Bård Røsok

Deregulation of the CD95/CD95L system in lymphocytes from patients with primary acute HIV infection

ObjectiveTo analyze the role of CD95/CD95 ligand (CD95L) expression and functionality in peripheral blood lymphocytes (PBL) during primary, acute HIV syndrome (AHS) and in the subsequent period. PatientsTwelve patients were studied during the acute phase of the viral infection and most were followed for some months. MethodsCell culture and cytotoxicity assays based upon 51Cr release and flow cytometry were used to evaluate cell killing via CD95 molecule, flow cytometry to assess surface antigens, enzyme-linked immunosorbent assay (ELISA) for the determination of soluble CD95 and CD95L plasma levels, quantitative competitive (QC) reverse transcription polymerase chain reaction (RT-PCR) with an original RNA competitor for the analysis of CD95L mRNA expression and QC RT-PCR for determining plasma viral load. ResultsThe analysis of PBL during this phase revealed that almost all cells, including CD8 T cells with a virgin phenotype, B lymphocytes and natural killer cells displayed CD95 molecules on the plasma membrane. Activation of CD95 on the surface of isolated lymphocytes by anti-CD95 monoclonal antibodies or binding to CD95L induced rapid apoptosis. However, CD95L mRNA was not expressed in PBL from these patients and was poorly inducible. Soluble CD95 was found in the plasma of all patients, but only in a few at high levels, even some months after seroconversion. In contrast, soluble CD95L was detected in only one patient, this occurring after the symptomatic period. For 10 of the 12 patients, expression of CD95 on the cell membrane or in the plasma did not correlate with the plasma viral load, which varied widely from patient to patient. Further, plasma levels of soluble CD95 were not altered by decreased lymphocyte activation or by efficient antiretroviral therapy. ConclusionsIn patients experiencing an acute, primary HIV infection, a prolonged deregulation of the CD95/CD95L system may exist, which is probably not entirely related to virus production but may contribute to the pathogenesis of the disease. The hypothesis can be put forward that a complex balance exists between proapoptotic events (increase in CD95 expression), probably triggered by the host as a method to limit viral production, and antiapoptotic events (decrease in CD95L expression) probably triggered by the virus as a way to increase its production and survival.

Normalization of CD4+ Cell Numbers and Reduced Levels of Memory CD8+ Cells in Blood and Tonsillar Tissue after Highly Active Antiretroviral Therapy in Early HIV Type-1 Infection

Antiretroviral therapy increases the number of both CD4+ and CD8+ T cells in the blood of HIV-1-positive patients with advanced disease. In the present study, we have examined the kinetics of CD4+ and CD8+ T cell restoration in blood and lymphoid tissue in asymptomatic HIV-1-positive individuals with high CD4+ cell counts during highly active antiretroviral treatment. Tonsillar biopsies and blood samples were collected at baseline and at regular intervals during the following 48 weeks and from HIV-1-negative controls. Mononuclear cells from blood and tonsils were phenotyped and quantified by three-color flow cytometry. After 48 weeks of therapy, blood CD4+ cell counts in the HIV-1-infected group were comparable to those found in uninfected controls. Naive CD4+ T cells in blood increased during the initial 2 weeks in parallel with reduced plasma viremia. Both naive and memory CD4+ T cells in blood reached normal numbers by week 48, whereas the CD4+ naive/memory cell ratio in tonsils was within normal range throughout the study. The level of memory CD8+ T cells in blood declined during the first 8 weeks in parallel with a reduction in the tonsillar memory CD8+ T cells. Naive CD8+ T cells in the blood increased after 4 weeks, while the level of naive CD8+ T cells in tonsils remained unaltered. Our data indicate that in the early stages of HIV-1 infection antiretroviral therapy normalizes CD4+ cell counts and causes a decrease in the level of memory CD8+ cells in blood and lymphoid tissue, suggesting reduced CD8+ cell turnover in response to reduced viral replication.

Activation and proliferation of CD8+ T cells in lymphoid tissues of HIV-1-infected individuals in the absence of the high-affinity IL-2 receptor.

Activation of CD8+ T cells may have important pathogenic implications in HIV-1 infection. Studies of this process have so far been confined to cells from the peripheral blood. In the present study, we have examined molecules involved in activation and proliferation of CD8+ T cells in lymphoid tissues from HIV-1-infected patients. Tonsillar tissue and blood samples from 13 HIV-1-infected patients and 6 seronegative controls were examined for cell surface expression and the presence of mRNA for CD69, CD25, and HLA-DR. Intonsillar tissue, the number of CD8+ T cells was increased several fold in HIV-1-infected patients compared with controls. The majority of these cells expressed CD69 and HLA-DR, but virtually no tonsillar CD8+ T cells were found to express CD25 on the cell surface or at the mRNA level. Following in vitro activation, however, almost all activated CD8+ T cells were found to express CD25. Tonsillar CD4+ T cell numbers were maintained or reduced compared with controls, and a considerable proportion expressed CD25. The data suggest that CD8+, but not CD4+ T cells proliferate extensively in lymphoid tissues in HIV-1-infected patients in the absence of the high-affinity interleukin-2 (IL-2) receptor.

