Refinement of the rKLi8.3-Based Serodiagnostic ELISA Allows Detection of Canine Leishmaniosis in Dogs with Low Antibody Titers.

The diagnosis of canine leishmaniasis (CanL) still represents a challenge due to the variable clinical manifestations and the large number of asymptomatic dogs. Serological tests are most commonly used to detect infected animals, revealing anti-Leishmania antibodies, mainly of the IgG isotype. Recently, a new diagnostic antigen, rKLi8.3, containing 8.3 kinesin tandem repeats (TR) from a Leishmania infantum strain from Sudan, has been shown to provide excellent specificity and sensitivity for the detection of Leishmania-infected humans and dogs. However, asymptomatic animals with very low antibody titers are often difficult to detect by serodiagnosis. Thus, we wondered whether the addition of an anti-IgG-enhancing step in the protein A/G-based rKLi8.3-ELISA will improve the diagnostic performance without decreasing the specificity. For this, parasitologically confirmed CanL cases with low or high clinical scores, uninfected healthy controls and dogs with other infections were tested by rKLi8.3-ELISA as well as two different immunochromatographic rapid tests, rKLi8.3-lateral flow test (LFT) and Dual Path Platform (DPP®) based on the rK28 antigen. Our results show that the diagnostic accuracies of the rKLi8.3-ELISA and LFT were similar to that of DPP, missing several asymptomatic animals. However, the addition of a secondary, amplifying anti-dog IgG antibody in the protein A/G-based rKLi8.3-ELISA enabled the detection of nearly all asymptomatic dogs without compromising its specificity.

Comparative Study of a Novel Lateral Flow Rapid Test with Conventional Serological Test Systems for the Diagnosis of Canine Leishmaniosis in Croatia and Brazil.

Control of canine infections with Leishmania infantum (L. infantum), a major zoonotic disease in Brazil and southern Europe, is becoming increasingly important due to its close proximity to humans, the increasing import of dogs from endemic regions and the impact of climate change on vector spreading. Simple, rapid and reliable diagnostic tests are therefore needed to detect infected dogs. Here, we re-evaluated different serological methods for the diagnosis of canine leishmaniosis (CanL) in Croatia and Brazil. The diagnostic performance of the indirect fluorescent antibody test (IFAT) and the VetLine® Leishmania ELISA (GSD Frankfurt, Germany) was compared with three rKLi8.3-based diagnostic test systems, the rKLi8.3 ELISA (GSD Frankfurt, Germany), the INgezim® Leishma CROM (GSD Madrid, Spain) lateral flow test (LFT) and the VetBlot®Leishmania LineBlot (GSD Frankfurt, Germany). CanL symptomatic dogs were efficiently diagnosed by all tests, except the VetLine® Leishmania ELISA, which is based on whole Leishmania antigens. The advantage of rKLi8.3 was also observed in oligo- and asymptomatic dogs from Brazil and Croatia, although with reduced diagnostic efficiency compared to symptomatic dogs. Similar to IFAT and rKLi8.3 ELISA, the LFT did not cross-react with other common canine pathogens; it showed very high specificity for healthy dogs from endemic regions in both countries and did not react with healthy, vaccinated dogs in Brazil. In conclusion, serodiagnostic tests based on the rKLi8.3 antigens are superior to whole parasite antigens, and the LFT has the advantage of providing a laboratory-independent, rapid and specific diagnosis of CanL.

Development of a Novel Enzyme-Linked Immunosorbent Assay and Lateral Flow Test System for Improved Serodiagnosis of Visceral Leishmaniasis in Different Areas of Endemicity.

