[Hepatitis B virus and the inflammatory/immunologic response (II): a new opportunity for the treatment of chronic hepatitis B and a suggestion for the treatment of other persistent viral diseases]. / Virus de la hepatitis B y la respuesta inflamatoria/inmunológica (II): Una nueva oportunidad de tratamiento de la infección crónica y una sugerencia para el tratamiento de otras enfermedades víricas persistentes.

With the immunologic rationale exposed in the first part of this paper, the authors analyze a new experimental treatment which includes the combination of an antiviral (ribavirin) and an immunomodulator (AM3) in a model of hepatotoxic viral-infection in mice. Rationale for this associated treatment is based on the ability of AM3 to restore the natural immunity through the induction of IL-12 and IFN-gamma. Furthermore, the treatment with AM3 decreases factor C3 of the complement system which appears to be implied in IL-12 downregulation. Experimental and clinical results showed herein suggest a new approach to the treatment of viral hepatitis which combines the use of antivirals in combination with immunomodulators able to restore the "immunologic chaos" induced by some viruses.

[Hepatitis B virus (HBV) and the inflammatory/immune response. I. The natural environment of the antigen presentation and immunologic chaos induced by the virus]. / Virus de la hepatitis B (VHB) y la respuesta inflamatoria/inmunológica (I): el entorno natural de la presentación antigénica y el caos inmunológico inducido por el virus.

In this paper, the authors update on the immunopathology of hepatitis B virus (HBV) infection, with special reference to the roles of inflammatory and natural immune responses (macrophages and NK cells) in the viral clearance. The role of specific immune responses being related to the influence of the environment of the antigen presentation (macrophages, NK cells, and their related cytokines IL-12 and IFN-gamma) on Th cells within the liver. The viral scape leading to chronic hepatitis B is thought to be due (a) to the suppressive actions of the virus on NK cells and IFN-gamma production (b) to the downregulation of IL-12/IL-15 production provoked by the inflammatory response (factor C3 of the complement system) on IL-12-producing macrophages: immunologic chaos.

Leukocyte Interleukin, Inj. (LI) augmentation of natural killer cells and cytolytic T-lymphocytes.

A serum free lymphokine preparation derived from human buffy-coat mononuclear cells [buffy coat interleukins (BC-IL)], also named Leukocyte Interleukin, Inj. (LI), trade name Multikine, containing glycosylated interleukin-2 (IL-2) among other interleukins, was tested in three head and neck cancer patients. They responded with tumor regressions associated with increased tumor infiltration of lymphocytes and tumor cell lysis indicating an LI Interleukin-2 induced tumor-specific immune response. To determine whether these responses elicited by LI were IL-2 driven, augmentation of natural killer cells (NKC) and cytolytic T cells (CTL), was tested both in vitro and in vivo. A single intraperitoneal (i.p.) injection of LI in adult BALB/c mice at doses of 3, 10, 30 and 100 of IL-2 equivalence International Units per mouse, led to significant (p < 0.01) augmentation of NKC cytotoxicity to YAC tumor cells. NKC cytotoxicity remained elevated for 7 days, peaking at 5 days post-treatment. Multiple treatments with LI did not increase NKC cytotoxicity above single injection, nor did it lead to NKC hyporesponsiveness. The most effective treatment routes leading to heightened NKC cytotoxicity were: intravenous(i.v.) > intraperitoneal (i.p.) > intramuscular (i.m.) > subcutaneous (sc). Significant (p < 0.05 to < 0.01) NKC cytotoxicity was achieved by all four routes. In vitro incubation of murine splenocytes with 30 and 100 International Units/ml (IU/ml) of IL-2 equivalent elevated NKC cytotoxicity significantly (p < 0.01) at all effector to target cell ratios tested and exceeded the response achieved with rhIFN gamma. NKC cytotoxicity of human peripheral blood lymphocytes (HPBL) against the K562 human tumor cell was also significantly elevated (p < 0.01) at the 30 and 100 IU/ml doses and (p < 0.05) at 3 and 10 IU/ml doses. Of particular interest was the significant increase of CTL response in HPBL generated by LI. Significant activity (p < 0.01) was achieved with levels of 10, 30 and 100 IU/ml at effector to target cell ratios as low as 6 to 1. These results indicate that the LI containing IL-2 led to the significant increase in NKC and CTL cytolytic activities. Relatively lower doses of LI were needed to attain equivalent cytolytic activities as achieved with rhIL-2 or rhIFN gamma.

Antiviral and immunomodulating inhibitors of experimentally-induced Punta Toro virus infections.

