Effect of Curcuma zedoaria crude extract against tumor progression and immunomodulation

The aim of the present work was to study the effect of the crude extract of Curcuma zedoaria on peripheral blood cells and tumor progression in C57Bl/6J mice injected with B16F10 murine melanoma cells. The intraperitoneal therapy showed a significant increase in total white and red blood cell counts, a decrease in peritoneal cell number and tumor volume reduction, whereas the oral administration revealed a noteworthy augmentation only in total leukocyte count. These results contribute to evaluate the importance of alternative treatments that employ phytotherapic compounds against tumor progression and its possible immunomodulation.

Haematopoietic effects induced in mice by the snake venom toxin jararhagin.

Venom toxins have been tested as anti-thrombotic, and anti-metastatic drugs in experimental models. The jararhagin toxin, from Bothrops jararaca snake venom, acts upon several biological processes, as inflammation, pain, platelet aggregation, etc. In this article, the systemic effects of intra-peritoneal injections of different jararhagin doses were determined in mice. About 50% significant decrease was observed in total blood leukocytes in the first (48 ng), and second (24 ng) weeks. The reduction of lymphocytes, monocytes and neutrophils accounted for this leucopoenia up to the sixth week. Significant increase in red blood cells was observed, especially on the third and fourth weeks (6 and 12 ng). A significant reduction in leukocyte infiltration was found in peritoneum (6, 12, 48 ng), whereas the infiltration was significantly increased in bronchial alveolar exudates (6 and 12 ng). The differential analysis of bone marrow cells showed significant increase, particularly of myelocytes (12 and 24 ng). These results show, at low doses, the toxin jararhagin induces red blood cells production, which is compensating the reduction of different leukocyte types. This severe leucopoenia suggests the occurrence of anti-proliferate activity or direct citotoxicity of jararhagin in the differentiation level of myeloid, and lymphoid stem precursor cells.

Antibody responsiveness during immunization and challenge of genetically modified antibody responder mice with murine hepatitis virus 3

The aim of this study was to evaluate some immunological patterns involved in natural and acquired resistance against MHV3 using the original model of genetically modified lines of mice selected for high (HIII) and low (LIII) antibody responsiveness. As previously shown, a lower pre-existing anti-MHV antibody level was found in susceptible HIII mice as compared to resistant LIII mice. Mortality rates of the F1 (H x L) hybrids and F2 and backcross segregants reflected co-dominance of both characters and the survivors had higher preexisting anti-MHV antibody titers. The present data show that both lines had the potential to synthesize antibodies and that the resistance acquired by the susceptible HIII mice paralleled the antibody synthesis. Nevertheless, higher antibody titers were necessary to confer resistance in HIII mice than in LIII ones. When compared to uvMHV3, a single immunization with a related infectious MHV strain induced a higher antibody synthesis and led the HIII mice to resist the MHV3 challenge. A direct correlation between the antibody level and resistance to infection was always observed in HIII mice. Although mounting a Th2 response as indicated by IgG1 responses, they were also able to readily synthesize large amounts of IgG2a antibodies after immunization or during infection, reflecting a Th1 response. The transfer of anti-MHV antibodies to susceptible HIII mice was capable of conferring resistance to MHV3, providing the antibodies were present before virus infection and in large amounts. The resistance and the survival time of these animals increased with the level of antibody administered. If these direct and clear data suggest that HIII mice can acquire resistance through antibodies, the basis of the resistance of the resistant LIII mice may rely on mechanisms less dependent on antibodies.

Down-regulation of Bgp1(a) viral receptor by interferon-gamma is related to the antiviral state and resistance to mouse hepatitis virus 3 infection.

Together with the evidence that the reduced virus growth and the antiviral state induced by interferon (IFN)-gamma, occurring only in macrophages from resistant animals, correlated with the decrease of MHV3 binding to macrophage membrane proteins, we show here the expression of cellular and viral genes in resistant (A/J) and susceptible (BALB/c) mouse macrophages after IFN-gamma activation/infection. The expression of interferon response gene 47 and interferon regulatory factor 1 genes takes place after IFN-gamma activation in both macrophages, indicating their activation. The expression of the biliary glycoprotein 1(a) (Bgp1(a), the main virus receptor) decreased only in IFN-gamma-activated A/J mouse macrophages, in contrast to the expression of the Bgp2 (alternative receptor), which was not influenced by IFN-gamma activation. The synthesis of both viral mRNA and virus particles was delayed only in IFN-gamma-activated A/J mouse macrophages compared with susceptible BALB/c macrophages. Besides the evidence that IFN-gamma may modulate the expression of the Bgp1(a) isoform of carcinoembryonic antigen family, these data show that IFN-gamma, which induces resistance against MHV3 infection, may be involved in the down-regulation of the main viral receptor expression, a key step forward in our understanding of the molecular basis of resistance against virus infection.