Visceral leishmaniasis (VL) is caused by protozoan parasites of the Leishmania donovani complex and is one of the most prominent vector-borne infectious diseases with epidemic and mortality potential if not correctly diagnosed and treated. East African countries suffer from a very high incidence of VL, and although several diagnostic tests are available for VL, diagnosis continues to represent a big challenge in these countries due to the lack of sensitivity and specificity of current serological tools. Based on bioinformatic analysis, a new recombinant kinesin antigen from Leishmania infantum (rKLi8.3) was developed. The diagnostic performance of rKLi8.3 was evaluated by enzyme-linked immunosorbent assay (ELISA) and lateral flow test (LFT) on a panel of sera from Sudanese, Indian, and South American patients diagnosed with VL or other diseases, including tuberculosis, malaria, and trypanosomiasis. The diagnostic accuracy of rKLi8.3 was compared with rK39 and rKLO8 antigens. The VL-specific sensitivity of rK39, rKLO8, and rKLi8.3 ranged from 91.2% over 92.4% to 97.1% and specificity ranged from 93.6% over 97.6% to 99.2%, respectively. In India, all tests showed a comparable specificity of 90.9%, while the sensitivity ranged from 94.7% to 100% (rKLi8.3). In contrast to commercial serodiagnostic tests, rKLi8.3-based ELISA and LFT showed improved sensitivity and no cross-reactivity with other parasitic diseases. Thus, rKLi8.3-based ELISA and LFT offer improved VL serodiagnostic efficiency in East Africa and other areas of endemicity. IMPORTANCE Reliable and field suitable serodiagnosis of visceral leishmaniasis (VL) in East Africa has until now been a big challenge due to low sensitivity and cross-reactivity with other pathogens. To improve VL serodiagnosis, a new recombinant kinesin antigen from Leishmania infantum (rKLi8.3) was developed and tested with a panel of sera from Sudanese, Indian, and South American patients diagnosed with VL or other infectious diseases. Both prototype rKLi8.3-based enzyme-linked immunosorbent assay (ELISA) and lateral flow test (LFT) showed improved sensitivity and no cross-reactivity with other parasitic diseases. Thus, rKLi8.3-based ELISA and LFT offer substantially increased diagnostic efficiency for VL in East Africa and other areas of endemicity, compared to currently commercially available serodiagnostic tests.

Performance of recombinant proteins in diagnosis and differentiation of canine visceral leishmaniasis infected and vaccinated dogs.

Control of canine visceral leishmaniasis (CVL), a major zoonotic disease in Brazil and many other tropical and subtropical countries, remains difficult as an accurate and reliable diagnosis is still missing. In endemic regions, infected dogs are the main parasitic reservoir host of human Visceral leishmaniasis (VL) infection. Vaccination of dogs against Leishmania infection constitutes an important strategy to prevent or to better control CVL, thus, a serological test that can discriminate between antibodies induced by immunization versus infection is highly desirable in order to improve and simplify diagnosis. Here, four recombinant proteins were evaluated for their ability to detect and differentiate between dogs that are infected with Leishmania or have been immunized with the anti-Leishmania vaccine Leish-Tec®. Receiver operating characteristic (ROC) curve analysis of the four Leishmania-specific IgG ELISA revealed superior performance of rK28, followed by rKLO8, rK39 and rLb6H. The rK28-based ELISA revealed not only the best accuracy against CVL, but also the lowest cross-reactivity with sera from Leish-Tec® immunized dogs. Our data show that the rK28-based ELISA is highly suitable for CVL screening as it shows high sensitivity with simultaneous low cross-reactivity. Further, the high specificity of the rKLO8 indicates its suitability for the confirmation of CVL diagnosis.

Diagnostic accuracy of rKLO8 versus rK26 ELISAs for screening of canine visceral leishmaniasis.