Cystatin A and HIV-1 p24 antigen expression in tonsillar lymphoid follicles during HIV-1 infection and during highly active antiretroviral therapy.

Summary: Cystatin A is a natural cysteine proteinase inhibitor and is found in a wide variety of normal cells. The physiologic role of Cystatin A is not fully known, however. Cystatin A is present in large amounts in follicular dendritic cells, which are important in HIV-1 pathogenesis. We analyzed Cystatin A expression in tonsillar sections from 20 patients at various stages of HIV-1 infection. There was a significant (P < 0.001) difference in Cystatin A fractions between patients and controls, with medians (ranges) of 0.61 (0.46-0.83) and 0.86 (0.78-0.90), respectively. Inverse correlations (Spearman &rgr;) existed between Cystatin A and the rate of follicular fragmentation (&rgr; = −0.658) and HIV-1 p24 antigen expression (&rgr; = −0.622) in germinal centers and the amount of HIV-1 RNA in tonsillar tissue (&rgr; = −0.765). The Cystatin A fraction declined from early chronic HIV-1 infection and was significantly lower in patients with a CD4 count below as compared with above 300 cells/&mgr;L of blood (P < 0.001), suggesting a favorable initiation of highly active antiretroviral therapy (HAART) at this level. Regeneration of Cystatin A to normal levels was shown in 11 patients 12 and 48 weeks after initiation of HAART, whereas the rate of follicular fragmentation was still elevated. Thus, we found Cystatin A to be a sensitive marker during HIV-1 infection and for regeneration of follicular lymphoid tissue during HAART.

Release of bicarbonate from damaged and restituted gastric mucosa in the cat

BACKGROUNDnGastric mucosal damage leads to luminal alkalinization, but its dependence on mucosal blood flow and acid secretory state of the mucosa is not known. This study examined release of bicarbonate to the gastric lumen and mucosal blood flow in cats after mucosal damage caused by 2 mol/L NaCl and during 90 minutes of epithelial restitution.nnnMETHODSnBicarbonate was calculated from measurements of pH and PCO2 in the luminal perfusate. Mucosal blood flow was measured with microspheres.nnnRESULTSnLuminal bicarbonate increased more than twofold after damage in pharmacologically nontreated, pentagastrin-treated, and omeprazole-treated animals (P < 0.001). Luminal bicarbonate thereafter decreased completely to pre-damage level in pentagastrin-treated, partly in nontreated, but remained elevated in omeprazole-treated animals. Mucosal blood flow increased about 100% 15 minutes after damage (P < 0.001), irrespective of secretory state. Bicarbonate availability (arterial [HCO(3-)] x mucosal blood flow) was significantly related to luminal release of bicarbonate from the newly damaged (P < 0.01) but not from the restituted mucosa.nnnCONCLUSIONSn(1) From the newly damaged mucosa, the luminal release of bicarbonate is related to availability of blood-borne bicarbonate. (2) From acid-stimulated restituted mucosa, the bicarbonate produced by the parietal cells is not released to the lumen, but either consumed within the mucosa by back-diffusing H+ or distributed to the systemic circulation.

Changes in tonsillar tissue in early HIV-1 infection and during 3 years of antiretroviral therapy

Tonsillar tissue from individuals in the early stages of HIV‐1 infection was studied during the natural course of infection and during antiretroviral therapy with and without a protease inhibitor in order to investigate markers of clinical progression and evaluate the effects of therapy. Tonsillar biopsies and blood samples were collected at regular intervals during 3 years and clinical observations were noted. Tonsillar morphology was evaluated and the fragmentation of the follicular dendritic cell network was quantified by standardised follicular fragmentation rate (FR) analysis. Lymphocyte subsets were phenotyped by flow cytometry, and viral load was calculated by limiting dilution assay. The FRs were higher in the HIV‐1‐infected individuals than in the uninfected controls, although tonsillar tissue from both groups contained follicular fragmentation. During HIV‐1 infection, the FR increased and the tonsillar CD4/CD8 ratio declined. During maximum viral suppression, FR approached that of controls while tonsillar T cell subsets and blood CD4 cell counts normalised. Even when virus suppression was incomplete, tonsillar improvements were observed in parallel with a resolution of the HIV‐1‐related dermatological disorders. However, persistent viral replication paralleled distortion of the tonsillar architecture. We suggest that a normalisation of the lymphoid tissue may have important functional and clinical implications in HIV‐1 infection.