DNA damage and nitric oxide synthesis in experimentally infected Balb/c mice with Trypanosoma cruzi

This study aimed to evaluate whether experimental Chagas disease in acute phase under benznidazole therapy can cause DNA damage in peripheral blood, liver, heart, and spleen cells or induce nitric oxide synthesis in spleen cells. Twenty Balb/c mice were distributed into four groups: control (non-infected animals); Trypanosoma cruzi infected; T. cruzi infected and submitted to benznidazole therapy; and only treated with benznidazole. The results obtained with the single cell gel (comet) assay showed that T. cruzi was able induce DNA damage in heart cells of both benznidazole treated or untreated infected mice. Similarly, T. cruzi infected animals showed an increase of DNA lesions in spleen cells. Regarding nitric oxide synthesis, statistically significant differences (p<0.05) were observed in all experimental groups compared to negative control, the strongest effect observed in the T. cruzi infected group. Taken together, these results indicate that T. cruzi may increase the level of DNA damage in mice heart and spleen cells. Probably, nitric oxide plays an important role in DNA damaging whereas benznidazole was able to minimize induced T. cruzi genotoxic effects in spleen cells.     

Pharmacological evaluation of anti-leishmanial activity by in vivo nitric oxide modulation in Balb/c mice infected with Leishmania major MRHO/IR/75/ER: an Iranian strain of cutaneous leishmaniasis

Cutaneous leishmaniasis with a variation in its clinical signs is still one of the health problems in the world, region and Iran. Immune responses against leishmania consist of cytokines, immune cells and mediators. Macrophages participate actively in the inflammatory response by releasing chemokines and mediators including nitric oxide (NO) and reactive oxygen and nitrogen intermediates. This study investigates whether NO had anti-leishmanial effects and/or mediated pathology in mice infected with Leishmania major MRHO/IR/75/ER (IR/75). NO inducer lipopolysaccharide (LPS) and NO donor S-nitrosoglutathione (SNOG) were used for their ability to increase RNI and to modify leishmania infection in susceptible Balb/c mice, in order to evaluate the effects of NO production on size and lesion macroscopy, delay of lesion formation and proliferation of amastigotes inside macrophages. Liver, spleen and lymph nodes were also studied as target organs to detect amastigotes. In addition to plasma, liver and spleen suspensions were investigated for NO induction by using Griess microassay. Statistical analysis of data revealed an association between increases in NO level with the pathology of disease in Balb/c mice infected with L. major IR/75. The survival of leishmania parasite inside the macrophages and its proliferation was affected by LPS and SNOG-treatments. An inconsistent relationship was evident between the NO modulation and pathological changes in treated Balb/c mice infected with L. major IR/75.     

Immune response to Leishmania (Leishmania) chagasi infection is reduced in malnourished BALB/c mice

Protein-energy malnutrition and micronutrient deficiencies may down-regulate immune response and increase morbidity and mortality due to infection. In this study, a murine model was used to study the effects of protein, iron and zinc deficiencies on the immune response to Leishmania (Leishmania) chagasi infection. Mice were initially fed a standard diet or with a diet containing 3% casein but deficient in zinc and iron. After malnutrition was established, mice were inoculated with L. chagasi and sacrificed four weeks later in order to evaluate liver and spleen parasite loads and serum biochemical parameters. Significant decreases in liver and spleen weight, an increase in the parasite loads in these organs and decreases in serum protein and glucose concentrations in malnourished animals were observed. Furthermore, the production of interferon-gamma by spleen cells from infected malnourished mice stimulated by Leishmania antigen was significantly lower compared with that in control diet mice. These data suggest that malnutrition alters the immune response to L. chagasi infection in the BALB/c model and, in association with the effects on biochemical and anatomical parameters of the host, favored increases in the parasite loads in the spleens and livers of these animals.     