Canine visceral leishmaniasis (CVL) represents an important public health issue. Despite numerous diagnostic tests available, CVL diagnosis still needs to be improved to achieve a more accurate detection rate. Recently, rKLO8, a new antigenic protein of Sudanese Leishmania donovani, was studied for the first time in diagnosis of human visceral leishmaniasis (HVL) and showed good performance. The present study aimed to evaluate serum reactivity to rKL08 and the reference antigen rK26, and to compare both diagnostic proteins with the combined DPP® CVL rapid test and ELISA (EIE-Bio-Manguinhos) confirmatory test, which are both recommended for the diagnosis of CVL in Brazil. Serum samples of dogs were grouped into: (I) DPP®/EIE negative (n=100) and (II) DPP®/EIE positive sera (n=100). Enhanced levels of IgG, mainly IgG2, to both rKLO8 and rK26 were found in group II. Sensitivity was 68% and 77% and specificity was 92% and 91%, for rKLO8 and rK26 antigens, respectively. Moreover, the combination of rKLO8 and rK26 antigens (rKLO8+rK26) exhibited higher sensitivity (85%) and specificity (93%). Thus, our results show that apart from the improved diagnostic power of rKLO8 in HVL, this new antigen is also suitable for the diagnosis of CVL. Further, the combination of rKLO8 and rK26 antigens increases the diagnostic accuracy of CVL.

Lack of endogenous IL-10 enhances production of proinflammatory cytokines and leads to Brucella abortus clearance in mice.

IL-10 is a cytokine that regulates the balance between pathogen clearance and immunopathology. Brucella abortus is an intracellular bacterium that causes chronic disease in humans and domestic animals. Here we evaluated the contribution of IL-10 in host immune response and pathology during B. abortus infection. To assess the role of IL-10 in vivo, IL-10 knockout (KO) or 129 Sv/Ev (wild-type) mice were infected with B. abortus and the number of viable bacteria from the spleen was determined at 1, 2, 3, 6 and 14-weeks postinfection. IL-10 KO mice showed reduced bacterial loads in the spleen when compared to wild-type mice during all time points studied. Additionally, at 14-weeks postinfection IL-10 KO mice had totally cleared the infection. This clearance was preceded by an enhanced IFN-γ, TNF-α and IL-17 responses in both the serum and the spleen of IL-10 KO mice. Additionally, dendritic cells from infected IL-10 KO mice produced elevated levels of IL-12 and TNF-α compared to wild-type animals. Histopathology analysis was performed and both KO and wild-type mice developed multifocal granulomas and necrosis in the liver. However, at six-weeks postinfection reduced numbers of granulomas was detected in IL-10 KO mice compared to wild-type animals. This reduced liver pathology at later stage of infection was accompanied by increased numbers of CD4+CD25+foxp3+ T cells and expression of TGF-ß in IL-10 KO splenocytes. Taken together, our findings demonstrate that IL-10 modulates the proinflammatory immune response to B. abortus infection and the lack of IL-10 increases resistance to Brucella infection.

Tumour necrosis factor receptors and apoptosis of alveolar macrophages during early infection with attenuated and virulent Mycobacterium bovis.

Apoptosis of macrophages has been reported as an effective host strategy to control the growth of intracellular pathogens, including pathogenic mycobacteria. Tumour necrosis factor-α (TNF-α) plays an important role in the modulation of apoptosis of infected macrophages. It exerts its biological activities via two distinct cell surface receptors, TNFR1 and TNFR2, whose extracellular domain can be released by proteolysis forming soluble TNF receptors (sTNFR1 and sTNFR2). The signalling through TNFR1 initiates the majority of the biological functions of TNF-α, leading to either cell death or survival whereas TNFR2 mediates primarily survival signals. Here, the expression of TNF-α receptors and the apoptosis of alveolar macrophages were investigated during the early phase of infection with attenuated and virulent mycobacteria in mice. A significant increase of apoptosis and high expression of TNFR1 were observed in alveolar macrophages at 3 and 7 days after infection with attenuated Mycobacterium bovis but only on day 7 in infection with the virulent M. bovis. Low surface expression of TNFR1 and increased levels of sTNFR1 on day 3 after infection by the virulent strain were associated with reduced rates of apoptotic macrophages. In addition, a significant reduction in apoptosis of alveolar macrophages was observed in TNFR1(-/-) mice at day 3 after bacillus Calmette-Guérin infection. These results suggest a potential role for TNFR1 in mycobacteria-induced alveolar macrophage apoptosis in vivo. In this scenario, shedding of TNFR1 seems to contribute to the modulation of macrophage apoptosis in a strain-dependent manner.

