Repeated immunization with recombinant gp160 human immunodeficiency virus (HIV) envelope protein in early HIV-1 infection: evaluation of the T cell proliferative response.

This longitudinal study was designed to evaluate cellular immunity in early-stage, asymptomatic human immunodeficiency virus (HIV)-1-infected persons (CD4 cell count,>400/mm3; median, 625/mm3) who were immunized with either recombinant (r) gp160 or placebo every 2 months for 5 years. Proliferative responses were assessed against rgp160, rp24, and a panel of recall antigens and mitogens. Despite good reactivity to recall antigens, at baseline approximately 33% had proliferative responses to gp160, and approximately 42% showed p24 gag responses. There was no statistical difference between vaccine and placebo groups for antigens or mitogens. After 1 year, approximately 73% of the subjects in the vaccine arm had new or boosted responses to gp160, versus approximately 18% in the placebo arm. Statistical significance was maintained throughout the study. Recurrent vaccination with recombinant gp160 was proven to be persistently immunogenic, increasing significantly the ability of HIV-1-infected persons to mount new proliferative responses to the vaccine.

Expansion of restricted cellular immune responses to HIV-1 envelope by vaccination: IL-7 and IL-12 differentially augment cellular proliferative responses to HIV-1.

The failure of immune effector mechanisms to control HIV-1 infection has important consequences for the human host. In a randomized cohort of HIV-infected patients, there was striking in vitro restriction of the proliferative response to HIV-1 envelope protein (Env), gp160; only 34% of patients recognized Env. Therapeutic vaccination with recombinant gp160 or gp120 (rgp160, rgp120) reversed the restriction in vitro, with Env recognition rising to 81%. Peripheral blood mononuclear cells (PBMC) from HIV-infected vaccine recipients, placebo recipients, and seronegative volunteers were cultured with exogenous IL-7 or IL-12 and either tetanus toxoid (TT) or gp160. IL-7 significantly augmented proliferative responses to TT and gp160, whereas IL-12 only affected proliferation to gp160. IL-7, but not IL-12, increased the number of HIV-infected placebo recipients who recognized rgp160. IL-12 had its greatest effect in the induction of rgp160-specific responses from seronegative individuals. The data suggest that these two cytokines have differential activity in the relief of restricted cellular immunity to Env; the predominant effect of IL-7 is in individuals who have been primed by exposure to antigen, while the effect of IL-12 is most evident in seronegative, unprimed individuals. Modification of restricted proliferative responses to Env by vaccination or cytokines in vitro suggests that strategies incorporating IL-7 or IL-12 as adjuvants may selectively boost cellular reactivity to HIV-1.

Consequences of stable transduction and antigen-inducible expression of the human interleukin-7 gene on tetanus-toxoid-specific T cells.

Interleukin-7 (IL-7) has previously been shown to increase antigen-specific immune responses; the effect of IL-7 on human antigen-specific T cell lines has not directly been addressed. A tetanus-toxoid (TT)-specific T cell line exhibited increased proliferation in the presence of exogenous IL-7, suggesting that IL-7 may be useful in the potentiation of immune responses to defined microbial antigens. Murine retroviral vectors encoding the human IL-7 gene and the neomycin phosphotransferase gene (neoR) were packaged into murine retroviral particles, and supernatants containing these retroviral vectors were used to infect a CD4+ lymphoblastoid cell line. Stable integration of the retroviral vector and constitutive expression of the IL-7 gene were observed. Successful IL-7 gene transduction into TT-specific T cells was also accomplished. Detection of neoR DNA sequences and expression of IL-7-specific mRNA increased with selection in geneticin. Production of IL-7 in these cells was induced by exposure to TT. Production of IL-4, IL-6, and interferon-gamma (IFN-gamma) was detected after antigenic stimulation; there was, however, no effect of IL-7 on the pattern or kinetics of cytokine production by these cells. Human IL-7 transduced cells showed greater proliferation to TT than control T cells, particularly at subthreshold TT concentrations. These dta imply that genetic modification of antigen-specific T cells may be a plausible strategy for the study and manipulation of the immune responses to microbial pathogens.

Association of interleukin-6 in the pathogenesis of acutely fatal SIVsmm/PBj-14 in pigtailed macaques.

Infection with a variant of simian immunodeficiency virus (SIVsmm/PBj-14) causes death in juvenile pigtailed macaques within 8 days of infection. The primary pathology is localized to the lymphoid tissues of the gut and spleen. Although the virus is present, the lesions are most consistent with acute reactive inflammation. We studied the serum and tissues for evidence of acute cytokine production often associated with acute inflammation. One factor, IL-6, was found to be significantly increased (> 1000-fold) over all other measured cytokines in all the pigtailed macaques who died acutely. Increased levels of IL-6 were found both in the serum and in the inflamed tissues. mRNA for IL-6 was found in the tissues with the highest protein levels of IL-6. The marked increase in IL-6 and IL-6 mRNA correlated with the virus levels in the tissues and serum as determined by viral isolation, immunohistochemistry, and Northern blot analysis. These findings suggest that the underlying pathogenesis of primary tissue damage, necrosis, and death by PBj-14 is the induction of cytokine production. Although the presence of the virus may be critical for the initiation of these events, the intense inflammatory reaction is associated with the cause of death.

Induction of interleukin-6 during human immunodeficiency virus infection.