Effect of the bacillus of Calmette-Guérin, Propionibacter acnes and avridine as immunomodulators in antirabies vaccination of mice using the Fuenzalida-Palacios mouse brain vaccine.

Using the laboratory mice, Fuenzalida-Palacios mouse brain human rabies vaccine was administered in groups of animals previously inoculated with rabies virus and then submitted to treatments with the immunomodulators onco-BCG, avridine and Propionibacterium acnes. Humoral and cellular immune responses were evaluated through the macrophage inhibition factor (MIF), intra-pad inoculation (IPI) and serum neutralization (SN) tests and by the detection of gamma-interferon (IFN-gamma). The IPI test was not effective in detecting the response of delayed-type hypersensitivity, contrary to MIF, which showed the immune cellular response. Higher levels of IFN-gamma were observed in the groups of mice vaccinated and treated with avridine and P. acnes. Although immunomodulating activities have been detected, the use of adjuvants with the Fuenzalida-Palacios type vaccine in mice did not reveal any encouraging results.

Effect of bacillus of Calmette-Guérin, avridine and Propionibacterium acnes as immunomodulators on rabies in mice.

The cellular and humoral immune responses of mice inoculated with rabies virus and treated with the Bacillus of Calmette-Guérin, Avridine and Propionibacterium acnes were evaluated in this paper. There was a higher percentage of surviving mice in groups submitted to P. acnes treatment. Lower levels of interferon-gamma (IFN-gamma) were found in infected mice. The intra-pad inoculation test (IPI) was not effective to detect cellular immune response, contrary to the results found in MIF reaction. The survival of mice did not present correlation with the levels of antirabies serum neutralizing (SN) antibodies titers, IFN-gamma concentration and MIF response.

Effect of bacillus of Calmette-Guerin, avridine and Propionibacterium acnes as immunomodulators on rabies in mice

Avaliou-se a resposta imune celular e humoral de camundongos inoculados com virus rabico de rua e submetidos aos imunomoduladores Onco-BCG, avridina e Propionibacterium acnes. Os animais submetidos ao tratamento com P. acnes apresentaram um maior percentual de sobrevivencia quando comparados aos dos demais tratamentos. Foram observados menores niveis de IFN-gama nos animais infectados, sugerindo imunossupressao viral. O teste do Coxim Plantar nao foi eficaz para a deteccao da resposta de hipersensibilidade retardada na metodologia utilizada, contrariamente ao MIF. A sobrevivencia dos animais nao apresentou correlacao com os niveis de anticorpos soroneutralizantes, concentracao de IFN-gama e resposta ao MIF

Effect of vaccination and the immunomodulators "bacillus of Calmette-Guérin, avridine and Propionibacterium acnes" on rabies in mice.

Responses of vaccination and treatment to immunomodulators against rabies in mice were evaluated through macrophage inhibition factor (MIF), intra-pad inoculation (IPI) and serum neutralization (SN) tests and by the detection of gamma-interferon (IFN-gamma). Onco-BCG, Avridine and Propionibacterium acnes were administered to groups of mice. Higher survival rates were found in animals treated with P. acnes. Lower levels of IFN-gamma were observed in the groups of infected and vaccinated mice. The IPI was not effective on detecting the response of delayed-type hypersensitivity. Vaccine induced in the infected animals a more intense response to MIF reaction.

Failure of protection induced by a Brazilian vaccine against Brazilian wild rabies viruses.

This report shows that the SMB vaccine currently used in Brazil for human immunisation provides different degrees of protection in mice, depending on the rabies virus strain used as challenge. Using the NIH and Habel potency tests to evaluate the protective activity of rabies vaccine, we observed that vaccinated mice showed a higher resistance to a challenge with a fixed rabies virus (CVS-Challenge Virus Strain). The vaccine potency using the Habel or NIH tests was respectively > 6.4 (log 10) and 1.0 (Relative Potency-RP) when the fixed rabies virus was used for challenge, and from 2.9 to 4.3 (log 10) or 0.13 to 0.8 (RP) when different wild rabies viruses were used for challenge. The presence of virus neutralising antibodies (VNA) could not explain the differences of susceptibility after vaccination, since sera of vaccinated animals had similar VNA levels against both fixed and wild strains before virus challenge (respectively, 5.6 +/- 0.24 and 5.0 +/- 0.25 IU/ml of VNA against the fixed rabies virus and the 566-M strain of wild rabies virus in sera of mice vaccinated with 0.2 units of vaccine). Only cell-mediated immunity parameters correlated with the protection induced by vaccination. The IFN gamma titers found in sera and brain tissues of animals challenged with CVS strain were higher (from 36.7 +/- 5.7 to 293.3 +/- 46.2 IU/ml) than those found in mice challenged with 566-M virus strain (from 16.7 +/- 5.8 to 36.7 +/- 5.8). The proliferation index of spleen cells obtained with CVS stimulation reached a maximal value of 15.1 +/- 0.7 while spleen cells from vaccinated mice stimulated with 566-M virus failed to proliferate. The implications of these data in human protection by vaccination are discussed.