Novel thalidomide analogues from diamines inhibit pro-inflammatory cytokine production and CD80 expression while enhancing IL-10.

Thalidomide is used to treat a variety of diseases including erythema nodosum leprosum, an inflammatory complication of leprosy. However, this drug has severe teratogenic activity and novel thalidomide analogues might be used to treat diseases without this severe side effect. A series of diamine compounds containing two hydrolyzed phthalimide units were chosen as analogues of thalidomide and evaluated regarding their capacity to regulate the production of molecules involved in inflammatory responses. TNF-α, IL-12 and IL-10 production, and the expression of CD80 and CD86 were investigated in LPS plus IFN-γ-stimulated J774A.1 cells by ELISA and flow cytometry, respectively. The expression of TNF-α and IL-10 mRNA was analyzed by real time RT-PCR. TNF-α, IL-6, IFN-γ, CXCL9 and CXCL10 production by human peripheral blood mononuclear cells (PBMC) were evaluated by flow cytometry. Compounds 3, 6 and 9 greatly inhibited TNF-α and IL-12 production while enhancing IL-10. In addition, CD80 expression was inhibited, but not CD86. The compounds inhibited TNF-α production by PBMC more than thalidomide and also had an inhibitory effect on the production of IL-6, IFN-γ, CXCL9 and CXCL10. Levels of mRNA for TNF-α were reduced after treatment with the compounds, suggesting post- transcriptional effects. The compounds had no effect on cell viability. Our results indicate that the novel diamine compounds 3, 6 and 9 inhibit critical pro-inflammatory cytokines and stimulate IL-10, which make them attractive candidate drugs for the treatment of certain inflammatory conditions and cancer.

Anthraquinone derivative O,O'-bis-(3'-iodopropyl)-1,4-dihydroxyanthraquinone modulates immune response and improves experimental autoimmune encephalomyelitis.

The present study investigated the effects of the anthraquinone derivative (O,O'-bis-(3'-iodopropyl)-1,4-dihidroxyanthraquinone - DIPDHAQ), mitoxantrone analog, in an experimental autoimmune encephalomyelitis (EAE) model. The results showed that DIPDHAQ treatment improved the clinical signs of the disease (n=10; vehicle: 3.8 ± 0.3; DIPDHAQ: 1.4 ± 0.9). The improvement was associated with a decrease of inflammatory cells, demyelination, IL-17, IFN-γ, IL-12p40, IL-6, TGF-ß, CCL5 and CCL20 levels in the spinal cord. DIPDHAQ presented a low cytotoxicity when in vitro assays were performed. Therefore, the findings suggest a major role for DIPDHAQ in multiple sclerosis, disease characterized as an autoimmune inflammatory disorder against myelin proteins of the brain and spinal cord. The attenuation of inflammation and consequently improvement of clinical signs, involving a decrease of pro-inflammatory cytokines and the low cytotoxicity of DIPDHAQ, suggest that this compound could be used as an alternative treatment for autoimmune diseases in the central nervous system.

Immunomodulatory effects and improved prognosis of experimental autoimmune encephalomyelitis after O-tetradecanoyl-genistein treatment.

