apoptosis and Fas expression are increased in incubated peripheral blood mononuclear cells obtained from HIV-1-infected patients and these phenomena are enhanced as disease progresses.

Activation of antigen-induced lymphocyte proliferation by interleukin-15 without the mitogenic effect of interleukin-2 that may induce human immunodeficiency virus-1 expression.

The newly identified cytokine, IL-15 enhanced antigen-induced proliferation of PBMC obtained from HIV-1-seropositive subjects. When compared to IL-2 which enhanced both spontaneous and antigen-induced lymphocyte proliferative responses, IL-15 rarely increased spontaneous lymphocyte proliferation. Additionally, in cultures of lymphocytes obtained from 15 HIV-1-infected patients with < 300 circulating CD4- lymphocytes/microliter IL-15 induced significant HIV-1 expression (46, 21, and 71 pg/ml) in only 3 of 15 experiments and IL-2 induced significant HIV-1 expression (range 16- > 5000 pg/ml) in 11 of 15 experiments (P < 0.01, Fischer's exact test). Simultaneous assays of cytokine-induced spontaneous lymphocyte proliferation and HIV-1 expression revealed similar dose-response relationships for induction of HIV-1 and lymphocyte proliferation by IL-2. Thus, IL-15 helps to correct the impaired proliferative response of CD4+ lymphocytes from HIV-1-infected persons without the mitogenic effect of IL-2 that also may induce HIV-1 expression.

HIV-1 Tat protein and its inhibitor Ro 24-7429 inhibit lymphocyte proliferation and induce apoptosis in peripheral blood mononuclear cells from healthy donors.

To explore further the effects of the HIV-1 transactivator protein Tat on human lymphoid cell function we examined the effects of Tat on lymphocyte proliferation and programmed cell death (apoptosis). We found that the HIV-1 Tat protein induced apoptosis and inhibited lymphocyte proliferative responses in lymphocytes obtained from healthy HIV-1 seronegative donors. Surprisingly, the Tat inhibitor Ro 24-7429 also induced apoptosis and inhibited antigen-induced lymphocyte proliferation. In checkerboard experiments, each agent could antagonize the effects of the other in both assays. These data suggest that Tat and its inhibitor may interact with a host element critically important in the processes of lymphocyte proliferation and programmed cell death. The HIV-1 Tat protein may affect both lymphocyte survival and function in HIV-1 infection, thereby contributing to the immune deficiency of HIV-1 disease.

Detection of apoptotic cells by selective precipitation of [3H]thymidine-labelled DNA.

Apoptosis is characterized by systematic fragmentation of high-molecular-weight DNA into oligonucleosome fragments. A sensitive method for detection of apoptotic cells involving [3H]thymidine-labelled DNA is presented. Cells from mid-log-phase cultures were labelled with [3H]thymidine for 15 to 18 h and then exposed to gamma irradiation to induce apoptosis. A modified Hirt method was used to separate low-molecular-weight DNA from high-molecular-weight DNA. The percentage of fragmented DNA and high-molecular-weight DNA were measured by scintillation spectrometry. This method was compared with an established flow cytometric method for detection of apoptotic cells by using propidium iodide staining of DNA. We observed a good correlation between these two methods in detecting apoptosis. Hence, expensive flow cytometric assays for detection of apoptosis in dividing cells may be replaceable by a method involving [3H]thymidine labelling of DNA and separation of low-molecular-weight DNA from high-molecular-weight DNA by precipitation.

HIV type 1 Tat protein induces apoptosis and death in Jurkat cells.

Jurkat cells stably expressing high levels of the HIV-1 Tat protein were generated after transfection with an Epstein-Barr virus-based episomal replicon and selection in hygromycin B. The Jurkat Tat transfectants exhibited a longer doubling time when compared to Jurkat cells or Jurkat cells transfected with the control parent plasmid. Cell cycle analysis revealed comparable durations of each phase of the cell cycle in the Tat and control transfectants. Flow cytometric analysis using Hoechst 33342 and propidium iodide staining revealed that the Tat transfectants exhibited a higher percentage of apoptotic cells when compared to the control transfectants (29.1 +/- 3.1 vs. 11.43 +/- 3.1%). Incubation of Jurkat cells with recombinant HIV-1 Tat protein resulted in induction of apoptosis. The HIV-1 Tat protein induces apoptosis in a CD4-positive T cell line. Tat-induced programmed cell death may contribute to the lymphocyte depletion seen in persons infected with HIV-1.

Oral zinc therapy in recurrent erythema nodosum leprosum: a clinical study.

Effect of oral zinc as an immunomodulator was studied clinically in patients with recurrent ENL over a period of one year. In this study, 40 leprosy patients with chronic ENL, requiring more than 30-40 mg of prednisolone/day for the control of their reactions, were given oral zinc sulphate for a period of four months, and, marked improvement in the frequency, duration and severity of reactions was observed after zinc therapy. Also evident was marked reduction in the steroid requirement after oral zinc therapy. It appears that zinc may be a good substitute for the present day anti-reaction treatment which is not free from disadvantages. Further investigations to know the precise action of zinc on immune-system may help to understand the role of zinc therapy and its optimum duration.

Sjögren-Larsson syndrome: report of a case in India.

The case of a six-year-old Indian girl with Sjögren-Larsson syndrome is reported. She displayed the classic triad of oligophrenia, ichthyosis, and spastic paraparesis. There was a history of parental consanguinity. The features that were absent were hypertelorism, pigmentary degeneration of the retina, simian creases of the palms, and aminoaciduria.

Comparison of two multidrug regimens in multibacillary leprosy.

One of the technical problems relating to the multidrug therapy of leprosy is the slow decrease in the bacteriological index (BI) in multibacillary patients. In this study we have compared a regimen containing rifampicin given daily for 9 months with the standard WHO multidrug regimen for multibacillary leprosy. We have found, at the end of two years, a significantly greater fall of BI in patients who had received the regimen containing daily rifampicin as compared to those who had received pulsed doses of rifampicin. The doses of dapsone and clofazimine were similar in these two groups. It appears that daily administration of rifampicin may be useful in treating multibacillary patients in whom reduction in the BI is slower than expected. However, in view of its high cost and the very much increased incidence of type-2 lepra reactions and hepatitis, daily rifampicin therapy cannot be recommended for a control programme.

Hyperkeratotic and verrucous skin lesions on lower extremities of leprosy patients.

Three morphological varieties of hyperkeratotic and verrucous skin lesions on the anterior aspect of ankle joints in patients with leprosy are described: (i) verrucous lesions with thread-like horny projections similar to filiform warts; (ii) irregular compact hyperkeratotic lesions with deep fissures in between; and (iii) hyperkeratotic lesions with linear fissures corresponding to the transverse creases on the anterior aspect of the ankle. Chemical cautery was useful for the treatment of the first two varieties, and a potent topical corticosteroid with salicylic acid was useful for the third.