Leishmania donovani recombinant iron superoxide dismutase B1 protein in the presence of TLR-based adjuvants induces partial protection of BALB/c mice against Leishmania major infection

In this study, we tested the protective efficacy of recombinant Leishmania donovani iron superoxide dismutase B1 (SODB1) against Leishmania major infection in BALB/c mice. Mice were challenged with L. major 3weeks after the second boost immunization with rSODB1 alone or in the presence of adjuvants. Injection of BALB/c mice with rSODB1 alone elicited both humoral and cellular immune responses. Administration of rSODB1 with CpG ODN or GLA-SE (a synthetic toll-like receptor 4 agonist) adjuvant resulted in the induction of anti-SODB1 IgG1, and more importantly of significantly high levels of IgG2a isotype. Immunization of mice with rSODB1 alone or with adjuvant induced the production of IFN-γ by splenocytes in response to stimulation with L. major soluble leishmanial antigens (SLA). Moreover, immunization protocols involving rSODB1 resulted in a significant decrease in IL-10 as compared to controls. The presence of CpG ODN or GLA-SE adjuvant in the immunization protocols resulted in a relative increase in IFN-γ in response to stimulation with rSODB1 in comparison to immunization with rSODB1 alone. Mice immunized with rSODB1 plus CpG ODN or GLA-SE, were able to partially control their Leishmania infections, as indicated by the reduction in footpad swelling and parasite numbers, compared to controls. These results suggest that immunization with recombinant SODB1 protein together with CpG ODN or GLA-SE can be potential vaccine candidate against leishmaniasis.     

Leishmania chagasi: effect of the iron deficiency on the infection in BALB/c mice

Iron deficiency and visceral leishmaniasis are serious problems of public health. The aim of this study was to evaluate the effect of iron deficiency, induced by the iron chelator desferrioxamine, on the course of the infection by Leishmania chagasi in BALB/c mice. Our data show that the iron chelator caused significant reduction in hemoglobin concentration of treated mice and reduction in parasite load in spleen and liver. Significant differences were not observed in the production of IFN-gamma and IL-4 among the experimental groups. In conclusion, the data reported in this paper suggest that iron deficiency may favor the host. If there is not enough iron available to the parasite, its multiplication may be reduced and infection attenuated.     

Immunogenicity and protective efficacy of recombinant iron superoxide dismutase protein from Bordetella pertussis in mice models

Whooping cough (pertussis) is a highly contagious respiratory infection caused by Bordetella pertussis. Although availability of effective pertussis vaccines reportedly decreases the incidence of the disease, B. pertussis circulation in populations has not been eliminated. Thus, it is necessary to find new protein candidates with greater immune protective capacities than the currently available acellular pertussis vaccines. In this study, iron superoxide dismutase (FeSOD) gene (sodB) was cloned, expressed in Escherichia coli and recombinant FeSOD protein thence purified. The recombinant protein (rFeSOD) was formulated with aluminum hydroxide (Alum) or monophosphoryl lipid A (MPLA) and injected intraperitoneally to immunize mice, after which IgG1, IgG2a and IFN‐γ titers were measured to assess humoral and cellular responses, respectively, to these immunizations. The extent of bacterial colonization in lungs of intranasally challenged mice was determined 5, 8 and 14 days post‐challenge. IgG1 and IgG2a responses were significantly stronger in mice that had been immunized with rFeSOD–MPLA than in those that had received rFeSOD‐Alum (P < 0.05). Additionally, IgG2a titers were higher in mice vaccinated with recombinant protein FeSOD (rFeSOD) formulated with MPLA, especially after the second immunization. Immunization with rFeSOD–MPLA also provided a modest, but significant decrease in bacterial counts in lungs of mice (P < 0.05). Antigen specific‐IFN‐γ responses were significantly stronger in the group vaccinated with rFeSOD–MPLA, which could account for the lower bacterial counts. These findings suggest that rFeSOD protein formulated with MPLA has potential as an acellular pertussis vaccine candidate component.     