Analysis of the expression of toll-like receptors 2 and 4 and cytokine production during experimental Leishmania chagasi infection

Toll-like receptors (TLRs) recognise pathogen-derived molecules and influence immunity to control parasite infections. This study aimed to evaluate the mRNA expression of TLRs 2 and 4, the expression and production of the cytokines interleukin (IL)-12, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, IL-17, IL-10 and transforming growth factor (TGF)-β and the production of nitric oxide (NO) in the spleen of mice infected with Leishmania chagasi. It also aimed to evaluate any correlations between mRNA expression TLR2 and 4 and cytokines and NO production. Infection resulted in increased TLR2-4, IL-17, TNF-α and TGF-β mRNA expression during early infection, with decreased expression during late infection correlating with parasite load. IFN-γ and IL-12 mRNA expression decreased at the peak of parasitism. IL-10 mRNA expression increased throughout the entire time period analysed. Although TGF-β, TNF-α and IL-17 were highly produced during the initial phase of infection, IFN-γ and IL-12 exhibited high production during the final phase of infection. IL-10 and NO showed increased production throughout the evaluated time period. In the acute phase of infection, there was a positive correlation between TLR2-4, TNF-α, IL-17, NO, IL-10 and TGF-β expression and parasite load. During the chronic phase of infection, there was a positive correlation between TLR2-4, TNF-α, IL-17 and TGF-β expression and parasite load. Our data suggest that infection by L. chagasi resulted in modulation of TLRs 2 and 4 and cytokines.     

Antigenic extracts of Leishmania braziliensis and Leishmania amazonensis associated with saponin partially protects BALB/c mice against Leishmania chagasi infection by suppressing IL-10 and IL-4 production

This study evaluated two vaccine candidates for their effectiveness in protecting BALB/c mice against Leishmania chagasi infection. These immunogenic preparations were composed of Leishmania amazonensis or Leishmania braziliensis antigenic extracts in association with saponin adjuvant. Mice were given three subcutaneous doses of one of these vaccine candidates weekly for three weeks and four weeks later challenged with promastigotes of L. chagasi by intravenous injection. We observed that both vaccine candidates induced a significant reduction in the parasite load of the liver, while the L. amazonensis antigenic extract also stimulated a reduction in spleen parasite load. This protection was associated with a suppression of both interleukin (IL)-10 and IL-4 cytokines by spleen cells in response to L. chagasi antigen. No change was detected in the production of IFN-γ. Our data show that these immunogenic preparations reduce the type 2 immune response leading to the control of parasite replication.     

Does toxoplasmosis cause DNA damage? An evaluation in isogenic mice under normal diet or dietary restriction

Toxoplasmosis is an anthropozoonotic widespread disease, caused by the coccidian protozoan parasite Toxoplasma gondii. Since there are no data regarding the genotoxicity of the parasite in vivo, this study was designed to evaluate the genotoxic potential of the toxoplasmosis on isogenic mice with normal diet or under dietary restriction and submitted to a treatment with sulfonamide (375 microg/kg per day). DNA damage was assessed in peripheral blood, liver and brain cells using the comet assay (tail moment). The results for leucocytes showed increases in the mean tail moment in mice under dietary restriction; in infected mice under normal diet; in infected, sulfonamide-treated mice under normal diet; in infected mice under dietary restriction and in infected sulfonamide-treated mice under dietary restriction. In liver and brain cells, no statistically significant difference was observed for the tail moment. These results indicated that dietary restriction and T. gondii were able to induce DNA damage in peripheral blood cells, as detected by the comet assay.     

Leishmanicidal activity of the Agaricus blazei Murill in different Leishmania species

