Prevention of SHIV transmission by topical IFN-ß treatment.

Understanding vaginal and rectal HIV transmission and protective cellular and molecular mechanisms is critical for designing new prevention strategies, including those required for an effective vaccine. The determinants of protection against HIV infection are, however, poorly understood. Increasing evidence suggest that innate immune defenses may help protect mucosal surfaces from HIV transmission in highly exposed, uninfected subjects. More recent studies suggest that systemically administered type 1 interferon protects against simian immunodeficiency virus infection of macaques. Here we hypothesized that topically applied type 1 interferons might stimulate vaginal innate responses that could protect against HIV transmission. We therefore applied a recombinant human type 1 interferon (IFN-ß) to the vagina of rhesus macaques and vaginally challenged them with pathogenic simian/human immunodeficiency virus (SHIV). Vaginal administration of IFN-ß resulted in marked local changes in immune cell phenotype, increasing immune activation and HIV co-receptor expression, yet provided significant protection from SHIV acquisition as interferon response genes were also upregulated. These data suggest that protection from vaginal HIV acquisition may be achieved by activating innate mucosal defenses.

Differential expression of interleukin-2 and gamma interferon in human immunodeficiency virus disease.

Subnormal T-cell production of interleukin-2 (IL-2) in human immunodeficiency virus (HIV) disease has been described; however, it is not clear whether failure to synthesize IL-2 represents a selective or global defect in T-cell cytokine production. We evaluated the intracellular production of gamma interferon (IFN-gamma) and IL-2 in CD4(+) cells that were stimulated with staphylococcal enterotoxin B or cytomegalovirus antigen. Strikingly, IFN-gamma and IL-2 are differentially regulated in T cells of HIV-infected patients such that the numbers of CD69(+) cells or IFN-gamma-positive cells that make IL-2 are proportionally decreased in CD4(+) T cells from HIV-infected patients. These findings demonstrate a selective defect in IL-2 production and suggest that enumeration of IFN-gamma-producing cells in response to T-cell receptor stimulation, while providing some estimate of antigen-reactive cell frequency, may not reflect or predict "normal" T-cell function in HIV-infected patients.

HIV-1 infection impairs cell cycle progression of CD4(+) T cells without affecting early activation responses.

Failure of CD4(+) T cells to proliferate in response to antigenic stimulation is a characteristic of HIV infection. Analysis of the proliferation defect has been hampered by an inability to identify CD4(+) cells with T cell receptor specificity for antigen. To focus only on cells that had been stimulated through the T cell receptor, CD4(+) T cells were stimulated with an anti-Vbeta3 Ab that activates approximately 3-5% of peripheral blood T cells. This approach revealed proliferation defects in cells from HIV-infected patients that were not appreciated using anti-CD3 Ab stimulation and provided the capacity to examine responses on a single cell basis. After anti-Vbeta3 Ab stimulation, CD4(+)Vbeta3(+) cells from HIV-infected patients demonstrated defects in expression of cell cycle-associated proteins, D-type cyclins, and cyclin A. However, the expression of early activation markers, CD69 and CD25, was not significantly impaired in cells from most patients. Thus, CD4(+) T cell proliferation failure in HIV disease is characterized by dysregulated activation that precludes cell cycle progression. This proliferation defect was most apparent in patients with diminished CD4(+) T cell numbers and higher plasma HIV RNA levels. CD4(+) T cell proliferation failure may be a key determinant of immune impairment in HIV disease.

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.

Differential activity of soluble versus cellular Fas ligand: regulation by an accessory molecule.

Fas ligand induces apoptosis by binding to its receptor Fas. This process has been shown to be important for activation-induced cell death of T lymphocytes, homeostasis of T cell numbers, cytotoxicity, and the maintenance of immunological privilege. Fas ligand is a type II membrane protein that is cleaved by a metalloproteinase to produce an active, soluble molecule. It has been found that a variety of target cells are differentially sensitive to soluble and membrane-associated forms of Fas ligand. However, the explanation for this differential activity has not been determined. One proposed explanation for this differential activity is that membrane-associated Fas ligand is more efficiently aggregated than soluble Fas ligand. Another possibility that we have investigated is that accessory molecules may act to enhance the activity of cellular Fas ligand. We have transfected cells to express membrane-associated Fas ligand and have characterized clones of these transfected cells in terms of Fas ligand and ICAM-1 surface expression. Enhanced activity was associated with enhanced levels of both Fas ligand and ICAM-1. Moreover, inhibition of ICAM-1 modulated the activity of membrane-associated Fas ligand so that its cellular specificity was similar to that of soluble Fas ligand. Thus, ICAM-1 plays a significant role in regulating Fas ligand activity, and this role explains, at least in part, the different functional attributes of the soluble versus the cell-associated molecule.