A major component of a US Army Medical Research and Development Command-supported program to discover and develop new drugs for the treatment of Rift Valley fever, sandfly fever, and Crimean-Congo hemorrhagic fever has been to study candidate test materials against hepatotropic infections of C57BL/6 mice induced by the related but less biohazardous Punta Toro virus (PTV). The effects of 75 compounds, some of which were considered immunomodulators in their primary mechanism of activity, were studied in the PTV infection model. Of these, ribavirin, ribamidine, ribavirin 2',3',5'-triacetate, tiazofurin, tiazofurin-5'-monophosphate, tiazofurin-2',3',5'-triacetate, selenazofurin, pyrazofurin, 3-deazaguanine, and 3-deazaguanosine were considered significantly inhibitory, acting against the infection by a direct antiviral (non-immunomodulatory) fashion. These compounds had therapeutic indices (TI) ranging from > or = 5 to 65, using increased survivors as the evaluation parameter. Immunomodulators considered significantly inhibitory to this infection were poly (ICLC), ampligen, human recombinant interferon-alpha-A/D, MVE-1, MVE-2, AM-3, AM-5, mannozym, bropirimine, CL246,738, phenyleneamine, and 7-thia-8-oxoguanosine. Utilizing increased survivor numbers as measure of activity, these inhibitors had TI ranging from > or = 16 to 1000. Other antiviral effects exerted by the active compounds included reduction of hepatic icterus, lowered serum glutamic oxaloacetic and pyruvic acid transaminases, and inhibition of recoverable serum and liver virus titers. The active immunomodulators were significantly effective when therapy was initiated as late as 48 h after virus inoculation, at a time when clinical signs of the PTV disease were being manifested in the animal.

Differential antiviral activity of two TIBO derivatives against the human immunodeficiency and murine leukemia viruses alone and in combination with other anti-HIV agents.

R82913 and R86183, two derivatives of tetrahydroimidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione (TIBO), were found to potently and selectively inhibit the replication and cell killing effects of a panel of biologically diverse laboratory and clinical strains of HIV-1. The two compounds exhibited significant activity in all human cell lines tested, as well as in fresh human peripheral blood lymphocytes and macrophages. One of these two compounds (R82913) was found to significantly inhibit the replication of a murine retrovirus (Rauscher murine leukemia virus) in both UV-XC plaque formation and virus yield reduction assays. R86183, despite differing from R82913 only in the positioning of a single chlorine molecule, was not active against the murine retrovirus but was 10-fold more potent in inhibiting HIV-1 replication. Combination antiviral assays with other reverse transcriptase inhibitors, including AZT, ddC, and carbovir, yielded synergistic anti-HIV activity with both TIBO derivatives. Additive to slightly synergistic results were obtained in combinations with ddI and phosphonoformic acid whereas additive to antagonistic activity was detected in combination with dextran sulfate.

Immunoregulatory biological response modifiers: effect of cytokines on septic shock.

Whole bacteria or bacterial components or their extracts were employed to restore or augment the immune system. Beneficial effects were attained with these agents in treating various diseases. These agents were named biological response modifiers (BRMs) because they regulated certain cellular components of the immune system. The cellular regulation induced by these BRMs was found to be due to cytokines. The cytokines were shown to act directly on the various cellular components and to provide therapeutic benefit in various autoimmune and immune deficiency diseases. Overproduction of specific cytokines however leads to a deleterious effect on the host. Overproduction of tumour necrosis factor (endotoxin, lipopolysaccharide) leads to septic shock. Bacteraemia is the leading cause of overproduction of tumour necrosis factor (TNF). Septic shock in many cases leads to death. Several monoclonal antibodies to lipopolysaccharide (LPS) and anticytokines have demonstrated protection against septic shock.

Significant anti-HIV activity of new modified polyanionic polymers in vitro.

The anti-HIV activities of two new polyanionic polymers (AM 242 and AM 612) were investigated in cell culture-based and biochemical antiviral assays. These compounds inhibited the reverse transcriptases from HIV-1 and HIV-2, using enzyme purified from virions and either a ribosomal RNA or gapped duplex DNA as the template. With the ribosomal RNA template, AM 242 and AM 612 had ID50 values of 1.1 and 0.10 micrograms/ml against the HIV-1 reverse transcriptase. In vitro cell based assays determined that both compounds significantly inhibited both the cytopathic effects associated with HIV-1 infection and the replication of virus in infected cells. AM 242 had an IC50 of approximately 1.0 micrograms/ml, while that of AM 612 was 0.19 micrograms/ml. These two active polyanionic polymers were effective in inhibiting the growth of a panel of HIV-1 isolates and were also active against HIV-2. Although the compounds were toxic at high concentration, they had antiviral activity over a wide range of nontoxic concentrations, yielding a high selectivity index. AM 612 was 100% protective for CEM cells from 320 ng/ml to 1 microgram/ml. Both compounds caused a significant increase in cellular proliferation as determined by the concentration-dependent increase in incorporation of radioactive precursors into cellular macromolecules.

