Inhibition of androstenediol-dependent LNCaP tumour growth by 17alpha-ethynyl-5alpha-androstane-3alpha, 17beta-diol (HE3235).

Androst-5-ene-3beta, 17beta-diol (AED) is an adrenal hormone that has been reported to sustain prostate cancer growth after androgen deprivation therapy (ADT). LNCaP cells express a mutated androgen receptor that confers the ability to respond not only to androgen but also to oestrogen and adrenal hormones such as AED, and thus provide a cell line useful for identifying compounds capable of inhibiting AED-stimulated cell growth. We sought to determine whether structurally related steroids could inhibit AED-stimulated LNCaP cell growth in vitro and tumour growth in vivo. We report here the identification of a novel androstane steroid, HE3235 (17alpha-ethynyl-5alpha-androstan-3alpha, 17beta-diol), with significant inhibitory activity for AED-stimulated LNCaP proliferation. This inhibitory activity is accompanied by an increase in the number of apoptotic cells. Animal studies have confirmed the cytoreductive activity of HE3235 on LNCaP tumours. The results suggest that this compound may be of clinical use in castration-resistant prostate cancer.

Propofol hemisuccinate suppression of experimental autoimmune encephalomyelitis.

Propofol hemisuccinate is a prodrug water soluble form of the lipophilic, phenolic compound propofol (2,6-di-isopropylphenol), that is the active ingredient in the widely used anesthetic agent Diprovan. Propofol binds to GABA(A) receptors but also has a phenolic structure that confers antioxidant properties to the molecule. The effects of propofol hemisuccinate in rat experimental autoimmune encephalomyelitis (EAE) were studied using different doses and time regimes. Propofol hemisuccinate, 100 mg/kg given three times a day from day 7 or day 12 until day 16 after disease initiation, significantly reduced maximal EAE score. Histology studies supported the clinical findings demonstrating reduction in the inflammatory response in the lumbar spinal cord in animals treated with propofol hemisuccinate. Decreased levels of nitrotyrosine and unchanged levels of induced nitric oxide synthase suggest propofol hemisuccinate crossed the blood brain barrier and exerted its effects by lowering reactive oxygen species levels. The results suggest that propofol hemisuccinate may provide an alternative mode of treatment for acute exacerbations of multiple sclerosis.

Antigenic and immunologic characterization of an allogeneic colon carcinoma vaccine.

We report the immunological characterization of three colon carcinoma cell lines, COLO 205, SW620 and SW403, which we selected to combine with cytokine-secreting fibroblasts for the development of an allogeneic tumour cell vaccine. The cell lines expressed HLA-A2 as well as shared tumour-associated antigens (TAAs) representative of colon carcinomas: CEA, Ep-CAM, MUC1, HER2/neu and MAGE antigens. They did not secrete high levels of the immunosuppressive factors TGF-beta, IL-10 or prostaglandins. The lines presented TAAs in a manner recognized by immune effector cells, which was demonstrated by the lysis of SW620 by HLA-A2-restricted anti-p53 cytotoxic T lymphocytes (CTL). COLO 205 and SW620 were genetically modified to express the co-stimulatory molecule CD80 (B7.1), which increased the ability of the cells to stimulate CTL in vitro. CTL clones derived from HLA-A2+ peripheral blood mononuclear cells stimulated with the CD80-expressing lines lysed the stimulator cell and an HLA-A2+ colon cancer cell line, but did not lyse an isogeneic fibroblast line or an HLA-A2- colon cancer cell line. CTL clones derived from colon carcinoma patients immunized with an allogeneic vaccine containing these lines demonstrated killing of autologous tumour cells, the vaccine cell lines and other HLA-A2+ colon cancer cell lines, but not fibroblasts isogeneic to certain of the target cell lines. Our studies demonstrate that these colon carcinoma cell lines express shared TAAs that can induce CTLs which recognize and lyse other colon carcinoma cells, and support the continued clinical evaluation of the CD80 gene modified allogeneic colon cell/cytokine-secreting fibroblast carcinoma vaccine.

Safety and immunogenicity of REMUNE in HIV-infected Thai subjects.