Leishmaniasis is a major public health problem, and the alarming spread of parasite resistance underlines the importance of discovering new therapeutic products. The present study aims to investigate the in vitro leishmanicidal activity of an Agaricus blazei Murill mushroom extract as compared to different Leishmania species and stages. The water extract proved to be effective against promastigote and amastigote-like stages of Leishmania amazonensis, L. chagasi, and L. major, with IC(50) (50% inhibitory concentration) values of 67.5, 65.8, and 56.8 μg/mL for promastigotes, and 115.4, 112.3, and 108.4 μg/mL for amastigotes-like respectively. The infectivity of the three Leishmania species before and after treatment with the water extract was analyzed, and it could be observed that 82%, 57%, and 73% of the macrophages were infected with L. amazonensis, L. major, and L. chagasi, respectively. However, when parasites were pre-incubated with the water extract, and later used to infect macrophages, they were able to infect only 12.7%, 24.5%, and 19.7% of the phagocytic cells for L. amazonensis, L. chagasi, and L. major, respectively. In other experiments, macrophages were infected with L. amazonensis, L. chagasi, or L. major, and later treated with the aforementioned extract, presented reductions of 84.4%, 79.6%, and 85.3% in the parasite burden after treatment. A confocal microscopy revealed the loss of the viability of the parasites within the infected macrophages after treatment with the water extract. The applied extract presented a low cytotoxicity in murine macrophages and a null hemolytic activity in type O(+) human red blood cells. No nitric oxide (NO) production, nor inducible nitric oxide syntase expression, could be observed in macrophages after stimulation with the water extract, suggesting that biological activity may be due to direct mechanisms other than macrophage activation by means of NO production. In conclusion, the results demonstrate that the A. blazei Murill water extract can potentially be used as a therapeutic alternative on its own, or in association with other drugs, to treat Visceral and Cutaneous Leishmaniasis.     

In vivo inhibition of inducible nitric oxide and evaluation of the brain tissue damage induced by Toxocara canis larvae in experimentally infected mice

Nitric oxide (NO) is known to be produced by macrophages, endothelial cells and neurons and synthesized by an enzyme called nitric oxide synthase (NOS). Various effector mechanisms and infections can affect the NO production. Excessive amount of NO will lead to biochemical reactions, which cause toxic effects. In this study the role of NO has been evaluated in larval toxocarosis, which is a systemic parasite infection caused by T. canis larvae. Infection was established in the Balb/c mice with or without inducible NOS (iNOS) inhibition and the effects of infection and NOS inhibition were observed according to the results of SOD and LPx measurements in brain tissue and NADPH-diaphorase (NADP-d) histochemistry. Results of NADPH-d histochemistry indicate that iNOS inhibition has protective effect on the brains of infected mice and that larval T. canis infection could be related to oxidative stress, and NO production and iNOS inhibition can protect the tissue from damage in this infection.     

Nitric oxide production by murine spleen cells stimulated with lipopolysaccharide from Actinobacillus actinomycetemcomitans

The aim of this study was to determine whether Actinobacillus actinomycetemcomitans lipopolysaccharide (LPS-A. actinomycetemcomitans) could induce murine spleen cells to produce nitric oxide (NO). Spleen cells derived from Balb/c mice were stimulated with LPS-A. actinomycetemcomitans or LPS from Escherichia coli for 4 days. The effects of N(G)-monomethyl-L-arginine (NMMA), polymyxin B, and cytokines (IFN-gamma and IL-4) on the production of NO were also assessed. The NO production from the carrageenan-treated spleen cells stimulated with LPS-A. actinomycetemcomitans or both LPS-A. actinomycetemcomitans and IFN-gamma was determined. The carrageenan-treated mice were transferred with splenic macrophages and the NO production was assessed from the spleen cells stimulated with LPS-A. actinomycetemcomitans or LPS-A. actinomycetemcomitans and IFN-gamma. The results showed that NO production was detectable in the cultures of spleen cells stimulated with LPS-A. actinomycetemcomitans in a dose-dependent fashion, but was lower than in the cells stimulated with LPS from E. coli. The NO production was blocked by NMMA and polymyxin B. IFN-gamma up-regulated but IL-4 suppressed the production of NO by the spleen cells stimulated with LPS-A. actinomycetemcomitans. The carrageenan-treated spleen cells failed to produce NO after stimulation with LPS-A. actinomycetemcomitans or both LPS-A. actinomycetemcomitans and IFN-gamma. Adoptive transfer of splenic macrophages to the carrageenan-treated mice could restore the ability of the spleen cells to produce NO. The results of the present study suggest that LPS-A. actinomycetemcomitans under the regulatory control of cytokines induces murine spleen cells to produce NO and that splenic macrophages are the cellular source of the NO production. Therefore, these results may support the view that NO production by LPS-A. actinomycetemcomitans-stimulated macrophages may play a role in the course of periodontal diseases.     

