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.

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.

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.

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.

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.

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.

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.

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.

Virus specificity of the antiviral state induced by IFN gamma correlates with resistance to MHV 3 infection.

A comparative study was carried out to investigate the correlation between the antiviral effect induced in macrophages by IFN gamma and the resistance of A/J and BALB/c mice to an experimental infection of MHV 3, MHV 4, and MHVA 59. Both mouse strains were resistant to intraperitoneal infection with MHV 4 or MHVA 59 and only the A/J mice showed resistance to MHV3, the BALB/c mice being fully susceptible to this virus infection. Comparable growth kinetics, for all three viruses, were observed in both mouse strains, except for the MHV3 growth in BALB/c mice, where the virus titre increased to a peak on day 2, remaining high until day 4 when the mice died of acute hepatitis. The IFN gamma titres in the peritoneum of mice preceded and correlated with the virus growth, higher titres being found in MHV 3 infected BALB/c mice. The highest titre was always observed 24 to 48 h after infection. Among viral strains grown in cultured macrophages, higher titres were always observed in cultures infected with MHVA 59, followed by MHV 3 and the lowest those infected with MHV 4. The macrophage activation by IFN gamma-induced a partial restriction of virus growth only in MHV 3 infected A/J mouse macrophages. A virus specificity of the IFN gamma-induced antiviral state was shown to be in direct correlation with the resistance of mice to MHV 3 infection.

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.

A comparative study of resistance to MHV3 infection in genetically homogeneous and heterogeneous mouse populations.

Resistance to MHV3 infection was investigated in genetically homogeneous inbred (A/J, BALB/c) and genetically selected (High, Low) mouse lines. The A/J and L lines are resistant and the BALB/c and H mice are susceptible. The genetic analysis was performed on the F1 hybrids, as well as on the genetically heterogeneous F2 populations and backcrosses bred from HxL and A/JxBALB/c lines. The mortality rates of the F1 hybrids showed codominance of susceptibility and resistance characters. The results indicate that the same MHV3 susceptibility genes are present in isogenic and selected lines and corroborate previous results showing that at least two major genes are involved in the control of this response.

A comparative study of resistance to MHV3 infection in genetically homogeneous and heterogeneous mouse populations

Resistance to MHV3 infection was investigated in genetically homogeneous inbred (A/J, BALB/c) and genetically selected (High, Low) mouse lines. The A/J and L lines are resistant and the BALB/c and H mice are susceptible. The genetic analysis was performed on the F1 hybrids, as well as on the genetically heterogeneous F2 populations and backcrosses bred from HxL and A/JxBALB/c lines. The mortality rates of the F1 hybrids showed codominance of susceptibility and resistance characters. The results indicate that the same MHV3 susceptibility genes are present in isogenic and selected lines and corroborate previous results showing that at least two major genes are involved in the control of this response

Macrophage activity in rabies virus infection of genetically selected high and low antibody responder lines of mice.

After infection with the Pasteur strain of fixed rabies virus, the onset of disease, mortality, interferon (IFN) synthesis and interaction of the virus with macrophages were investigated in high (HI) and low (LI) antibody responder lines of mice. The HI mice were shown to be more resistant than the LI mice, and resistance was age-dependent, since mice from both mouse lines were fully susceptible up to 2 weeks of age. IFN synthesis studies of the serum indicated that, after rabies infection, HI mice produced a slightly higher amount of IFN, which was determined to be predominantly IFN-gamma. In the brains of LI mice, only IFN-alpha/beta was found, in contrast to the mixture of IFN-alpha/beta and IFN-gamma observed in the brains of HI mice. Although macrophages from the two mouse lines expressed the same degree of extrinsic activity, their intrinsic activities were quite different; the LI mice showed a greater ability to uptake and process the virus or ingest C3 (IgM) sheep red blood cells. The present findings attribute the higher antibody response and IFN-gamma synthesis observed in HI mice during rabies infection to slower processing of the rabies antigen in their macrophages, thus conferring upon them a greater ability to present it to the immune system, leading to a higher degree of resistance to rabies infection.

Macrophage activity in rabies virus infection of genetically selected high and low antibody responder lines of mice

After infection with the Pasteur strain of fixed rabies virus, the onset of disease, mortality, interferon (IFN) synthesis and interaction of the virus with macrophages were investigated in high (HI) and low (LI) antibody responder lines of mice. The HI mice were shown to be more resistant than the LI mice, and resistance was age-dependent, since mice from both mouse lines were fully susceptible up to 2 weeks of age. IFN synthesis studies of the serum indicated that, after rabies infection, HI mice produced a slightly higher amount of IFN, which was determined to be predominantly IFN-gamma. In the brains of LI mice, only IFN-alpha/beta was found, in contrast to the mixture of IFN-alpha/beta and IFN-gamma observed in the brains of HI mice. Although macrophages from the two mouse lines expressed the same degree of extrinsic activity, their intrinsic activities were quite different; the LI mice showed a greater ability to uptake and process the virus or ingest C3 (IgM) sheep red blood cells. The present findings attribute the higher antibody response and IFN-gamma synthesis observed in HI mice during rabies infection to slower processing of the rabies antigen in their macrophages, thus conferring upon them a greater ability to present it to the immune system, leading to a higher degree of resistance to rabies infection.

Phagocytic activity of human colostrum macrophages.

1. We evaluated the ability of human colostrum adhering cells to phagocytize sheep red blood cells (E) incubated with rabbit anti-E IgG antibody (A) and zymosan particles incubated with fresh human serum or with the aqueous phase of colostrum. 2. The cells were found to have considerably intense phagocytic ability, i.e., 96.8% phagocytized EA particles, 83.2% phagocytized zymosan particles opsonized with fresh human serum, and 73.3% phagocytized zymosan particles opsonized with the aqueous phase of colostrum. Thus, the aqueous phase of colostrum can opsonize zymosan particles, an activity attributed to the complement system. 3. Total hemolytic complement (CH50) and the C3 component in a pool of normal human serum were two-fold higher than in a pool of the aqueous phase of colostrum. 4. These results indicate the existence of Fc gamma and C3 receptors on the membrane of human colostrum macrophages and suggest that these cells may be biologically active.

Phagocytic activity of human colostrum macrophages

1. We evaluated the ability of human colostrum adhering cells to phagocytize sheep red blood cells (E) incubated with rabbit anti-E IgG antibody (A) and zymosan particles incubated with fresh human serum or with the aqueous phase of colestrum. 2. The cells were found to have considerably intense phagocytuc ability, i.e., 96,8% phagocytized EA parcicles, 83.2% phagocytized zymosan particles opsonized with fresh human serum, and 73.3% phagocytized zymosan particles opsonized with the aqueous phase of colostrum. Thus, the aqueous phase of colostrum can opsonize zymosan particles, an activity attributed to the complement system. 3. Total hemolytic complement (V+CH50) and the C3 component in a pool of normal human serum were two-fold higher than in a pool of the aqueous phase of colostum. 4. These results indicate the existence of Fcgama and C3 receptors on the membrane of human colostum macrophages and suggest that these cells may be biologically active