Technical note: Aberrant detection of cell surface Fas ligand with anti-peptide antibodies.

Polyclonal rabbit Abs raised against peptides from the C-terminal region (the extracellular domain) of human Fas ligand were produced for the detection of the molecule in Western blot analysis and immunohistochemistry. These Abs have been used by several groups of investigators to assess cell surface Fas ligand via flow cytometry, but we show that these polyclonal rabbit Abs do not detect cell surface Fas ligand by that technique.

Viral regulation of CD95 expression and apoptosis in T lymphocytes.

T lymphocyte apoptosis has been observed in vivo in association with viral infections. One mechanism known to mediate T cell apoptosis is the CD95 (Fas/APO-1) Ag apoptotic pathway. CD95 is a cell surface protein that is expressed on activated T cells and is capable of relaying an intracellular apoptotic signal upon binding with CD95 ligand. CD95-dependent apoptosis has been shown to be involved in the homeostatic control of peripheral T cell numbers; however, the functional significance of the CD95 apoptotic pathway in the context of viral infections has not been clearly elucidated. We show that exposure to a variety of viral pathogens causes enhanced CD95 expression in naive peripheral blood cell T lymphocytes without leading to enhanced susceptibility to CD95-dependent apoptosis. Productive viral infection was not required for this effect. Furthermore, using a T cell line and the K562 tumor cell line, we demonstrate that cells normally resistant to CD95-mediated killing become susceptible when productively infected with virus in a manner that does not rely on increased CD95 surface expression. These findings demonstrate a regulatory influence of viral infection on CD95 expression and activity and suggest that in addition to having a role in T cell homeostasis, the CD95 apoptotic pathway might also function to eliminate virus-infected T cells.

Fas ligand deficiency in HIV disease.

Apoptosis is postulated to be involved as an anti-viral immune mechanism by killing infected cells before viral replication has occurred. The Fas-Fas ligand interaction is a powerful regulator of T cell apoptosis and could potentially act as a potent anti-viral immune mechanism against T cell tropic virus such as human immunodeficiency virus (HIV). We investigated the status of Fas ligand in peripheral blood mononuclear cells (PBMCs) obtained from persons infected with HIV. We found that monocytes in freshly isolated PBMCs from healthy individuals possess cell surface Fas ligand. In contrast, monocytes in freshly isolated PBMCs from HIV-infected patients had no detectable Fas ligand on the cell surface. Consistent with these findings of surface expression, Fas ligand activity was deficient in the cells from HIV-infected persons. The effect of replacing Fas ligand activity on HIV production by patients' cells was assessed in an in vitro assay. The addition of a functional anti-Fas antibody to PBMCs from HIV-infected individuals inhibited viral production by greater than 90% without affecting lymphocytic function. These findings suggest the possibility of a new therapeutic modality for the treatment of HIV-infected individuals based on the reconstitution of Fas ligand activity.

Herpes simplex virus type 2 inhibition of Fas ligand expression.

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are common human pathogens. In this report we demonstrate the capacity of HSV-2, but not HSV-1, to inhibit the activity and cell surface expression of Fas ligand, an important molecule involved in T-cell apoptosis and cell-mediated cytotoxicity. Cells infected with HSV-2 retained Fas ligand intracellularly instead of expressing it on the cell surface. Addition of anti-Fas antibodies markedly inhibited HSV-2 viral production, suggesting that the capacity of the virus to regulate Fas ligand expression, and thereby programmed cell death, may represent a powerful mechanism for the virus to enhance viral replication.

The role of interleukin-10 in the inhibition of T-cell proliferation and apoptosis mediated by parainfluenza virus type 3.

We have previously demonstrated that parainfluenza virus type 3 (PIV3), a significant respiratory pathogen, can markedly inhibit T-cell function in vitro. We now report that the virus potently induces interleukin-10 (IL-10) production by peripheral blood mononuclear cells. The IL-10 produced contributes to viral inhibition of T-cell proliferation and protects T cells from PIV3-mediated apoptosis. These findings suggest that IL-10 is likely to play an important immunoregulatory role in PIV3 infections.

Specific inhibition of granzyme B by parainfluenza virus type 3.

