Homeostasis of naive and memory T cell subpopulations in peripheral blood and lymphoid tissues in the context of human immunodeficiency virus infection.

To understand the nature of naive and memory T cell depletion in human immunodeficiency virus (HIV) immunopathogenesis, their homeostasis in peripheral blood (PB) and lymph node (LN) compartments of HIV-infected patients was examined. Although the percentage of naive CD4+ cells was higher in LN than in PB mononuclear cells (LNMC and PBMC, respectively), the memory cells were higher in PBMC than in LNMC. The ratio of naive:memory CD4+ cells from PB positively correlated with that in LNs and with the absolute CD4+ cell counts and recall antigen responses, and the ratio inversely correlated with the cellular virus load from the corresponding compartment. These findings indicate that although the pattern of naive and memory cells in the LN and PB compartments appear divergent, their relationship is nonrandom and is significant. The naive&rcolon;memory ratio in PB appears to reflect the lymphoid microenvironment and may potentially be useful as a surrogate marker for treatment efficacy and immune reconstitution.

Chemokine/CD4 receptor density ratios correlate with HIV replication in lymph node and peripheral blood of HIV-infected individuals.

OBJECTIVES: Lymphoid tissue is a major reservoir for virus replication in HIV-infected subjects. The relationship of CCR5 and CXCR4 coreceptor density and HIV replication in peripheral blood mononuclear cells (PBMC) and lymph node (LN) mononuclear cells (LNMC) of HIV-infected subjects was examined. METHODS: PBMC and cervical LNMC from 12 HIV-infected patients were examined for virological and immunological parameters including chemokine receptor density, HIV plasma and cellular viral load, coreceptor usage and CD38/HLA-DR expression. RESULTS: The number of CCR5 and CXCR4 molecules on CD4 lymphocytes in the LN were significantly higher than in PBMC. In contrast the number of CD4 molecules/CD4 T cell was higher in PBMC than in LNMC. The CXCR4/CD4 and CCR5/CD4 ratios in the LN were significantly higher than in the PBMC. This was associated with a cellular viral load in the LN that was approximately 110-fold higher than in PBMC. The absolute number of coreceptor molecules per cell did not correlate with the viral load. However, the CCR5/CD4 and CXCR4/CD4 ratios in the LN positively correlated with HIV cellular and plasma RNA. Characterization of the viral isolates suggested an association between clinical isolates using a distinct coreceptor and the upregulation of the corresponding chemokine receptor. CONCLUSIONS: The ratios of chemokine receptors to CD4 molecules in CD4 T cells from LN is higher than in PBMC and may account for the relative difference in cellular viral load in these compartments. Additionally, the coreceptor/CD4 ratios, particularly in the lymphoid tissue, were highly related to HIV replication.

Emerging treatments for viral retinitis.

Cytomegalovirus is the most frequent cause of viral retinitis. It rarely causes disease in immunocompetent individuals, but is an important cause of retinitis in immunocompromised patients. Therapy with ganciclovir or foscarnet may result in a decrease in the morbidity related to cytomegalovirus, but problems with drug toxicity and development of clinical and viral resistance mandate additional therapeutic approaches. Preliminary phase I/II clinical trials suggest that human neutralising monoclonal antibodies to cytomegalovirus plus ganciclovir or foscarnet may be more effective than either ganciclovir or foscarnet alone for treating cytomegalovirus retinitis and in prolonging the time to cytomegalovirus disease progression in patients with AIDS. An approach based on an antisense oligonucleotide complementary to cytomegalovirus messenger RNA also shows promise.

Human cytomegalovirus-induced immunosuppression. Relationship to tumor necrosis factor-dependent release of arachidonic acid and prostaglandin E2 in human monocytes.

Cytomegalovirus (CMV) has been associated with immunosuppression. Previously CMV was reported to interfere with signal transduction pathways in T cells. In this report the mechanisms underlying CMV-mediated immunosuppression were examined. Supernatants of CMV (Strains C-87, AD-169)-infected primary human monocyte (MO) cultures inhibited mitogenic T cell proliferative responses by > 95%. The inhibitory activity was observed 24 h through day 7 postinfection. The infection of MO was associated with a sustained elevation of intracellular levels of cAMP and the release of arachidonic acid (AA) and its metabolite PGE2 (activator of adenylate cyclase) in culture supernatants. The AA release was incidentally associated with TNF-alpha production. Monoclonal antibodies to TNF-alpha and pentoxyphylline (inhibitor of TNF synthesis) inhibited both AA and PGE2 release. The release of AA required protein synthesis and occurred under conditions consistent with the expression of CMV immediate early genes. Treatment of MO cultures at time of infection with 100 microM indomethacin or 1 microg of TNF-alpha mAb abolished the CMV-induced T cell inhibitory activity of the supernatants by 100%. These data suggest that TNF dependent release of AA and PGE2 contributes to CMV-induced immunosuppression.