The safety and immunogenicity of REMUNE, an HIV-specific immune based therapy for HIV infection, was evaluated in a cohort of 30 HIV infected subjects in Thailand. This therapy utilizes a gp120 depleted inactivated virus (HZ321), which exhibits a high degree of conservation with the core antigens of both type B' and E strains of HIV, the predominant Thailand isolates. The treatment was well tolerated, with no serious adverse events reported over the course of the 4-month trial. Treatment in which four doses were administered with REMUNE appeared to boost HIV-specific immune responses, with approximately 75% of the treated subjects demonstrating an increase in either the repertoire or the intensity of the serological response to HIV as measured by Western blot. CD4%, viral load, and weight remained stable over the course of the 4-month study relative to baseline values. Viral subtyping of this cohort revealed a predominance of type 'E'. These data suggest that REMUNE is safe and immunogenic in seropositive Thai subjects and supports further study of the therapeutic potential of REMUNE to treat HIV-1 infection.

Characterization of highly purified, inactivated HIV-1 particles isolated by anion exchange chromatography.

This report characterizes inactivated, gp120 depleted, HIV-1 particles purified by an anion exchange chromatography production process. This antigen formulated with incomplete Freund's adjuvant constitutes Remune, which is being evaluated in a phase III clinical endpoint trial to determine the effect of this immune-based therapy on clinical progression of HIV-1 seropositive patients. Multiple production lots of the inactivated HIV-1 antigen strain HZ321, isolated by anion exchange chromatography, exhibit purity of > 95% by gel filtration. These findings are corroborated by thin section electron microscopy showing a homogenous field of intact particles. Analyses of the purified virus particles for protein, lipid, carbohydrate and RNA show structural retention of the envelope proteins, lipid bilayer and core components after large scale processing. The qualitative identification of at least 85% of total HIV-1 protein is determined by ELISA, Western blot, HPLC and amino acid sequencing analyses. Quantitative values are assigned to 50% of these proteins. The data confirm the presence of virally encoded proteins p6, p7, pI15, p17, p24, p32, pI39Gag, gp41, pp55Gag, p66/51, Vpr, Vif and Nef. Excellent consistency between production lots and equivalency to HIV-1 preparations purified by sucrose density gradient sedimentation has been established for protein and lipid composition, and overall purity. These findings further establish that non-viral encoded proteins and lipids are integral structural components of the intact virion and are not contaminants unique to a particular isolation method. The data confirm the presence of multicomponent antigens in the viral particles for stimulating a broad HIV-1 specific immune response. Finally, the work demonstrates that the two inactivation procedures (beta-propiolactone and gamma irradiation), which achieve efficient viral inactivation meeting US FDA guidelines, do not damage the protein antigens of the viral particles.

HIV-1-Specific Functional Immune Measurements as Markers of Disease Progression.

The impairment of lymphocytes to proliferate to HIV antigen is a relatively early functional defect of cell-mediated immunity found in HIV-infected individuals. The finding of strong proliferative responses in nonprogressive HIV disease as well as its inverse association with viral load and clinical manifestation of AIDS supports the further use of this marker as a surrogate of disease progression. The observation that HIV-specific lymphocyte proliferation is associated with the production of CD8-derived HIV suppressive factors such as the beta-chemokines further supports this conclusion. These functional immune measurements provide an additional marker to monitor disease progression in HIV-infected individuals, along with the current standards of CD4 counts and viral load. Copyright 1997 S. Karger AG, Basel

Association of ERK2 mitogen-activated protein kinase with human immunodeficiency virus particles.

Here we report the presence of a protein kinase activity associated with human immunodeficiency virus type 1 (HIV-1) particles. We observed phosphorylation of five major proteins by the endogenous protein kinase activity. Phosphoamino acid analysis revealed phosphorylated serine and threonine residues. In addition, we observed autophosphorylation of two proteins in the presence of gamma-ATP in an in-gel phosphorylation assay. These two proteins are not linked by a disulfide bond, suggesting that two different protein kinases are associated with HIV-1 virions. Our results indicate the presence of ERK2 mitogen-activated protein kinase and of a 53,000-molecular-weight protein kinase associated with virions. Moreover, the use of different HIV strains derived from T cells and promonocytic cells, as well as the use of human T-cell leukemia virus type 1 particles, demonstrates that ERK2 is strongly associated with retrovirus particles in a cell-independent manner. Exogenous substrates, such as histone proteins, and a viral substrate, such as Gag protein, are phosphorylated by virus-associated protein kinases.