Immunomodulators: current and future development and application.

It has been amply demonstrated that immunomodulators have a place in the armamentarium with other therapeutic modalities for the treatment of various diseases. They presently are, and in the future will be, most effective in preventing diseases which cause, or are the result of, immunodeficiencies. For future development the biological agents that are potent immunomodulators can be more purified and their molecular structures defined and synthesized, such as muramyldipeptides are products of BCG, etc. The active moiety of Picibanil (OK 432), a very powerful immunostimulator should be defined. Further investigations in isolating and characterizing biological agents as immunomodulators should continue in view of the success that has been achieved with BCG in treating superficial transitional cell bladder carcinoma. This mode of treatment is much less toxic to the patient than treatment with the cytotoxic agents thiotepa, mitomycin C. Chemically defined immunomodulators have been used successfully when combined with other therapeutic modalities. Levamisole and its additive therapeutic effect when combined with 5-FU in the treatment of Stage C colorectal carcinoma establishes the potential usefulness of chemicals which specifically augment the immuno response. The explosive growth of cytokine research has led to many technical advances which were key to give cloning and the availability of recombinant cytokines which have extended and modified our concepts of cytokines. The capability of cloning to provide considerable quantities of pure cytokines, permitted studies of immunological, physiological, and therapeutic roles of cytokines. All three classes of immunomodulators: biologicals; chemical; and cytokines will continue to play a major role in advancing and improving the quality of treatment of several of human as well as animal diseases.

A TIBO derivative, R82913, is a potent inhibitor of HIV-1 reverse transcriptase with heteropolymer templates.

R82913, (+)-S-4,5,6,7-tetrahydro-9-chloro-5-methyl-6-(3-methyl-2-butenyl)- imidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione (a TIBO derivative), inhibited the replication of thirteen different strains of HIV-1 in CEM cells with a median IC50 of 0.15 microM. The concentration of compound that killed 50% of the cells was much higher (46 microM), indicating that R82913 has a high selectivity index. R82913 was 20-fold more potent than AZT-TP in the inhibition of HIV-1 reverse transcriptase in an assay using a naturally occurring template (ribosomal RNA) that more accurately resembles native viral RNA than a synthetic homopolymer. With this template, R82913 inhibited HIV-1 reverse transcriptase with an ID50 (0.01 microM) that is equal to, or lower than, the IC50 for this compound in all of our cell culture assays (0.01-0.65 microM). R82913 has no effect on the replication of HIV-2 in CEM cells and does not inhibit the reverse transcriptase from this virus.

Inhibition of NK cell generation by Corynebacterium parvum.

Treatment of mice with Corynebacterium parvum (Cp) resulted in a substantial decrease of splenic NK activity associated with a reduced number of LGL. Cp also inhibited in vitro augmentation of NK cytotoxicity by IFN or IL-2 as well as generation of LAK activity. Localization experiments by using radiolabelled LGL indicated that the lower number of LGL in the spleen was not attributable to a Cp-induced alteration of LGL homing. Finally Cp was found to affect the ability of bone marrow cells to reconstitute NK activity in lethally irradiated mice, indicating that it can interfere with development of NK cells from bone marrow progenitors.

Imexon and biological response modifiers in murine models of AIDS.

The Rauscher murine leukemia retrovirus system provides an in vivo model of the human acquired immune deficiency syndrome for testing the ability of antiviral agents and biological response modifiers (BRM) to suppress viremia and retroviral disease. In the present report we examined three agents in the Rauscher retrovirus model: imexon, Ampligen and poly[I,C]-LC. Imexon reduced splenomegaly, viremia, and serum reverse transcriptase levels even when treatment was not initiated until 7 days after virus infection. Imexon also significantly prolonged the survival of infected mice. Thus it proved to be an effective antiviral agent in this system, although imexon did not completely eliminate retroviral infection in treated mice. Poly[I,C]-LC and Ampligen had immunomodulatory effects. Both of these BRM augmented the cytolytic activity of splenic natural killer (NK) cells in infected animals when treatment was initiated 24 h after infection. Poly[I,C]-LC had antiretroviral activity when administered on this schedule. In order to examine the role of NK cell augmentation in the antiviral activity of poly[I,C]-LC, we attempted to deplete NK activity by treatment with rabbit antibody to asialo GM1, a ganglioside on the surface of murine NK cells. Combined treatment of infected mice with poly[I,C]-LC and anti-asialo GM1 decreased the antiviral activity of poly[I,C]-LC. This finding suggests that NK cells may be involved in the antiviral effect of this BRM.