HIV-induced TNF-alpha regulates arachidonic acid and PGE2 release from HIV-infected mononuclear phagocytes.

Arachidonic acid (AA) has been shown to interact with transmembrane signaling pathways involved in T-cell activation. The latter have been shown to be impaired in lymphocytes obtained from HIV-infected patients. In the present study, AA and its metabolite, PGE2, released from differentiating human mononuclear phagocytes in response to HIV infection, and their relationship to HIV replication and TNF-alpha production were examined. The macrophage (M phi) cultures were more permissive for HIV replication than monocyte (MO) cultures. AA release in response to HIV infection was observed in both MO and M phi with a peak at 24 hr postinfection (p.i.). This AA release was 3.8- and 6-fold that of uninfected MO and M phi cultures, respectively. Supernatants from MO and M phi cultures at the peak of AA production inhibited [3H]thymidine uptake of peripheral blood mononuclear cells in response to PHA by 45 and 54%. At 24 hr p.i., PGE2 production was increased in both MO and M phi cultures. This increase was associated with a 1.2- and 20-fold inhibition of IL-1 production, respectively. TNF release, however, increased through day 14 p.i. Treating mock-infected MO with recombinant TNF-alpha induced AA release. Monoclonal antibodies to TNF inhibited this release by 80%. TNF (0.01-0.4 microgram/ml) added exogenously to MO produced a biphasic pattern of AA release; while low concentrations were stimulatory, higher concentrations were inhibitory. Treating monocyte and macrophage cultures with mAb to TNF-alpha inhibited the HIV-induced release of AA and PGE2. These findings indicate that HIV-induced TNF-alpha regulates the release of AA and PGE2, which might provide insight into the mechanisms involved in the pathogenesis of HIV-related disorders.

Protein kinase C and intracellular free Ca++: relationship to human immunodeficiency virus (HIV)-induced cellular hyporesponsiveness.

Protein Kinase C (PKC) and Ca++ are both involved in the chain of events leading to T-cell activation. An impairment of the immune response is characteristic of T cells obtained from patients with HIV infection. In this report, the involvement of PKC and Ca++ in HIV-mediated cellular hyporesponsiveness was examined. Infection of peripheral blood mononuclear cells (PBMC)s from HIV-seronegative normal donors with HIV strain HTLV IIIB, or two fresh patient isolates produced a 1.4-, 10.7-, and 11.4-fold enhancement in PKC activity at 1 hr postinfection (PI) and a 1.8-, 2.3-, and 3.8-fold enhancement at 12 hr PI, respectively. A marked decrease of PKC content, as determined by Western Blot analysis, was observed in HIV-infected cells by Day 4 and 7 PI compared with mock-infected control cells. Furthermore, PKC synthesis was also inhibited in cells from immunosuppressed AIDS patients. PKC activity of PBMCs from HIV-infected patients did not change in response to 1 microM of phorbal myristate acetate (PMA). In contrast, the same dose enhanced the activity by 50%-100% in PBMCs from normal HIV-seronegative donors. A 40%, 50%, and 125% increase in intracellular free Ca++ in response to HIV infection was observed 12 hr PI in MT4, JURKAT, and PBMCs, respectively. However, the increase in intracellular free Ca++ in HIV-infected PBMCs obtained from normal donors in response to PHA was 56% and 17% compared with an increase of 100% and 120% in mock infected cells at 12 hr and 1 week PI, respectively. Comparing the Ca++ response to PHA in PBMCs from HIV-infected patients showed that patients with < 250 absolute T4 cells/mm3 had an impaired Ca++ response. These data suggest that there is a relationship between intracellular free Ca++ and PKC and HIV-induced T-cell hyporesponsiveness.

Cytomegalovirus DNA polymerase activity and an 80 kDa-associated polypeptide: a potential diagnostic tool for CMV disease.