Effect of immunization with an inactivated gp120-depleted HIV-1 immunogen on beta-chemokine and cytokine production in subjects with HIV-1 infection.

OBJECTIVE: To measure beta-chemokine and cytokine production in HIV-1-infected subjects undergoing treatment with HIV-1 immunogen (REMUNE). DESIGN: Open label treatment study. METHODS: beta-Chemokine and cytokine production in peripheral blood mononuclear cell (PBMC) culture. RESULTS: Interferon-gamma production (p = 0.04) and lymphocyte proliferation (p = 0.001) to HIV-1 antigen-stimulated PBMCs increased after immunization with the HIV-1 immunogen. A correlation was demonstrated after immunization between HIV-1 antigen-stimulated lymphocyte proliferation and interferon-gamma levels (r = 0.53, p = 0.04). No significant change after immunization was seen for interleukin-4 production. A significant increase in mean levels of HIV-1 antigen-stimulated RANTES (i.e., regulated upon, activation normal T-cell expressed and secreted), was evident 1 month after immunization (p = 0.002) and remained elevated 3 months after immunization. RANTES production was decreased in CD8-depleted PBMC cultures. Mean serum HIV-1 RNA copy numbers and CD4 cell counts remained stable after immunization (p > 0.5). A correlation was demonstrated between HIV-1 antigen-stimulated interferon-gamma and RANTES production (r = 0.54, p = 0.002). CONCLUSIONS: This report describes an augmentation of beta-chemokines and TH1-type cytokines from PBMCs after immunization with the HIV-1 immunogen.

Combination therapies against HIV-1 infection: exploring the concept of combining antiretroviral drug treatments with HIV-1 immune-based therapies in asymptomatic individuals.

The deleterious effect of HIV on the immune system begins at the time of infection. At seroconversion the virologic and immunologic factors that ultimately will dictate the rate of disease progression are believed to be already in place. The concept developed in this paper implies that, to impact significantly on the progression of disease, anti-HIV therapies should be initiated as early as possible in asymptomatic individuals. Published results have shown that combination drug therapies are potent in reducing HIV-1 RNA load in plasma in asymptomatic individuals, and that some HIV-1 immune-based therapies have a positive impact on immunological markers of disease progression, including HIV-1 cell-mediated immunity (CMI) and CD4 percent. The strategy discussed is to test a combination of antiretroviral therapy with HIV-1 immune-based therapy, such as the inactivated HIV-1 immunogen preparation, in asymptomatic individuals. The goal of this combination approach is to overcome the limitations of each therapy alone. Preliminary data suggest that antiretroviral therapy and the HIV-1 Immunogen can be combined with no noticeable interference and/or added toxicity in a broad range of HIV-1-infected individuals. Combining both therapies may enhance and expand the impact on key surrogate markers of disease progression, although they likely achieve this impact through different mechanisms. Thus, the primary question remains: Can these effects be synergistic?

Viral load, CD4 percentage, and delayed-type hypersensitivity in subjects receiving the HIV-1 immunogen and antiviral drug therapy.

Two trials of subjects inoculated with the inactivated, gp120-depleted HIV-1 Immunogen are reported. In one study, in which 19 subjects received ZDV and 8 subjects received ddI, treatment with the HIV-1 Immunogen did not affect the pharmacokinetic parameters of the antiviral drugs. In another study, 65 subjects who were previously immunized with the HIV-1 Immunogen over a mean period of 4.0 years (range, 1.2-5.4 years) received inoculations at 0 and 6 months. At some point during this 48-week study, 72% of the subjects (47/65) were receiving antiviral drug therapy. The HIV-1 DNA load in CD4 cells and CD4 percentage were found to be stable over the 48-week period. Delayed-type hypersensitivity to HIV-1 antigens increased after two inoculations with the HIV-1 Immunogen. In these two trials, no serious treatment-related adverse events were documented in the subjects. The two studies presented herein are the first to suggest that an immune-based therapy such as the HIV-1 Immunogen can be combined safely with antiviral drugs, supporting further study to evaluate the clinical utility of this approach.