Experimental autoimmune encephalomyelitis (EAE) is a murine autoimmune disease used to study multiple sclerosis (MS), a human inflammatory demyelinating disease of the central nervous system. Genistein, an isoflavonoid phytoestrogenic compound found in soy, is known to reverse clinical signs of EAE. Although genistein has some potential in clinical application, it has some disadvantages related to its chemical structure, such as rapid in vivo metabolism and a fast decline in serum after oral administration. The present work investigates the treatment of EAE by using 7-O-tetradecanoyl-genistein (TDG), a more lipophilic analog of genistein obtained by esterification. The clinical course of EAE was investigated in C57Bl/6 mice immunized with myelin oligodendrocyte glycoprotein peptide (MOG)(35-55) in complete Freund's adjuvant supplemented with Mycobacterium tuberculosis H37RA. After 14 days of MOG immunization, mice were treated with TDG for seven days. Numbers of IL-17-producing cells and Foxp3 by CD4(+) T cells and CTLA-4 expression by CD3(+) T cells from brain were determined by flow cytometry. Levels of IL-6, IFN-γ and IL-10 were evaluated by ELISA. Brain sections were stained by hematoxylin and eosin method. The data obtained indicate that TDG treatment ameliorates the clinical signs of EAE, which correlates with a decrease of IL-17-producing cells and an increase in Foxp3(+)CD4(+) cells in the brain. TDG is also shown to enhance IL-10 production and CTLA-4 expression and to reduce IFN-γ and IL-6. Altogether, these findings suggest an immunomodulatory therapeutic role for TDG in EAE and multiple sclerosis.

Synthesis of lipophilic genistein derivatives and their regulation of IL-12 and TNF-α in activated J774A.1 cells.

Genistein modulates inflammatory responses in part by reducing the production of the pro-inflammatory cytokines IL-12, TNF-α, and nitric oxide, by activated macrophages in response to lipopolysaccharide stimulus. Previous studies have shown that synthetic lipophilic genistein glycosides were significantly more active than hydrophilic glycosides. The aims of this study were to synthesize and to evaluate the effect of novel lipophilic genistein derivatives on IL-12, TNF-α, and nitric oxide production by J774A.1 cells. The results show that the modification of genistein enables the generation of non-cytotoxic compounds with increased IL-12 inhibition. However, these derivatives failed to inhibit TNF-α. The nitric oxide production was notably inhibited by the monoester (2, 3) and monoether (6, 7) compounds in a dose-dependent manner.

Splenectomy increases atherosclerotic lesions in apolipoprotein E deficient mice.

BACKGROUND: Atherosclerosis is an inflammatory immune disease associated with lipid accumulation in the intima layer of arteries. The spleen plays an important immune function, but its influence in development of atherosclerosis remains unclear. Evaluation of the role of the spleen in atherosclerosis is justified due to the high frequency of total splenectomies. In this work, the effect of splenectomy on the development of atherosclerosis in apolipoprotein E (ApoE) deficient mice was investigated. METHODS: ApoE deficient mice were divided into a sham-operated control group (CT) and a splenectomized group (SP). Thirty days after surgery, animals were fed a high fat western diet. After 8 wk, mice were euthanized and their blood, heart, and aorta were subjected to analysis. Atherosclerotic lesion areas in the aortic root were stained with hematoxylin-eosin and quantified by morphometry. The atherosclerotic lesions in the thoracic and abdominal portions of aorta were determined by assessing the percentage of the luminal surface area stained by Sudan IV. Total serum cholesterol and anti-oxidized LDL antibodies were measured. RESULTS: Levels of total serum cholesterol did not vary significantly after splenectomy. Anti-oxidized LDL IgG antibodies were similar between groups. However, compared with the control group, lesions in the aortic root were significantly larger in splenectomized mice (P<0.01). These data were confirmed by the increase of atherosclerotic area in the thoracic and abdominal portions of aorta in splenectomized mice. CONCLUSIONS: These data indicate that splenectomy increases atherosclerotic lesions in ApoE deficient mice fed an atherogenic diet, suggesting an atheroprotector role of the spleen.

Human T cell and antibody-mediated responses to the Mycobacterium tuberculosis recombinant 85A, 85B, and ESAT-6 antigens.