Effect of vaccination and the immunomodulators "bacillus of Calmette-Guerin, Avridine and Propionibacterium acnes" on rabies in mice

Responses of vaccination and treatment to immunomodulators against rabies in mice were evaluated through macrophage inhibition factor (MIF), intra-pad inoculation (IPI) and serum neutralization (SN) tests and by the detection of g-interferon (IFN-g). Onco-BCG, Avridine and Propionibacterium acnes were administered to groups of mice. Higher survival rates were found in animals treated with P. acnes. Lower levels of IFN-g were observed in the groups of infected and vaccinated mice. The IPI was not effective on detecting the response of delayed-type hypersensitivity. Vaccine induced in the infected animals a more intense response to MIF reaction.

Polygenic control of antibody production and correlation with vaccine induced resistance to rabies virus in high and low antibody responder mice.

The amplification of "high" (H) and "low" (L) multispecific antibody responses achieved respectively by H and L lines of selection GP represents a valuable tool in the genetic study of host-infection interactions. These lines were obtained by bidirectional selective breeding for high (HGP) or low (LGP) antibody production to natural complex antigens. HGP and LGP parental lines and reciprocal F1 hybrids, as well as their F2 segregants and backcrosses were submitted to immunization and challenge with rabies virus CVS strain. Acquired resistance was 1000-fold higher in HGP than LGP mice, with a dominance effect to low antibody production observed in F1 hybrids. An association between high antibody response and acquired resistance (P < 0.001) in F2 segregant mice was noticed. The genetic study was performed in these several populations, with a single dose of 104.5-fold LD50 CVS. We could demonstrate 3 independent loci regulating the anti-rabies antibody production, that are distinct, at least in part, from the 10 genes controlling the antigen selection response (sheep erythrocytes) of selection GP.

Interferon-gamma levels during the course of Trypanosoma cruzi infection of Calomys callosus (Rodentia-Cricetidae) and Swiss mice.

Serum levels of interferon-gamma (IFN-gamma) were evaluated in Calomys callosus and Swiss mice during the course of infection by four strains of Trypanosoma cruzi. All strains stimulated the production of this interleukine; however, the timing of its onset and permanence varied among strains and between the two animal models. When chronically infected animals with no detectable serum IFN-gamma were challenged with the homologous strain, they produced quantities comparable with those obtained during the acute phase of infection. In C. callosus there was a correlation between H2O2 liberation by peritoneal macrophages and serum IFN-gamma levels, whereas no such correlation was found in mice. C. callosus had a higher capacity to heal histopathological lesions, whereas lesions in mice were progressive. The results obtained suggest that C. callosus develops well-adapted immune mechanisms that may be important for its role as a reservoir of T. cruzi.

Specific T-cell response correlates with resistance of genetic heterogeneous mouse populations to mouse hepatitis virus 3 infection.

In a recently published study [Vassão RC, Mello IGC, Pereira CA (1994) Arch Virol 137: 277-288] we have shown that the genetically selected high antibody responder mice (HIII) are susceptible and the low antibody responder counterparts (LIII) are resistant to death induced by experimental infection with mouse hepatitis virus 3 (MHV3). This report shows that the MHV3 titers in the peritoneal exudate (PE) of HIII mice, 3 days after infection, were more than 2 log greater than in the resistant LIII mice, the interferon gamma (IFN gamma) titers in the PE of both mouse populations being not significantly different. The treatment with monoclonal antibodies (mAb) against CD4+ or CD8+ T cells induced susceptibility among LIII mice. The depletion of CD4+ T-cell subset in LIII mice was evidenced by, and led to a significant reduction in, the IFN gamma synthesis in their PEs with a 100 fold increase in MHV3 titers. When lymph node cells (LNC) were harvested from MHV3-infected mice and stimulated "in vitro" with MHV3 inactivated by ultraviolet radiation (uv-MHV3), only LNC from LIII mice were capable of proliferating and synthesizing significant amounts of interleukin 2 (IL-2). The LNC proliferation and IL-2 synthesis were inhibited by treatment with mAbs against CD4 or CD8 molecules. The MHV3 infection induced in both lines of mice a profound depression of the mitogenic response of LNC to phytohemaglutinin (PHA). A correlation between the specific T-cell response and the resistance to MHV3 infection is discussed.