Interleukin-6 (IL-6), a multifunctional cytokine produced in monocytes, fibroblasts, and other cell types, is induced by a variety of stimuli, including bacteria, viruses, and other cytokines. When normal monocyte cultures were exposed to a monocytotropic strain of human immunodeficiency virus (HIV), HTLV-IIIBa-L, significant levels of IL-6 bioactivity were detected in the culture supernatants after 12 to 43 days of incubation, at a time when there was associated evidence of HIV production. Similarly, when normal monocyte cultures were cocultured with peripheral blood mononuclear cells from HIV-infected individuals, HIV replication in these cultures was associated with production of IL-6. In further studies, we determined that mean serum levels of IL-6 bioactivity were abnormally elevated in HIV-seropositive individuals with stage 1/2 infection (25.2 x/divided by 1.8 U/mL) and stage 3/4 infection (46.1 x/divided by 1.7 U/mL) when compared with normals (1.6 x/divided by 1.2 U/mL). In contrast mean serum IL-6 levels were not different from normal in stage 5/6 infection (2.7 x/divided by 1.6 U/mL). A selected group of 12 HIV-seropositive individuals (stages 1, 2, and 3) who harbored HIV capable of replicating in T cells but not in monocyte cultures had a mean serum IL-6 level of 5.3 U/mL (x/divided by 1.5), a value significantly lower (P less than .004) than that measured in control HIV-seropositive individuals infected with monocytropic HIV (39 x/divided by 1.9 U/mL). In addition, serum IL-6 levels in HIV-seropositive individuals (stages 1 through 6) correlated directly with serum immunoglobulin G (IgG) levels (R = .74, P less than .001). Monocytes but not T cells are capable of a high level IL-6 production in vitro, and monocyte-derived IL-6 stimulates Ig production in activated B cells. Thus, HIV-seropositive individuals who often are infected with monocytotropic HIV and often display abnormally elevated serum IgG levels may exhibit these abnormalities as a consequence of abnormally elevated IL-6 levels induced by HIV.

Organoid structure of normal rat bladder in unilaminar and bilaminar histophysiologic gradient culture : methods and observations.

In lining epithelium of mammals certain recurrent architectural patterns are recognized that may be critical for epithelial organization in culture. Among these structural imperatives are three dimensional growth, restricted migration of cells, histophysiologic gradients, and continuity of epithelial membranes. Histophysiologic gradient culture procedures have been developed to comply with requirements suggested by normal tissue architecture. In a small chamber, 5 mm diam, epithelium grows attached to a thin permeable transparent collagen membrane or sandwiched between two apposed collagen membranes. The chamber is held in a plastic capsule so that metabolic exchange is limited to substances that diffuse across the collagen membranes to the adherent basal layer of epithelium. On a single membrane after 2 wk of growth, normal urothelium appears as a diffusely hypercellular urothelium, 6 to 10 cells thick. As the culture period is extended by 2 or more wk, multiple nodules of urothelium extend from the basal surface into the subepithelial space between the epithelium and the collagen membrane. Normal bladder, cultured between two apposed collagen membranes, gives rise in a few days to confluent epithelium that contains many extracellular cysts. Through an apparent merging of cysts, after 2 wk the urothelium appears as a highly organoid structure, a flattened cyst lined by completely stratified polarized urothelium. Such microbladders consist of a stratified epithelium without interruption of continuity. With histophysiologic gradient culture, processes in carcinoma and precursor lesions are accessible to study at the level of tissue organization.

Separation on Percoll density gradients of cells derived from malignant ascites of mice.

In an attempt to separate malignant from normal and reactive stromal cells, we fractionated ascites cells from BALB/c mice bearing a transplantable myeloma (MPC-11) by isopyknic centrifugation in continuous density gradients of povidone-coated silica gels (Percoll). Cells from different fractions were then analyzed by morphologic and immunologic criteria. The ability of cells from the different fractions to form colonies in soft agar and to produce tumors in BALB/c mice was also examined. Although most fractions contained morphologically identifiable plasma cells, colony-forming cells (CFC), derived from multiply passaged tumors, separated in a sharp peak at 1.072 g/ml. CFC peaked at 1.078-1.082 g/ml for tumors passed less than three times and were invariably markedly depleted from the low-density portions of the gradients. Cells recovered from different fractions of the gradients were cultured in soft agar and inoculated sc into syngeneic mice. In these experiments, a highly significant correlation was observed between the ability of cells to form colonies in soft agar and to form tumors in vivo. This correlation suggests that CFC and tumorigenic cells have similar distributions.

Effect of enzymatic disaggregation on proliferation of human tumor cells in soft agar.

Cell suspensions were prepared by either mechanical or enzymatic disaggregation methods from biopsy specimens from 54 patients with various tumors. The biologic activities of cells derived from the two suspensions were then examined. Biopsy specimens of solid tumors were minced, and one-half of each specimen was further processed by being teased with needles, whereas the other half was exposed to a combination of collagenase, hyaluronidase, and DNase. The enzymatic disaggregation method yielded fewer cells per gram of tissue than the mechanical method. However, the percentage of dye-excluding cells was increased by the enzymatic procedure in 93% of the cases. Cells obtained by enzymatic means also had higher cloning efficiencies than those obtained by mincing. The histologic types of cells present in the initial cell suspensions were the same for cells obtained by the enzymatic or mechanical disaggregation methods. The number of colonies obtained was linearly related to the number of cells plated in both cases. The tritiated thymidine suicide indices (estimates of the percentage of cells in the S-phase of the cell cycle) were the same for the two cell populations obtained by the two methods. The results indicate that cells obtained from solid tumors by enzymatic dissociation methods did not differ significantly from cells obtained by the more conventional mechanical techniques. However, cell viabilities and cloning efficiencies were significantly improved by the enzymatic technique.