Antigenicity of Mycobacterium paratuberculosis superoxide dismutase in mice

Mycobacterium paratuberculosis (MPT) is the etiologic agent of paratuberculosis. The disease is prevalent in cattle worldwide, and exacts a heavy financial toll. Effective control requires the development of acellular vaccines offering a better protection than the current available vaccines without side effects and allowing the discrimination between infected and vaccinated animals. We studied the immune response of mice to the MPT superoxide dismutase (SOD) alone or adjuvanted by Ribi. We cloned, overexpressed and purified this antigen in Escherichia coli. Spleen cells from immunized mice, after exposure to recombinant MPT SOD (MPT rSOD), produced significant levels of IFNgamma, TNFalpha and IL-6. IFNgamma and TNFalpha production was increased by the addition of Ribi. In contrast, low levels of NO, IL-4 and IL-10 were secreted by spleen cells culture from immunized mice. The immunoglobulin isotype distribution analysis showed that Ribi adjuvant clearly induced a significantly higher anti-MPT rSOD antibody production of all classes tested and decreased the IgG1/IgG2a ratio thus improving the Th1 response. Delayed-type hypersensitivity responses in mice footpads were observed only in mice immunized with MPT rSOD emulsified in Ribi. Vaccination of MPT rSOD emulsified with Ribi induced both a Th2 and Th1 type of immune response with the later slightly more pronounced. The results presented here on the immunogenicity of MPT SOD suggest that this antigen should be further tested as a candidate antigen for a future acellular vaccine against paratuberculosis.     

Generation of superoxide radicals in alkaline solutions of hydrogen peroxide and the effect of superoxide dismutase on this system

Superoxide radicals in high concentrations were generated from alkaline H₂O₂ without using catalysts or irradiation. The dependence of the intensity and parameters of the superoxide radical EPR spectrum on pH, temperature, viscosity and H₂O₂ concentration were studied. The observed changes are explained on the base of matrix effects. The addition of superoxide dismutase to alkaline H₂O₂ led initially to a drop in the EPR spectrum intensity, followed by an increase in the concentration of superoxide radicals.     

Macrophage subsets harbouring Leishmania donovani in spleens of infected BALB/c mice: localization and characterization

The purpose of the current study was to characterize parasite-containing cells located in spleens of BALB/c mice infected with Leishmania donovani. In particular, expression of MHC class II molecules by these cells was examined to determine whether they could potentially act as cells capable of immunostimulating Leishmania-reactive CD4+ T lymphocytes. To this end, an immunohistological analysis of spleens taken at various time points after infection was undertaken. Using this approach, we observed, in the red pulp, the formation of small cellular infliltrates containing heavily infected macrophages that could be stained with the monoclonal antibodies MOMA-2 and FA/11. All of them expressed high levels of MHC class II molecules. Parasites were also detected in the white pulp, especially in MOMA-2+, FA/11+ and MHC class II+ macrophages of the periarteriolar lymphocyte sheath and in MOMA-2+ marginal zone macrophages. Infected cells were further characterized by fluorescence microscopy after their enrichment by adherence. All infected mononuclear cells recovered by this procedure could be stained with MOMA-2 and FA/11 and thus very probably belonged to the mononuclear phagocyte lineage. Furthermore, all of them strongly expressed both MHC class II as well as H-2M molecules, regardless of the time points after infection. Analysis of the parasitophorous vacuoles (PV) by confocal microscopy showed that these compartments were surrounded by a membrane enriched in lysosomal glycoproteins lamp-1 and lamp-2, in macrosialin (a membrane protein of prelysosomes recognized by FA/11) and in MOMA-2 antigen. About 80% of the PV also had MHC class II and H-2M molecules on their membrane. Altogether, these data indicate that in the spleens of L. donovani-infected mice, a high percentage of amastigotes are located in macrophages expressing MHC class II molecules and that they live in PV exhibiting properties similar to those of PV detected in mouse bone marrow-derived macrophages exposed to a low dose of interferon gamma (IFN-gamma) and infected in vitro.     

Effect of temperature and htpR on the biosynthesis of superoxide dismutase in Escherichia coli

The synthesis of Mn- and FeSODs in response to temperature changes was examined in strains of Escherichia coli with different mutations in sod and htpR genes. Growth at or shift to elevated temperatures induced FeSOD but not MnSOD. The induction of FeSOD by heat was inhibited by chloramphenicol and was independent of the heat shock (htpR-controlled) regulon. FeSOD was more stable at 42 degrees C than was MnSOD.     

Antileishmanial effect of free and encapsulated sinefungin against Leishmania donovani infections in BALB/c mice

Sinefungin, a C-nucleoside antibiotic, displays a high antiparasitic action. The effect of free and microencapsulated sinefungin was compared on BALB/c mice infected with Leishmania donovani; the encapsulated form proved more effective by one order of magnitude.     