Antiviral activity of interferon gamma in vivo during mouse hepatitis virus infection.

A/J mice became resistant to experimental MHV3 infection after immunization with UV-inactivated MHV3 (0% mortality, 0/10). Depletion of interferon (IFN) gamma-producing CD4+ T lymphocytes with monoclonal antibodies to CD4+ led to susceptibility to virus infection (60% of mortality, 6/10). The resistance to MHV3 infection of CD4+ T lymphocyte-depleted-A/J mice was restored by treatment with 1000 U of IFN gamma on days -1, 0, 1, 2, 3 and 4 (10% of mortality, 1/10). The low virus titers observed in resistant mice (controls or CD4+ depleted plus IFN gamma treated) were cleared 6 days after infection and the virus titers observed among susceptible mice (CD4+ depleted) increased gradually and peaked on day 6, when the animals died. Previous data, taken together with the direct evidence presented in this paper, provide strong evidence supporting the concept of an in vivo antiviral role of IFN gamma through a central action on the mechanisms of resistance to MHV3 infection.

Antiviral activity of interferon gamma in vivo during mouse hepatitis virus infection

A/J mice became resistant to experimental MHV3 infection after immunization with UV-inactivated MHV3 (0 percent mortality, 0/10). Depletion of interferon (IFN) gamma-producing CD4+ T lymphocytes with monoclonal antibodies to CD4+ led to susceptibility to virus infection (60 percent of mortality, 6/10). The resistance to MHV3 infection of CD4+ T lymphocyte-depleted-A/J mice was restored by treatment with 1000 U of IFN gamma on days -1, 0, 1, 2, 3 and 4 (10 percent of mortality, 1/10). The low virus titers observed in resistant mice (controls or CD4+ depleted plus IFN gamma treated) were cleared 6 days after infection and the virus titers observed among susceptible mice (CD4+ depleted) increased gradually and peaked on day 6, when the animals died. Previous data, taken together with the direct evidence presented in this paper, provide strong evidence supporting the concept of an in vivo antiviral role of IFN gamma through a central action on the mechanisms of resistance to MHV3 infection

Gamma-IFN and macrophage respiratory burst in Calomys callosus challenged with Trypanosoma cruzi bloodstream and metacyclic forms.

Parasitemia levels of Calomys callosus inoculated with a high dose (HBT) of 4 x 10(3) Trypanosoma cruzi strain M226 bloodstream trypomastigotes (BT) exceeded those with the same inoculum of metacyclic trypomastigotes (MT) while a similar parasitemia was obtained with a low dose (LBT) of 5 x 10(2) of BT. Serum IFN-gamma levels during the acute phase of infection were higher in the LBT inoculated group when compared with the group inoculated with HBT, while the IFN-gamma levels in MT inoculated animals were close to uninfected controls. Spontaneous liberation of H2O2 of peritoneal macrophages explanted from animals on days 21 and 28 after infection was comparable to that of controls for HBT and LBT groups while that of the MT inoculated group was significantly higher. Phorbol Myristate Acetate (PMA) stimulation resulted in high H2O2 liberation specially in the infected groups. In vitro challenge with BT suppressed the small amount of spontaneous H2O2 release, while MT challenge stimulated this release to a limited degree in infected groups. In this animal model, interacting with a parasite strain isolated from the same host, macrophage activation as measured by H2O2 release was low, while the same strain had been previously observed to result in hyperactivation of mouse macrophages. We suggest that this distinctive behavior may be due to a host-parasite adaptation.

Role of macrophages, interferon gamma and procoagulant activity in the resistance of genetic heterogeneous mouse populations to mouse hepatitis virus infection.