T-cell-mediated cytotoxicity is an important means of defense against viral pathogens; however, several viruses possess mechanisms to disrupt cytotoxicity, thereby allowing them to avoid immune clearance. These viruses have been shown to inhibit cytotoxicity by interfering with the capacity of T lymphocytes to specifically recognize infected cells. An alternative mechanism for virally induced cytotoxic dysfunction is identified in this report. We show that parainfluenza virus type 3, a negative-stranded RNA virus, can inhibit cytotoxicity by causing a defect in the cytotoxic effector apparatus. This defect is identified as a selective inhibition of granzyme B mRNA.

Infection and immunoregulation of T lymphocytes by parainfluenza virus type 3.

Human parainfluenza virus type 3 (HPIV3) is a major cause of disease in newborns and infants. It also has a striking potential to reinfect individuals throughout their lives, suggesting that HPIV3 does not induce lifelong immunity; however, the operative mechanism for the failure to prevent reinfection is not known. We have assessed the potential of the virus to infect nontransformed human T lymphocytes and have found that T cells are readily infected by the virus. Productive infection requires activation of the T cells and results in a marked inhibition of proliferation. Furthermore, our results indicate that exposure to the virus, even without overt expression of viral proteins as detected by immunohistology, profoundly alters the functional capacity of the T cells. The capacity of the virus to regulate T-lymphocyte function may play an important role in the failure of the virus to induce lifelong immunity.

Radioimmunoassay of inhibin based on synthetic human inhibin alpha-chain peptide.

Polyclonal rabbit antisera were produced against cyclic human inhibin [(Cys6, Tyr7) alpha-(6-30)NH2] peptide, covalently conjugated to bovine serum albumin. The tyrosine residue introduced at position 7 facilitated the oxidative incorporation of radiolabel (125I) to yield a tracer with specific activity of 73.9 Ci/g. These reagents were used to develop a homologous equilibrium radioimmunoassay for human inhibin, with polyethylene glycol, 200 g/L, serving as the separation phase. At a detection limit of 2 micrograms/L (n = 7), immunoactive inhibin was detectable in human pre-ovulatory follicular fluid (128 micrograms/L), seminal plasma (2374 micrograms/L), amniotic fluid (66 micrograms/L), and placental extract (347 micrograms/L). We also demonstrated inhibin immunoreactivity in biological fluids from other mammalian species: macaque, chimpanzee, porcine, and bovine, but not rodent (guinea pig). Although the antisera were raised against a nonbioactive inhibin peptide, immunoglobulins fractionated on Protein A-Sepharose neutralized the bioactivity of human ovarian inhibin. Further characterization of inhibin immuno- and bioactivity was undertaken with immobilized heparin, divalent metal cations, and dye ligands. Only heparin-Sepharose distinguished between immuno- and bioactive inhibin.

Ovarian stimulation for in vitro fertilization using pure follicle-stimulating hormone with and without gonadotropin-releasing hormone agonist in high-responder patients.

There is a distinct pattern of response to gonadotropin stimulation in some patients marked by high peak estradiol (E2) levels, multifollicular ovarian response, and elevated basal luteinizing hormone (LH)/follicle-stimulating hormone (FSH) ratios. We reviewed the stimulation profiles of five such high-responder patients who failed to conceive during in vitro fertilization with ovarian stimulation using pure FSH. All patients had baseline LH/FSH greater than 1.5 and peak E2 greater than 800 pg/ml. One cycle was canceled prior to hCG administration because of marked ovarian response (E2 greater than 2500 pg/ml, multiple small follicles). In a subsequent cycle, all patients were pretreated with the gonadotropin releasing-hormone agonist (GnRHa) leuprolide acetate for 10-14 days prior to initiation of FSH for ovarian stimulation. Leuprolide was continued until the day of hCG administration. During cycles using GnRHa, there was a statistically significant decrease (P less than 0.05) in serum FSH on day 3 (less than 5 vs 8.3 mIU/ml), serum E2 on day 3 (14.6 vs 34.6 pg/ml), and peak serum E2 (1197.6 vs 1923.0 pg/ml). Patients during cycles with GnRHa had a greater number of preovulatory (8.6 vs 3.0) and total (12.4 vs 6.0) oocytes retrieved (P less than 0.05). The fertilization rate of preovulatory oocytes was also higher during cycles using GnRHa (83 vs 64%). Two pregnancies occurred in the cycles pretreated with GnRHa. These preliminary data indicate that in high-responder patients, a combination of GnRHa and pure FSH results in lower E2 levels during the stimulation cycle and a greater number of total and mature oocytes retrieved and fertilized.