Expression of manganese superoxide dismutase is not altered in transgenic mice with elevated level of copper-zinc superoxide dismutase

In evaluating the relative expression of copper-zinc and manganese superoxide dismutase (CuZnSOD and MnSOD) in vivo in states like Down syndrome in which one dismutase is present at increased levels, we measured activities of both enzymes, in tissues of control and transgenic mice constitutively expressing increased levels of CuZnSOD, during exposure to normal and elevated oxygen tensions. Using SOD gel electrophoresis assay, CuZnSOD and MnSOD activities of brain, lung, heart, kidney, and liver from mice exposed to either normal (21%) or elevated (>99% oxygen, 630 torr) oxygen tensions for 120 h were compared. Whereas CuZnSOD activity was elevated in tissues of transgenic relative to control mice under both normoxic or hyperoxic conditions, MnSOD activities in organs of transgenic mice were remarkably similar to those of controls under both conditions. To confirm the accuracy of this method in quantitating MnSOD relative to CuZnSOD expression, two other methods were utilized. In lung, which is the organ exposed to the highest oxygen tension during ambient hyperoxia, a sensitive, specific ELISA for MnSOD was used. Again, MnSOD protein was not different in transgenic relative to control mice during exposure to air or hyperoxia. In addition, lung MnSOD protein was not changed significantly by exposure to hyperoxia in either group. In kidney, a mitochondrion-rich organ, SOD assay, before and after inactivation of CuZnSOD with diethyldithiocarbamate, was used. MnSOD activity was not different in organs from air-exposed transgenic relative to control mice. The data indicated that expression of MnSOD in vivo was not affected by overexpression of the CuZnSOD and, therefore, the two enzymes are probably regulated independently.     

Protective immunity against Leishmania major induced by Leishmania tropica infection of BALB/c mice

Leishmania (L.) tropica is a causative agent of human cutaneous and viscerotropic leishmaniasis. Immune response to L. tropica in humans and experimental animals are not well understood. We previously established that L. tropica infection induces partial protective immunity against subsequent challenge infection with Leishmania major in BALB/c mice. Aim of the present study was to study immunologic mechanisms of protective immunity induced by L. tropica infection, as a live parasite vaccine, in BALB/c mouse model. Mice were infected by L. tropica, and after establishment of the infection, they were challenged by L. major. Our findings shows that L. tropica infection resulted in protection against L. major challenge in BALB/c mice and this protective immunity is associated with: (1) a DTH response, (2) higher IFN-γ and lower IL-10 response at one week post-challenge, (3) lower percentage of CD4⁺ lymphocyte at one month post-challenge, and (4) the source of IFN-γ and IL-10 were mainly CD4⁻ lymphocyte up to one month post-challenge suggesting that CD4⁻ lymphocytes may be responsible for protection induced by L. tropica infection in the studied intervals.     

Use of superoxide dismutase and catalase producing lactic acid bacteria in TNBS induced Crohn's disease in mice

Reactive oxygen species are involved in various aspects of intestinal inflammation and tumor development. Decreasing their levels using antioxidant enzymes, such as catalase (CAT) or superoxide dismutase (SOD) could therefore be useful in the prevention of certain diseases. Lactic acid bacteria (LAB) are ideal candidates to deliver these enzymes in the gut. In this study, the anti-inflammatory effects of CAT or SOD producing LAB were evaluated using a trinitrobenzenesulfonic acid (TNBS) induced Crohn's disease murine model. Engineered Lactobacillus casei BL23 strains producing either CAT or SOD, or the native strain were given to mice before and after intrarectal administration of TNBS. Animal survival, live weight, intestinal morphology and histology, enzymatic activities, microbial translocation to the liver and cytokines released in the intestinal fluid were evaluated. The mice that received CAT or SOD-producing LAB showed a faster recovery of initial weight loss, increased enzymatic activities in the gut and lesser extent of intestinal inflammation compared to animals that received the wild-type strain or those that did not receive bacterial supplementation. Our findings suggest that genetically engineered LAB that produce antioxidant enzymes could be used to prevent or decrease the severity of certain intestinal pathologies.     