CMV has been reported to be associated with a DNA polymerase activity (DPA). In this communication its purification, characterization and potential diagnostic value were examined. CMV DNA polymerase was prepared from cell free supernatants of CMV (AD 169) infected cultures. Separation and purification of the enzyme was accomplished by column chromatography of the purified, lysed virus. CMV DPA was measured on an oligo (dT)-poly (dA) template primer. SDS-PAGE and western blot analysis under reducing conditions using an anti-CMV early antibody showed an 80 kDa protein band that was associated with the peak of polymerase activity. However, CMV isolates and CMV from urines from CMV retinitis patients immunoblotted by the same Ab revealed 140 kDa and 80 kDa bands under non-reducing and reducing conditions respectively, the latter was also associated with a 58 kDa band. The diagnostic value of the CMV associated DAP was tested using CMV positive urines. The latter demonstrated high PAA-sensitive DPA activity, compared to normal, HSV positive urines and urines from HBSAg positive patients. Taken collectively, these findings indicate the potential usefulness of CMV-associated DNA polymerase activity in the diagnosis and follow-up of patients with CMV-related illnesses.

Involvement of cAMP and protein kinase C in cytomegalovirus enhancement of human immunodeficiency virus replication.

Cytomegalovirus (CMV) infection constitutes a serious threat to patients with acquired immune deficiency syndrome. Recently we reported that human immunodeficiency virus (HIV) infection of CD4+ cells was associated with sustained elevation of cellular levels of cAMP. Moreover, cyclic nucleotide modulators enhanced HIV replication by increasing intracellular levels of cAMP. In this study, the effect of CMV on HIV replication in CMV/HIV mixed infection and its relationship to cAMP were examined. MT-4 cells, CMV strain AD169, and HIV strain IIIB were used. Optimal enhancement (4.4-fold increase) of HIV replication was observed when MT-4 cells were infected with CMV at Day 0 followed by HIV on Day 4 after infection, as determined by reverse transcriptase activity on Day 11 after infection. cAMP (measured by radioimmunoassay) levels in cells infected with CMV alone, HIV alone, or CMV/HIV together were 2-, 3-, and 5-fold above untreated cells, respectively. CMV also enhanced the replication of UV-irradiated HIV 4-fold and this was associated with a 2-fold increase in cAMP as well. Moreover, UV-irradiated CMV enhanced HIV replication 8.8-fold. The same dose of viable and UV-irradiated CMV used in the above experiments increased protein kinase C activity in these cells 3.0- and 8.0-fold, respectively. These findings might suggest that cAMP and protein kinase C are involved in CMV enhancement of HIV replication. These findings may have relevance to the identification of novel target sites for development of antiviral therapeutics.

Human immunodeficiency virus replication: modulation by cellular levels of cAMP.

HIV infection is associated with qualitative and functional immune deficiencies. It has been shown that the in vitro infection of CD4+ cells with HIV was associated with sustained elevation of cAMP and cGMP. In the present report the role of cAMP on HIV replication in MT-4 cells was investigated. The MT-4 cells were infected with HIV (strain 3b), in the presence or absence of agents that increase intracellular levels of cAMP, through different mechanisms. At selected times postinfection, HIV replication was measured by reverse transcriptase activity or HIV P24Ag in culture supernatants. Forskolin (FK, an activator of adenylate cyclase 1-100 microM), Isobutyl-methylxanthine (IBMX, a phosphodiesterase inhibitor, which indirectly increases intracellular levels of cAMP, 30-100 microM) and dibutyryl (db) cAMP (0.1-10 microM) enhanced HIV replication, in a dose-dependent manner. FK, IBMX, and db cAMP enhanced HIV replication by 2- to 10-fold, 4- to 7-fold, and 2- to 6-fold, respectively. Intracellular levels of cAMP were measured by radioimmunoassay and were also enhanced. Since cAMP exerts its catalytic effects through activation of protein kinase (PK) A the effect of H-8 (a specific inhibitor of the cAMP dependent PK A) on HIV replication was simultaneously examined. The H8 at doses of 0.1 to 10 microns inhibited HIV replication by 25 to 99.9%. Moreover H9 inhibited HIV replication in peripheral blood mononuclear cells by more than 90%. The replication of HIV appears to be a cAMP-dependent event, and PK A could possibly be a target for the development of anti-HIV therapies.

Recombinant human interferon beta ser protects against zidovudine-induced genetic damage in AIDS patients.