Effect of imexon treatment on Friend virus complex infection using genetically defined mice as a model for HIV-1 infection.

Imexon (4-imino-1,4-diazobicyclo-3.1.0-hexan-2-one) was moderately effective in the treatment of a retroviral infection in a genetically defined murine model. The animal model consisted of a Friend virus complex (FV) infection in a hybrid mouse strain, (B10.A x A/WySn)F1, which has similarities with acquired immune deficiency syndrome (AIDS). Intraperitoneal imexon initiated 1 or 3 days after FV inoculation and continued through 13 days after inoculation significantly reduced splenomegaly, splenic cell-free virus titers and viral RNA. Viral infectious centers/10(6) splenocytes and FV titers in the plasma were reduced, though not to a statistically significant level. The effect of imexon on survival was not statistically significant which suggested that the antiviral effects were only transiently effective. Phytohemagglutinin-induced blastogenesis and percent of total T cells, T helper cells and T suppressor/cytotoxic cells in the spleens were increased, and the percentage of B cells decreased by imexon treatment of both FV-infected and uninfected mice. The splenic natural killer cell activity and interleukin-1 production were not markedly affected. Virus specific neutralizing antibody developed in both imexon- and placebo-treated FV-infected mice, although titers were lower in the imexon-treated animals.

Protein deficiency reduces natural antitumor immunity.

Clinical data have shown that neoplastic diseases and/or related therapies frequently result in protein depletion of tumor-bearing patients. Depressions of acquired and specific immunity caused by protein depletion are well known. In an experimental model protein depletion was induced by lack of nutritional protein in otherwise isocaloric conditions in BALB/c and C57BL/6 mice over various time periods (max. 35 days). The results show that natural immune effector cells, natural killer cells, and monocyte/macrophages also during treatment with biological response modifiers (BRM) are depressed in their cytotoxic potentials in vitro and in vivo. Substantial and critical reductions of bone marrow cellularity (bone marrow nucleated cells) were also observed. In contrast, preliminary results show that if, following protein depletion, mice were treated parenterally with amino acids (Neo-aminomel, Boehringer-Ma. Co., FRG) complete restoration of immune parameters takes place. Adequate protein status is shown to be a crucial factor for natural immunity and therapy with BRM.

Antiproliferative activity of picolinic acid due to macrophage activation.

Activation and subsequent enhancement of cytotoxicity of mouse peritoneal macrophages (M phi) by picolinic acid (PLA) in vivo have been reported previously by the authors' group. The optimum dose was found to be 100 mg/kg. PLA-stimulated M phi lysed different tumor targets in vitro, MBL-2 lymphoma cells and Madison 109 lung carcinoma cells, with equal efficiency. Treatment with PLA was performed daily for 5 consecutive days with a dose of 100 mg/kg intraperitoneally in C57/BL mice, which previously had been inoculated with MBL-2 tumor cells. Treatment was initiated on the first day after tumor inoculation. Oral treatment with PLA (200 mg/kg) dissolved in the drinking water was also performed for 7 days. In addition, some groups received PLA treatment 1 or 2 days before tumor implantation but not afterwards, to elucidate the in vivo efficacy of M phi activation. Intraperitoneal therapy with PLA after tumor inoculation resulted in a highly significant increase in lifespan (46%); intraperitoneal pretreatment caused a significant increase (15%); orally administered PLA was without effect. Thus intraperitoneal treatment with PLA was found to have protective and therapeutic effects against the MBL-2 ascites tumor in vivo. These effects are most likely caused by macrophage activation.

Cell regulatory and immunorestorative activity of picibanil (OK432).

Picibanil (OK432), a pharmaceutical preparation of a low virulent Su strain of Streptococcus pyogenes, possesses cell regulatory activity particularly in its ability to augment natural killer (NK) cell activity and to activate macrophages to exert a tumoricidal effect both in vitro and in vivo. It is effective in retarding and/or inhibiting the growth of three different tumors: MBL-2 lymphoma, M109 alveolar adenocarcinoma, and B16 melanoma. The antitumor effect is mediated through regulation of NK cells and macrophages, possibly by its ability to stimulate the production and secretion of interferon and interleukin 1 and 2. It is a very effective adjuvant for tumor cell vaccines that elicit cytotoxic T-cell responses. Following cytoreductive chemotherapy (Cytoxan) Picibanil treatment leads to an earlier reconstitution of both bone marrow cellularity and differentiation to granulocyte-macrophage colonies.