Initial studies on active immunization of HIV-infected subjects using a gp120-depleted HIV-1 Immunogen: long-term follow-up.

In 1987, exploratory clinical studies were initiated to determine whether the development of AIDS in HIV-infected individuals might be delayed or prevented by immunization with an inactivated HIV preparation. Preclinical studies had shown the preparation to be safe and immunogenic. Twenty-three patients with biopsy-confirmed persistent generalized lymphadenopathy (CDC III) and two with asymptomatic HIV infection and CD4 lymphocyte counts between 135 and 769/mm3 were studied, of whom eight (32%) had additional HIV-related symptoms. Over a 3-year period, they received a median of eight open-label inoculations of 100 micrograms of inactivated gp 120-depleted HIV-1 Immunogen in incomplete Freund's adjuvant (IFA). Clinical, general laboratory, immunologic, and virologic parameters were followed for up to 6 years. No serious treatment-related adverse experiences were reported, nor was accelerated HIV disease progression seen. Twelve patients developed a delayed-type hypersensitivity response (HIV-DTH) to the immunogen and nine showed fourfold or greater increases in anti-p24 antibody titers. In the follow-up period, 10 of the 25 patients developed AIDS and one with Kaposi's sarcoma (KS) at baseline progressed. Of the 12 patients who became HIV-DTH-responsive, one developed an opportunistic infection (OI), occurring approximately 5 years from study onset, and subsequently died. One additional HIV-DTH responder developed KS. Of the 13 patients who remained HIV-DTH-nonresponsive, nine (69%) progressed to AIDS and seven of these have died. Differences were also observed in terms of HIV-DNA copy number, CD4 percentages, and anti-p24 antibody patterns between the HIV-DTH-responsive and -nonresponsive groups, suggesting a more favorable clinical course in the former. HIV-1 Immunogen in IFA appears to be safe and immunogenic. Further studies are indicated to determine clinical efficacy of the HIV Immunogen as well as the significance of the apparent correlation between HIV-DTH responsivity and a more favorable clinical course.

Autoproliferation in HIV-1-infected patients undergoing active HIV-1-specific immunotherapy.

We have observed a treatment-associated autoproliferative response in cultured peripheral blood mononuclear cells (PBMC) of asymptomatic HIV-1-infected subjects receiving a gp120-depleted, inactivated HIV-1 antigen in incomplete Freund's adjuvant (IFA; HIV-1 Immunogen). The frequency and magnitude of the autoproliferative response appeared to be dose-related (P < 0.05), and was not observed in subjects receiving IFA alone. Immunophenotyping of the proliferating cells demonstrated the presence of both CD4+ and CD8+ lymphocytes, with the CD4+ blasts almost exclusively expressing the CD45RO+ phenotype. A comparison of this response with the HIV-1-specific antigen stimulation responses in this cohort revealed a significant correlation between increases in HIV-1-specific cell-mediated immunity and autoproliferation (r2 = 0.61, P < 0.001). These findings suggest that immunization with the HIV-1 Immunogen induces an autoproliferative response that may reflect changes in HIV-1-specific cell-mediated immunity in infected individuals.

Safety and immunogenicity of a gp120-depleted, inactivated HIV-1 immunogen: results of a double-blind, adjuvant controlled trial.

We report the safety and immunogenicity results of a double-blind adjuvant controlled trial of the human immunodeficiency virus type 1 (HIV-1) immunogen. Healthy, asymptomatic HIV-1-seropositive individuals received either three 100 microgram doses of the inactivated HIV-1 antigen in incomplete Freund's adjuvant (IFA) or three doses of IFA alone. The results of this study show that this HIV-1 immunogen is safe, with mild and transient adverse events. No difference in the number or type of adverse events was noted between the treatment groups. Rises in HIV-1-specific humoral and cell-mediated immune (CMI) responses were also noted, favoring the HIV-1 immunogen-treated group. The results of this study confirm and extend the safety and immunogenicity profile of this HIV-1 immunotherapeutic agent. The observation that this treatment can augment HIV-1-specific CMI is encouraging because this immunological marker may represent a key surrogate end point in HIV-1 infection and disease.

HIV-1 immunogen induction of HIV-1-specific delayed-type hypersensitivity: results of a double-blind, adjuvant-controlled, dose-ranging trial.