Oxidative stress of liver in hamsters infected with Leishmania (L.) chagasi

Oxidative stress as a mediator of hepatic tissue damage concurrent with Leishmania (L.) chagasi infection was investigated. Chemiluminescence in liver supernatant of hamsters infected with Leishmania (L.) chagasi showed a ratio of 1.53/ mg protein and 2.10/liver weight 90 days after infection when compared with the control. The malondialdehyde (MDA) levels also increased significantly both with and without addition of Fe3+/ascorbic acid in the reaction mixture, with a ratio of 2.12 and 1.55/mg protein or 2.91 and 2.12/liver weight, respectively. The parasite burden in the spleen, as a measure of infection severity, was 9.1+/-1.33 x 10(8) parasites/organ. On the 10th day of infection, the chemiluminescence also was significantly higher in infected hamsters than in the controls (ratio = 1.36/mg protein or 1.34/liver weight); however, the MDA levels were not different from those of controls. After 90 days of infection, significant correlations were observed between chemiluminescence and MDA concentration with and without the presence of Fe3+/ascorbic acid (r = 0.54, P = 0.0001; r = 0.56, P = 0.0001; respectively). The high infection/control ratio of both chemiluminescence and MDA concentration and the significant correlation between those events strongly indicate the occurrence of oxidative stress and lipid peroxidation as a mechanism of liver damage in cases of chronic infection by L. chagasi. The significant increase in chemiluminescence at 10 days of infection demonstrates that oxidative stress occurs very early, first consuming the antioxidants and then inducing lipid peroxidative damage later in the chronic stage of this disease.     

Changes in T-cell subpopulations in mice during prolonged experimental secondary infection withEchinococcus granulosus

Balb/c mice were infected intraperitoneally with protoscoleces ofEchinococcus granulosus. After 15 months of infection, and by means of flow cytometry, the expression of T-cell markers CD3, CD4, and CDS on T cells from peripheral blood, spleen, and thymus was analyzed and compared with that of age-matched controls. Infected mice had higher percentages of CD3+, and CD4+ cells in peripheral blood, and higher percentages of CD8+ cells in the spleen, when compared with control mice. CD4+ and CD8+ cells in peripheral blood and CD8+ cells in thymus also showed higher percentages of expression of interleukin-2 receptor. The results infer a role for interleukin-2 in experimental secondary echinococcosis.     

Prostaglandins mediate suppression of lymphocyte proliferation and cytokine synthesis in acute Trypanosoma cruzi infection

Suppression of host lymphoproliferative responses to mitogens and Ag is characteristically seen during acute infection with the protozoan parasite Trypanosoma cruzi. We investigated the reciprocal regulation of prostaglandins (PG), TNF-alpha, and nitric oxide (NO) production and their effects on cytokine production and lymphoproliferative responses to parasite Ag and to Con A by spleen cells (SC) from T.-cruzi-infected mice. Large amounts of PGE2, TNF-alpha, and NO were produced during infection. TNF-alpha stimulated PG and NO synthesis, while both mediators inhibited TNF-alpha synthesis. Blocking PG also reduced NO synthesis indicating that PG stimulate NO production. Treatment with indomethacin or NMLA stimulated lymphoproliferation on days 6 and 22 of infection; on day 14, when suppression of proliferation and NO production was maximal, combined inhibition of NO and PG production restored parasite Ag specific and Con A proliferative responses. Blocking PG or NO production increased IL-2, IFN-gamma, and TNF-alpha, but not IL-12 production by SC; IL-10 levels were not reduced. Indomethacin-treated infected mice had higher mortality compared to untreated infected animals. The data indicate that PG, together with NO and TNF-alpha, participate in a complex circuit that controls lymphoproliferative and cytokine responses in T. cruzi infection.     