Genetic heterogeneous mouse populations selected for high (HIII) and low (LIII) antibody response were used to study some aspects of mouse hepatitis virus 3 (MHV3) infection, such as the resistance pattern, virus replication in the liver and peritoneal exudate or in cultured peritoneal macrophages, the interferon (IFN) synthesis in the serum and peritoneal exudate and the procoagulant activity (PCA) of the peritoneal exudate (PEC) and spleen cells (SC). The HIII mice, when compared to their LIII mice counterparts, were susceptible to MHV3 infection showing higher virus titres in the liver and peritoneal exudate, comparable IFN alpha/beta or IFN gamma titres in the peritoneal exudate or in the serum, and higher levels of PCA of PEC and SC. A higher virus titre was detected in the supernatants of HIII mouse macrophages infected with MHV3. The activation of HIII mouse macrophages with LPS, IFN alpha/beta or IFN gamma, in contrast to that of LIII mouse macrophages, did not induce an antiviral effect with partial restriction of the MHV3 replication. The LPS antiviral activity was shown to be partially exerted by IFN alpha/beta synthesis. The IFN gamma was shown to be more effective in inducing an antiviral state in LIII macrophages, when compared to IFN alpha/beta. The data obtained are consistent with the notion that the resistance mechanisms to the MHV3 infection involve the PCA and the sensitivity of macrophages to IFN.

A genetic analysis of macrophage activation and specific antibodies in relation to the resistance of heterogeneous mouse populations to MHV3 infection.

The genetically selected high antibody responder mice (HIII) are susceptible and the low antibody responder mice (LIII) are resistant to the experimental infection with Mouse Hepatitis Virus 3 (MHV3). The mortality rates of the F1 hybrids and of the F2 segregants showed the codominance of the susceptible and resistant characters. The direct individual intrapopulation correlation between the induction of antiviral state in macrophages activated by IFN gamma and the resistance to the virus infection, showed that an antiviral state could be induced in resistant mouse macrophages, whereas in susceptible mouse macrophages no restriction of virus replication could be observed. A direct inter- and intrapopulation correlation of pre-existing antibody titres against MHV3 with the mortality and a direct interpopulation correlation of those titres with the mean survival time of susceptible animals was shown. The data indicate, among the mechanisms of resistance against the virus infection, a role of IFN gamma macrophage-activation and of antibodies against MHV3 which may delay the mean survival time in susceptible animals.

TNF alpha, IL-1 and O2- release by macrophages do not correlate with the anti-mouse hepatitis virus 3 effect induced by interferon gamma.

Macrophages have been described to be important in determining the resistance of A/J mice or the susceptibility of BALB/c mice to the experimental infection with Mouse Hepatitis Virus 3 (MHV3). The interferon gamma (IFN gamma) activation of A/J and BALB/c mouse macrophages was shown to partially restrict the MHV3 replication only in macrophages from the resistant A/J mice. The activation by IFN gamma and/or infection with MHV3 showed that BALB/c mouse macrophages were capable of releasing tumor necrosis factor alpha (TNF alpha), interleukin 1 (IL-1) and anion superoxide (O2-), and A/J mouse macrophages were capable of releasing TNF alpha and IL-1 but not O2-. Comparable amounts of TNF alpha or IL-1 were released by IFN gamma-activated A/J or BALB/c mouse macrophages. Following MHV3 infection or IFN gamma activation and MHV3 infection, BALB/c mouse macrophages were always capable of releasing higher amounts of TNF alpha, IL-1 or O2- than A/J mouse macrophages, which correlated with their susceptibility to the virus infection. The data indicate that the anti-MHV3 effect induced by IFN gamma in A/J mouse macrophages is not related to the studied extrinsic activities of these cells.

Resistance of genetically selected mice to MHV3 infection is not dependent on the H2O2 release by macrophages.

The genetically selected high antibody responder line of mice (HIII) for a natural immunogen were fully susceptible to mouse hepatitis virus 3 (MHV3) and the corresponding low antibody responder mice (LIII) were fully resistant, regardless of whether they were previously treated or not with the bacillus Calmette-Guérin (BCG). The resistance or susceptibility correlated with the virus growth in the peritoneum of mice. Peritoneal cells isolated from resistant mice released higher amounts of H2O2 after phorbol myristate acetate (PMA) stimulation than susceptible mice. No spontaneous in vitro H2O2 release by peritoneal exudate cells from HIII mice, shown to be mostly macrophages, were observed during the MHV3 infection. In contrast, the spontaneous in vitro H2O2 release by these cells from MHV3-infected LIII mice increased gradually, reaching a maximal value 3 days after infection, and decreased in parallel to the virus clearance from the peritoneum. The BCG treatment primed the macrophages of HIII mice for the production of H2O2 during the MHV3 infection, but did not confer resistance against the virus infection. The data obtained suggest that the acquired capability of macrophages to release H2O2 does not participate in the anti-MHV3 activity or resistance against the MHV3 infection.