OBJECTIVE: To investigate the capacity of an HIV-1 immunogen to induce or augment HIV-1-specific delayed-type hypersensitivity (DTH) over a range of doses in asymptomatic HIV-1-seropositive adults. DESIGN: A single center, double-blind, adjuvant-controlled, dose-ranging trial involving 48 HIV-1-seropositive asymptomatic patients. Each dose group consisted of 12 subjects, eight receiving HIV-1 immunogen and four incomplete Freund's adjuvant (IFA). The doses studied were 50, 100, 200, or 400 micrograms (total protein). The HIV-1 immunogen was administered intramuscularly every 4 weeks for 36 weeks, with dosing contingent on the lack of an HIV-1 immunogen DTH response. A maximum of six doses was permitted. METHODS: Immunogenicity was assessed every 4 weeks by DTH skin testing to the inactivated HIV-1 antigen in saline with > 9 mm induration representing a response to immunization. Changes in p24-antibody levels were determined by endpoint titration using an enzyme-linked immunosorbent assay and Western blot. RESULTS: At doses of > or = 100 micrograms, all treated patients demonstrated significant differences in the ability to mount an HIV-1-specific cell-mediated response relative to adjuvant controls. Dose-related response patterns were observed in the period between doses and the occurrence of rises in HIV-1 DTH. Treatment appeared to increase p24-antibody titers as well as reactivities to other HIV-1 antigens as determined by Western blots. The HIV-1 immunogen was well tolerated. CONCLUSIONS: The minimum dose of the HIV-1 immunogen in IFA required to induce HIV-1 DTH relative to the IFA control group was 100 micrograms in this patient population.

Effect of immunization with inactivated gp120-depleted human immunodeficiency virus type 1 (HIV-1) immunogen on HIV-1 immunity, viral DNA, and percentage of CD4 cells.

A 1-year study measured the effect of administration of an inactivated human immunodeficiency virus type 1 (HIV-1) immunogen in incomplete Freund's adjuvant (IFA) on HIV-1-specific immunity and viral burden in asymptomatic HIV-1-infected patients. A total of 103 patients were enrolled in this double-blind, randomized study comparing immunogen in adjuvant with adjuvant alone. This study was conducted at nine medical centers throughout the United States. Significant differences in cell-mediated immune responses to HIV-1 and p24 core antigen were observed between the treated and control groups (P < .01). Compared with controls, treated patients as a group had a significantly lower rate of increase in viral DNA as determined by quantitative HIV-1 DNA-polymerase chain reaction (P = .016). Significant differences in the rate of percent CD4 cell decline were also observed between the 2 groups (P = .024). These data suggest that augmentation of HIV-1-specific immunity via immunotherapy may alter the rate of increase of HIV-1 DNA in peripheral blood mononuclear cells and stabilize the percent of CD4 cell decline in relatively healthy HIV-1-infected persons.

Inactivated HIV-1 Immunogen: impact on markers of disease progression.

The pursuit of valid markers of disease progression in human immunodeficiency virus type 1 (HIV-1) infection is especially relevant considering the potential treatment alternatives that presently are under evaluation. Because HIV-1 infection results in a virally induced immune suppression characterized by the loss of cell-mediated immunity (CMI), depletion of CD4+ cells, loss of core antibody, and an increase in viral burden, these markers seemed to be appropriate to monitor in a controlled study. We monitored a number of virologic, immunologic, and cytologic markers of disease progression in 103 subjects who were enrolled in a 12-month, double-blind, randomized, adjuvant-controlled study of the HIV-1 inactivated Immunogen. The markers included HIV-1 DNA, HIV-1 RNA, CD4 percent, p24 antibody, and lymphocyte proliferation. Analysis of HIV-1 DNA with a quantitatively polymerase chain reaction (PCR) assay indicated a treatment effect on viral burden in the HIV-1 Immunogen-treated group. Analysis of HIV-1 RNA revealed a similar trend favoring the Immunogen-treated group. In addition, a significant effect was shown on CD4 percent and CMI in the Immunogen-treated group. An analysis of CMI that used stimulation indices underrepresented the immunogenicity of the Immunogen. Further examination revealed that the lymphocytes of the HIV-1 Immunogen-treated patients were proliferating in vitro without exogenous antigen. Although the clinical significance of this phenomenon currently is unknown, it may be a relevant prognostic marker for assessment of HIV-1 therapy. The data presented here support the concept that immunotherapy with the HIV-1 Immunogen may slow disease progression.(ABSTRACT TRUNCATED AT 250 WORDS)

T cell receptor V beta repertoire in HIV-infection individuals: lack of evidence for selective V beta deletion.