Human prostate tissue antigens defined by murine monoclonal antibodies.

BALB/c mice were hyperimmunized against a membrane preparation derived from a pool of transurethral resection specimens which included three benign prostatic hyperplasia and one prostate adenocarcinoma tissue samples. The activated lymphocytes were fused with the NS-1 mouse myeloma cell line, and supernatants from immunogen-reactive hybridomas were screened for antibody binding activity using a solid-phase radioimmunoassay against the Calu-1 human lung adenocarcinoma cell line and several membrane preparations derived from various normal human tissues. Hybridoma cultures secreting antibodies which did not appear cross-reactive were doubly cloned by limiting dilution and screened against a large panel of membrane preparations derived from normal prostate, benign prostatic hyperplasia, and prostate adenocarcinoma tissues as well as samples obtained from a variety of normal human tissues. The monoclonal antibodies were also evaluated against 24 normal, virally transformed, and malignant human cell lines. Two monoclonal antibodies were isolated which demonstrated a restricted binding activity to prostate antigens and were not widely cross-reactive with nonprostate normal tissues or cell lines. These antibodies were designated TURP-27 (IgG3, k) and TURP-73 (IgG2a, k). Both of these monoclonal antibodies were reactive against formalin-fixed, paraffin-embedded tissues in the immunoperoxidase assay and were subsequently tested against a variety of normal, hyperplastic, and malignant human tissues. These studies indicated that TURP-27 may be directed against a new prostate organ-associated marker and that TURP-73 is directed against an antigen expressed on prostate and a limited number of other tissues.

Antiprostate carcinoma monoclonal antibody (D83.21) cross reacts with a membrane antigen expressed on cytomegalovirus-transformed human fibroblasts.

Virological and epidemiological studies have implicated human cytomegalovirus (HCMV) as a possible etiological agent of prostate cancer. Because of the suspected associations, this laboratory tested the reactivity of a prostate-associated monoclonal antibody with HCMV-transformed cells. This mouse monoclonal antibody, D83.21, reacts with a membrane antigen on prostate and bladder tumor cells and does not bind to a variety of other malignant or normal cells. The results of this study indicated that the prostate-associated antibody bound to a membrane antigen on HCMV-transformed cells as detected by radioimmunoassay, immunofluorescence, and complemented-dependent cytotoxicity. This cross-reactivity appeared to be specific for HCMV-transformed cells and did not react with HCMV-infected cells or those transformed by other viruses. Antibody affinity chromatography, used to isolate the D83.21-reactive protein, revealed two peptides of 60 and 28 kd on both prostate tumor and HCMV-transformed cells. The results suggest that D83.21 reacts with a common cell surface protein expressed on HCMV-transformed cells and urogenital tumors.

Immunohistochemical localization of prostate carcinoma-associated antigens.

The immunoperoxidase technique was used to study the localization and distribution of antigens reactive with two monoclonal antibodies, D83.21 and P6.2, produced against cultured prostate tumor cells, in formalin-fixed, paraffin-embedded histological sections of human tissues. Monoclonal D83.21 reacted with 11 of 19 (58%) primary prostate carcinomas and 1 of 6 (17%) metastatic tumors, whereas monoclonal P6.2 reacted with 14 of 19 (68%) primary and 4 of 6 (67%) metastatic prostate tumors. Neither antibody reacted with five primary prostate tumors and one metastatic prostate tumor. In some tumor cells, the antigens recognized by these monoclonals were localized in either the cytoplasm or cell membrane, while in other tumor cells, both diffuse cytoplasmic and membrane or focal staining patterns were observed. In addition to the variable staining patterns, antigenic heterogeneity was also noted within most prostate tumors examined. Two types of staining variability were observed: (a) tumor cells in one area of the tissue section stained positive, but in another area they did not react with the antibody; and (b) both stained and unstained tumor cells were adjacent to each other. These results would suggest that a panel of monoclonals will be required to detect the different subpopulations of prostate tumor cells. Neither antibody reacted with 6 normal or 12 benign prostate tissues, nor any of a variety of other normal human tissues except for staining of the proximal tubules of normal kidneys. The antigen detected by P6.2 demonstrated a wider tissue distribution being found on bladder, breast, lung, and pancreatic tumors, whereas the antigen recognized by D83.21 was restricted to prostate and bladder carcinomas. These antibodies may have clinical applicability for the identification of prostate tumor cells in biopsy specimens and for immunohistopathological classification of prostate carcinomas.