Leishmania aethiopica: strain identification and characterization of superoxide dismutase-B genes

This study was performed to characterize the genes that code for superoxide dismutase (SOD) in Leishmania aethiopica. It involved three main steps: specimen collection and parasite isolation, species identification, and molecular characterization of the SOD genes. Out of 20 skin slit specimens cultured and processed from suspected cutaneous leishmaniasis patients enrolled in the study, five (25%) were found to be positive for motile promastigotes. Isoenzyme electrophoresis and PCR-RFLP results confirmed that the isolates were L. aethiopica. Superoxide dismutase-B (SODB) genes were identified from L. aethiopica for the first time. Iron superoxide dismutase-B genes amplified from promastigotes of L. aethiopica (LaeFeSODB) were similar in size to the SODB genes of other Leishmania species. Nucleotide sequences of LaeFeSODB1 showed 95.4, 93.5, and 97.3% identity with L. donovani SODB1 (LdFeSODB1) L. major SODB1 (LmFeSODB1) and L. tropica SODB1 (LtrFeSODB1), respectively. Similarly, LaeFeSODB2 showed 95.9 and 94.1 and 97.6% identity with LdFeSODB2 and LmFeSODB2 and LtrFeSODB2, respectively. On the other hand, predicted amino acid sequence comparison indicated that LaeFeSODB1 had 91.3, 89.8, and 93.9% identity with LdFeSODB1, LmFeSODB1, and LtrFeSODB1, respectively. The difference in nucleic acid sequence of LaeFeSODB from that of LmFeSODB and LtrFeSODB can be utilized to develop specific molecular methods that help differentiate these species in places where there is an overlap in the distribution of these species. In addition, the data provide information about the situation of L. aethiopica with respect to SODB genes.     

Identification of iron Superoxide dismutase and a copper/zinc Superoxide dismutase enzyme activity within the marine cyanobacterium Synechococcus sp. WH 7803

Abstract Three constitutive forms of Superoxide dismutase activity have been demonstrated in the cyanobacterial marine picoplankter Synechococcus sp. WH 7803 using polyacrylamide gel activity staining techniques. A protein which gave a positive non-haem iron stain on native polyacrylamide gels exhibited N-terminal similarity to both the iron Superoxide dismutase and the manganese Superoxide dismutase of Escherichia coli . The metal prosthetic group of each of the three activity bands was characterised by analysing their differential sensitivities to 5 mM H₂O₂, 2 mM cyanide and 2 mM of the copper chelator diethyldithiocarbamate. Three distinct Superoxide dismutase activities were observed, an iron Superoxide dismutase, a copper/zinc Superoxide dismutase and a third form which has not been identified. Growth of Synechococcus cells in ASW medium containing no added iron resulted in no alteration in the activity of the iron Superoxide dismutase. Growth of cultures in the absence of copper or zinc resulted in differential changes in the activities of the copper/zinc Superoxide dismutase and the unidentified Superoxide dismutase.     

Cultivation of Leishmania donovani and Leishmania braziliensis in Defined Media: Nutritional Requirements

SYNOPSIS Nutritional requirements of promastigotes of Leishmania donovani and Leishmania braziliensis were studied in modifications of a simple defined culture medium. "Continuous growth," considered as propagation through 10 successive passages, was supported by inorganic salts, 14 l -amino acids (arginine, cysteine, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, valine), glucose, adenosine, and a mixture of 11 vitamins and related growth factors. Purified defatted bovine serum albumin proved beneficial. The nutritional needs of the above species of Leishmania differ from those of 2 other hemoflagellate species, Leishmania tarentolae and Crithidia fasciculata , for which glucose, proline and glutamine were found to be nonessential. It is suggested that lower hemoflagellates may be capable of synthesizing these substrates de novo. Leishmania donovani and L. braziliensis required higher levels of folic acid than L. tarentolae , probably due to the fact that folates are involved as cofactors in the biosyntheses of pyrimidines and serine. Although the mixtures reported here cannot be regarded as "minimal essential" media, they are considerably less complex than the ones employed so far for cultivating hemoflagellates, and are therefore well suited for studies related to nutrition and biosynthetic capabilities of Trypanosomatids.     

Expression of the Cu,Zn superoxide dismutase of Aspergillus fumigatus as determined by immunochemistry and immunoelectron microscopy

Abstract A polyclonal antibody against purified Cu,Zn superoxide dismutase (SOD) from the pathogen Aspergillus fumigatus was raised in a sheep. This antibody recognised purified A. fumigatus SOD, together with a single band of 19 kDa in A. fumigatus cytoplasmic antigen, by immunodevelopment of Western blots. The polyclonal serum did not recognise either the manganese or iron containing forms of the enzyme; however, it was reactive against putative Cu,Zn SODs in other members of the genus Aspergillus . Immunofluorescent staining of A. fumigatus cultures demonstrated expression of the Cu,Zn SOD in conidia and hyphae, with the cell wall staining particularly intensely. Conidiophores were stained in an uniformly intense pattern. Immunoelectron microscopy confirmed that the SOD was present within the hyphal cell wall, although there was also labelling in the cytoplasm. SOD may protect Aspergillus against oxidants produced by immune effector cells and these observations demonstrate that the enzyme is available to perform its antioxidant function within the cell wall.     