Plasmodium berghei: the influence of blood volume changes on the malaria-induced anemia in Balb/C mice

Malaria-induced anemia exceeds that attributable to direct parasite destruction of erythrocytes. Since spleen and liver weights increase significantly, hemodilution may account for part of this excessive anemia. To determine the role of hemodilution in the etiology of anemia, vascular volumes were measured with Evans blue and isotope dilution techniques. The Evans blue dilution technique showed that blood volume increased 20%, in infected Balb/C mice. However, when blood volume was measured with Evans blue bound to protein prior to injection or with iodinated albumin and chromium-labelled red blood cells no significant change was detected. Evidently the permeability of the vasculature and/or erythrocytes of infected mice was increased so that injected Evans blue occupied a space larger than the vascular volume before becoming bound to plasma proteins. We conclude that hemodilution is not involved in the excessive anemia of Plasmodium berghei-infected Balb/C mice.     

BCG expressing LCR1 of Leishmania chagasi induces protective immunity in susceptible mice

Cellular immune responses are required for protective immunity against Leishmania chagasi. Immunization strategies using live intracellular bacteria (e.g., bacille-Calmette Guerin strain of Mycobacterium bovis) expressing recombinant antigens can induce cellular immune responses to these antigens. Previous studies demonstrated that the L. chagasi antigen LCR1 stimulates IFN-gamma production from T cells of infected BALB/c mice, and immunization with recombinant LCR1 partially protects against L. chagasi infection. To determine whether live bacteria could enhance the immunization potential of LCR1, we engineered BCG expressing LCR1 (BCG-LCR1). Subcutaneous immunization with BCG-LCR1, but not with BCG containing plasmid only (BCG-pMV261), elicited better protective immunity against L. chagasi infection than LCR1 protein alone. BCG-LCR1 administered intraperitoneally did not protect. Splenocytes from mice immunized s.c. with either BCG-LCR1 or BCG-pMV261 and then infected with L. chagasi promastigotes had increased antigen-induced IFN-gamma and reduced IL-10 production compared to splenocytes of control mice. We propose that BCG-LCR1 promotes a Th1-type protective immune response, and it may be a useful component of a Leishmania vaccine.     

Leishmania amazonensis infection does not inhibit systemic nitric oxide levels elicited by lipopolysaccharide in vivo

Leishmaniasis is a parasitic disease that leads to chronic inflammation. Macrophages, depending on their activation state, are either hosts or killers of the parasites. Downregulation of nitric oxide (NO) synthesis by the parasite infecting the macrophages has been proposed to be an important evading mechanism based on in vitro studies. We confirmed inhibition of NO release by macrophages infected with Leishmania amazonensis in vitro. To examine the role of the parasite in regulating NO production in vivo, we monitored systemic NO levels elicited by challenging naive and L. amazonensis-infected BALB/c mice with lipopolysaccharide (LPS). Animals were challenged after 1, 2, 6, and 9 wk of infection. NO production was monitored by electron paramagnetic resonance spectroscopy as the levels of hemoglobin nitrosyl complexes (HbNO) present in the animal's blood. No significant differences in HbNO levels were observed between LPS-treated naive and inoculated mice at any time during infection. To control for increased macrophage numbers in infected mice, naive mice were injected with a macrophage cell line before LPS challenge; this treatment did not increase produced NO levels. The results argue against a major role for the parasite in downregulating NO production in vivo.     

Immune responses to T-dependent and T-independent antigens during visceral leishmaniasis in mice: evidence for altered T-cell regulation of immune responses to non-parasite antigens