The gradual decline of CD4+ T lymphocytes in HIV-infected individuals culminates in the lethal immunosuppression of AIDS. The mechanism of CD4+ T cell loss is currently unknown, but has recently been suggested to occur as a result of an HIV-encoded superantigen which facilitates a selective deletion of T cells expressing specific V beta genes. To verify and extend such observations, peripheral blood leucocytes (PBL) from 15 HIV+ individuals, 10 of which had very low CD4 T cell counts (< 200/mm3), were analysed for T cell receptor (TCR) V beta gene expression. In contrast to a recent study, the results presented here fail to provide evidence that selective loss of V beta-bearing T cells occurs in HIV+ individuals. Furthermore, when PBL from HIV+ individuals were stimulated with Staphylococcal enterotoxin B (SEB), T cells expressing V beta subfamilies known to engage this superantigen were expanded, indicating that such cells were not deleted and were responsive to stimulation by a bacterial superantigen. Collectively, these data suggest that CD4 loss in HIV patients does not occur in a V beta-selective, superantigen-mediated fashion.

Cell-mediated immunity to HIV-1 in Walter Reed stages 1-6 individuals: correlation with virus burden.

Cell-mediated immunity (CMI) to human immunodeficiency virus-1 (HIV-1) was assessed in a blinded fashion for a patient group (n = 79) representing Walter Reed (WR) stages 1-6. At the same time, viral load was quantitatively measured by two different methods, specifically, virus isolation and HIV viral DNA copy number as measured by the polymerase chain reaction (PCR). After unblinding it was determined that the ability to generate a lymphoproliferative response to an inactivated gp120-depleted HIV (HIV-ag) and tetanus toxoid diminished with advancing WR staging, with complete anergy to HIV-ag and tetanus at stage 6. As a group, individuals whose peripheral blood mononuclear cells (PBMC) proliferated to HIV-ag were either virus isolation negative or produced low levels of virus as measured by p24 antigen (< 250 pg p24) on day 7. Similarly, HIV DNA copy number in the HIV-ag responders was low (< 200 copies/4 x 10(5) PBMC). In contrast, antigen proliferation to tetanus toxoid did not correlate with virus load. Thus, clinical progression as defined by the WR staging system appears to coincide with a loss of CMI to HIV. More importantly, the low viral load measured in HIV-ag responders suggests a link between viral burden and CMI to HIV which might be exploited in the design of immunotherapies for HIV-infected individuals.

Characterization of endogenous and recombinant proviral elements of a highly tumorigenic AKR cell line.

As an approach to evaluating the contribution of classes of endogenous viral sequences to leukemogenesis, a genomic library was prepared from the highly tumorigenic AKR SL12.3 cell line and screened for env-containing proviruses. An extensive battery of virus-derived probes and specific oligonucleotide probes were used to segregate 83 positive clones into related groups. The nonecotropic endogenous retroviruses were identified as members of the polytropic, modified polytropic, or xenotropic groups. At least three unique xenotropic proviruses were detected that differed from the published xenotropic sequence within a variable region of the 5' portion of env. Changes among the xenotropic proviruses included relative insertions and/or deletions that maintain an open reading frame and hence the potential to encode viable envelope gene products. Several recombinant viruses were also detected. Recombination was not random and primarily involved the formation of mink cell focus-inducing class I retroviruses via recombination between polytropic elements and ecotropic virus. One other recombinant was detected which contained ecotropic virus sequences in the 5' region encoding p15 of an otherwise xenotropic provirus. An interesting observation was the finding that certain clones contained more than one provirus within the average 20-kb cloned insert. This would not be expected if integration were totally random. The de novo recombinant proviruses identified here provide a series of potential candidates to be evaluated for their contribution to the tumorigencity of the SL12.3 cell line.