This study was conducted to evaluate the in vivo genotoxicity of zidovudine (ZDV) in patients with Acquired Immune Deficiency Syndrome (AIDS). Patients with this disease who were non-smokers and on ZDV (1200 mg/day) as their only medication for 4 weeks to 7 months were studied. Patients with AIDS who had not received ZDV served as a negative control. Whole blood cultures were initiated by conventional methods with PHA 1:50 dilution. In addition, for each culture there was an untreated control and a recombinant interferon-beta (rIFN-beta)-treated culture. The IFN-treated cultures were exposed to 10, 100, 1000, or 10000 units of rIFN-beta for the entire incubation period. The cells were harvested at 72 h and stained with a fluorescence plus Giemsa method which permits the determination of the number of division cycles a cell has completed. One hundred metaphases from first division cells were scored from each culture for chromosome aberrations that were mainly from the chromatid-type, i.e. chromatid, chromosome, and isochromatid breaks. The frequency of breaks in the ZDV and no ZDV group was 8.29 +/- 2.65 and 0.5 +/- 0.29 per 100 cells respectively (P less than 0.05). Cultures from ZDV patients that were incubated with 100 and 1000 units of rIFN-beta, however, showed a frequency of 1.3 +/- 0.71 and 1.9 +/- 1.08 respectively, which was significantly lower than observed in the cultures not exposed to IFN (P less than 0.05). At the highest dose of rIFN-beta utilized no aberrations were detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Patterns of interferon-gamma production by peripheral blood mononuclear cells from patients with human immunodeficiency virus infection.

A number of immune parameters have been described as impaired in AIDS patients. The patterns of interferon-gamma (IFN-gamma) production by peripheral blood mononuclear cells (PBMC) from AIDS-related complex (ARC) and AIDS patients in response to specific and nonspecific mitogens and their relationship to proliferative responses, interaction with exogenous interleukin 2 (IL-2), and absolute CD4 cell counts were studied. The PBMC were exposed to phytohemagglutinin (PHA) or cytomegalovirus (CMV) antigen (Ag) and/or 10 units of IL-2. At selected times, culture supernatants were tested for IFN-gamma production by radioimmunoassay and at identical times proliferative responses were determined by [3H]thymidine uptake. IFN-gamma production in response to PHA or CMV Ag was inhibited significantly and appeared dependent on absolute CD4 cell counts. Proliferative responses were also similarly decreased. While IFN-gamma production to PHA was severely inhibited in PBMC only from patients with less than 100 CD4 cells/mm3, equivalent inhibition in response to CMV Ag was observed only in those with CD4 counts less than 600/mm3. IL-2 enhanced the PHA and CMV Ag-induced IFN production significantly by 2.8- and 5.3-fold respectively. Therefore, the administration of IL-2 might improve certain cell-mediated immune responses.

Differential reconstitution of zidovudine-induced inhibition of mitogenic responses by interleukin-2 in peripheral blood mononuclear cells from patients with human immunodeficiency virus infection.

Zidovudine (ZDV), an anti-human immunodeficiency virus (HIV) therapy, has been associated with reduction in mortality and improvement of patients with acquired immunodeficiency syndrome (AIDS). The ZDV recipients, however, experience a multitude of side effects of which bone marrow suppression is the most noteworthy, especially among patients with low CD4 cell counts. The effect of ZDV and interleukin-2 (IL-2) on phytohemagglutinin (PHA)-induced proliferative response of peripheral blood mononuclear cells (PBMs) from patients with HIV infection was investigated. ZDV 0.5 micrograms inhibited 40% of PHA-induced thymidine uptake in PBMs from healthy donors or patients with HIV, irrespective of their CD4 cell counts. However, IL-2 (10 U/ml) had differential effect on PHA-induced thymidine uptake that appeared to be dependent on absolute CD4 cell counts. While PBMs from patients with CD4 cell counts of 400/mm3 or more did not respond to IL-2 (low responders), IL-2 enhanced the PHA-induced thymidine uptake in PBMs from patients with CD4 cell counts less than 400/mm3 at an average of 60% (high responders). Moreover, IL-2 restored the ZDV-induced inhibition by almost 100% in the high responder group while it did not affect counts in the low responder group. The production of IL-2 in vitro, in response to PHA or recall antigens, was equivalently inhibited in both groups. These data suggest that ZDV and IL-2 could have an additive effect on immune parameters in certain groups of patients infected with HIV. The differential effect of IL-2 was independent of IL-2 receptor expression.