Interleukin-18 enhances a Th2 biased response and susceptibility to Leishmania mexicana in BALB/c mice

Interleukin-18 deficient mice on a BALB/c background display increased resistance to cutaneous infection with Leishmania mexicana, with reduced lesion progression and reduced parasite burdens compared with wild-type mice. Infected IL-18-/- mice had lower antigen specific IgG1 levels and total IgE levels and conversely higher antigen specific IgG2a levels than similarly infected wild-type mice. Splenocytes isolated from infected IL-18-/- mice produced significantly lower levels of antigen induced IL-4 and higher levels of IFN-gamma than wild-type animals. Consequently IL-18 during L. mexicana infection of BALB/c mice promotes a Th2 biased response and thereby has a disease exacerbating role.     

Dominant role of copper in the kinetic stability of Cu/Zn superoxide dismutase

Mutations in Cu/Zn superoxide dismutase (SOD) are involved in some cases of familial amyotrophic lateral sclerosis, and it appears that misfolding and aggregation, perhaps mediated by abnormal binding or loss of copper (Cu) and/or zinc (Zn), may play a pathological role. It is known that the absence of both metals kinetically destabilizes wild type and mutant SOD leading to a 60-fold increase in their rate of unfolding. Here, the individual contributions of Cu and Zn to the kinetic stability of SOD were investigated, and the results show that Cu plays a greater role. Thus, the deficiency of Cu or Zn, especially the former, will compromise the kinetic stability of SOD, thereby increasing the probability that pathogenic mutants and even the WT protein may misfold and self-assemble into toxic species.     

Inhibitory effects of superoxide dismutase 3 on IgE production in B cells

Immunoglobulin E (IgE) functions as a first-line defense against parasitic infections. However, aberrant production of IgE is known to be associated with various life-threatening allergic diseases. Superoxide dismutase 3 (SOD3) has been found to suppress IgE in various allergic diseases such as allergic conjunctivitis, ovalbumin-induced allergic asthma, and dust mite-induced atopic dermatitis-like skin inflammation. However, the role of SOD3 in the regulation of IgE production in B cells remains elusive. In this study, we investigated the effect of SOD3 on LPS/IL-4 and anti-CD40/IL-4-mediated secretion of IgE in murine B cells. Our data showed that SOD3 can suppress both LPS/IL-4 and antiCD40/IL-7-induced IgE secretion in B cells isolated from both wild-type (SOD3^+/+) and SOD3 knock-out (SOD3^?/?) mice. Interestingly, B cells isolated from SOD3^?/? mice showed higher secretion of IgE, whereas, the use of DETCA, a known inhibitor of SOD3 activity, reversed the inhibitory effect of SOD3 on IgE production. Similarly, SOD3 was found to reduce the proliferation, IgE isotype switch, ROS level, and CCL17 and CCL22 productions in B cells. Furthermore, SOD3 was found to suppress both LPS/IL-4 and anti-CD40/IL-4-mediated activation of downstream signaling such as JAK1/JAK3, STAT6, NF-κB, p38, and JNK in B cells. Taken together, our data showed that SOD3 can be used as an alternative therapy to restrict IgE-mediated allergic diseases.     

Leishmania infantum: mixed T-helper-1/T-helper-2 immune response in experimentally infected BALB/c mice

The main goal of the present study was to characterise the course of infection and immunological responses developed by Leishmania infantum infected BALB/c mice. Parasite load was determined by Real-time TaqMan PCR while cytokine and Immunoglobulin G (IgG) production were assessed by ELISA. Leishmania DNA was detected in spleen and liver as soon as day 1 post-inoculation (pi) and the parasitism was sustained until the end of the experiment. The cytokine kinetics in spleen and liver was generally associated with the oscillations of parasite load. Overall, it was not observed a distinct Th1 or Th2 pattern of cytokine production during the time of experiment. The infected mice developed a mixed immune response, with concomitant production of IFN-gamma, IL-4 and IL-10, both in spleen and liver, and both IgG isotypes. However, our results suggest that, compared to liver, the spleen is more susceptible to L. infantum infection.