Antibody responses to T-dependent and T-"independent" antigens were studied in disease-susceptible (BALB/c and C57BL/10) and disease-resistant (A/J) mice infected with Leishmania donovani chagasi. Disease-susceptible mice but not disease-resistant mice showed a transient decrease in PFC responses to TNP on a T-dependent carrier (BGG) during the period of 4-8 weeks after infection. Infected disease-susceptible animals also showed increased responses to TNP on a type II T-independent carrier (Ficoll), which persisted until at least 14 weeks after infection. The increased responses were associated with a significant increase in anti-TNP antibody of the IgG2b subclass. When T-enriched spleen cells from infected mice and B-enriched spleen cells from uninfected mice were transferred to irradiated recipients immunized with TNP-Ficoll, increased anti-TNP PFC were observed over numbers seen in irradiated recipients which received both B and T cells from uninfected mice. Increased responses to TNP-Ficoll were also induced by prior administration of soluble leishmania extract in CFA. Infected mice immunized with TNP-LPS, a T-independent type I antigen, also had increased anti-TNP antibody responses, but had normal anti-LPS antibody responses. The elevated antibody production which occurred in response to the T-"independent" antigens could not be attributed to the relatively low polyclonal response which occurred in both disease-resistant and disease-susceptible mice infected with L. donovani chagasi. The observations are consistent with leishmania induced, transient alterations in some T-cell functions including response to haptens on T-dependent carriers, and a lack of down regulation of T-"independent" responses. Subtle lesions in immunoregulation may be important correlates of successful protozoal infection and may be responsible for some of the immunologic manifestations of the disease.     

Nematospiroides dubius: passive transfer of protective immunity to mice with monoclonal antibodies

Nine hybridoma cell lines secreting monoclonal antibodies specific for Nematospiroides dubius were produced by fusion of the mouse myeloma cell line NS-1 to either spleen cells or mesenteric lymph node cells from mice repeatedly infected with N. dubius. Seven of the antibodies were identified as IgM and two as IgG1. Each monoclonal antibody bound to polypeptide epitopes on both infective larvae (L3) and adult worms. However, five antibodies bound preferentially to L3 and three to adult worms. All nine antibodies reacted with high molecular weight protein antigens. Passive protective immunity in Balb/c mice was demonstrated with monoclonal antibodies Nd2 and Nd3 in ascites fluid which stunted both male and female worms and reduced parasite fecundity.     

CTLA-4 blockage increases resistance to infection with the intracellular protozoan Trypanosoma cruzi

Recent studies have revealed an important role for CTLA-4 as a negative regulator of T cell activation. In the present study, we evaluated the importance of CTLA-4 to the immune response against the intracellular protozoan, Trypanosoma cruzi, the causative agent of Chagas' disease. We observed that the expression of CTLA-4 in spleen cells from naive mice cultured in the presence of live trypomastigote forms of T. cruzi increases over time of exposure. Furthermore, spleen cells harvested from recently infected mice showed a significant increase in the expression of CTLA-4 when compared with spleen cells from noninfected mice. Blockage of CTLA-4 in vitro and/or in vivo did not restore the lymphoproliferative response decreased during the acute phase of infection, but it resulted in a significant increase of NO production in vivo and in vitro. Moreover, the production of IFN-gamma in response to parasite Ags was significantly increased in spleen cells from anti-CTLA-4-treated infected mice when compared with the production found in cells from IgG-treated infected mice. CTLA-4 blockade in vivo also resulted in increased resistance to infection with the Y and Colombian strains of T. cruzi. Taken together these results indicate that CTLA-4 engagement is implicated in the modulation of the immune response against T. cruzi by acting in the mechanisms that control IFN-gamma and NO production during the acute phase of the infection.     

Experimental infection parameters in Galea spixii (Rodentia: Caviidae) with Leishmania infantum chagasi

In order to better understand the epidemiological transmission network of leishmaniasis, an endemic disease in Northeast Brazil, we investigated the susceptibility of Spix yellow-toothed cavies (Galea spixii) to the Leishmania infantum chagasi parasite. Nine cavies were experimentally infected, separated into three groups and monitored at 30, 90 and 180 days, respectively. Amastigotes were identified in the spleen slides of two cavies killed 180 days after infection. Antibodies against the L. i. chagasi were identified in one of the cavies. This demonstrates that G. spixii is in fact capable of maintaining a stable infection by L. i. chagasi without alterations in biochemical and hematological parameters of the host and without perceivable micro and macroscopic lesions.