Tuberculosis remains a major health problem throughout the world causing large number of deaths. Effective disease control and eradication programs require the identification of major antigens recognized by the protective responses against M. tuberculosis. In this study, we have investigated humoral and cellular immune responses to M. tuberculosis-specific Ag85A, Ag85B, and ESAT-6 antigens in Brazilian patients with pulmonary (P, n = 13) or extrapulmonary (EP, n = 12) tuberculosis, patients undergoing chemotherapy (PT, n = 23), and noninfected healthy individuals (NI, n = 7). Compared to NI, we observed increased levels of IgG1 responses to Ag85B and ESAT-6 in P and PT groups. Regarding cellular immunity, Ag85A and ESAT-6 were able to discriminate P, PT, and EP patients from healthy individuals by IFN-γ production and P and PT groups from EP individuals by production of TNF-α. In summary, these findings demonstrate the ability of Ag85A, Ag85B, and ESAT-6 to differentiate TB patients from controls by IgG1, IFN-γ and TNF-α production.

Increased IgG1, IFN-gamma, TNF-alpha and IL-6 responses to Mycobacterium tuberculosis antigens in patients with tuberculosis are lower after chemotherapy.

Detection of specific antibodies may represent an additional tool in diagnosis of tuberculosis (TB). Herein, levels of serum IgG antibodies against early secreted antigenic target (ESAT-6), culture filtrate antigen-10 (CFP-10) and 16 kDa Mycobacterium tuberculosis antigens were measured in 33 active pulmonary TB patients (0M-TB), in 47 patients after 1-3 months of treatment (3M-TB) and in 22 patients who had completed 6 months of chemotherapy (6M-TB). The control group consisted of 38 BCG-vaccinated healthy controls (HC). In addition, IFN-gamma, tumor necrosis factor (TNF)-alpha, IL-6, IL-2, IL-4 and IL-10 production in PBMC cultures from 20 patients were measured following stimulation with the M. tuberculosis-specific fusion protein ESAT-6/CFP-10. Elevated levels of IgG against ESAT-6, CFP-10 and 16 kDa antigens were detected in 0M-TB and 3M-TB patients in comparison to the HC and 6M-TB groups. Receiver operating characteristic analysis indicated sensitivity of 85, 94 and 61% and specificity of 89, 87 and 89% for serum IgG against ESAT-6, CFP-10 and 16 kDa, respectively. A predominant IgG1 response to ESAT-6 and CFP-10 was observed in 0M-TB patients, together with ESAT-6/CFP-10-specific IFN-gamma, TNF-alpha and IL-6 that were produced at lower levels in the 6M-TB group. These data indicate that a T(h)1 phenotype against early phase Mtb antigens appears to be dominant in the peripheral blood of patients with active pulmonary TB that is reduced after chemotherapy. Taken together, ESAT-6/CFP-10 cytokine tests together with detecting IgG antibodies specific to ESAT-6 and CFP-10 may be the useful TB disease biomarkers in monitoring treatment success.

Mycobacterium tuberculosis Rv1419 encodes a secreted 13 kDa lectin with immunological reactivity during human tuberculosis.

In this study, we have identified a secreted 13 kDa lectin from Mtb (Mtb, Mycobacterium tuberculosis; sMTL-13) by homology search of a non-redundant lectin database. Bioinformatic analysis revealed that sMTL-13 belongs to the ricin-type beta-trefoil family of proteins containing a Sec-type signal peptide present in Mtb complex species, but not in non-tuberculous mycobacteria. Following heterologous expression of sMTL-13 and generation of an mAb (clone 276.B7/IgG1kappa), we confirmed that this lectin is present in culture filtrate proteins from Mtb H37Rv, but not in non-tuberculous mycobacteria-derived culture filtrate proteins. In addition, sMTL-13 leads to an increased IFN-gamma production by PBMC from active tuberculosis (ATB) patients. Furthermore, sera from ATB patients displayed high titers of IgG Ab against sMTL-13, a response found to be decreased following successful anti-tuberculosis therapy. Together, our findings reveal a secreted 13 kDa ricin-like lectin from Mtb, which is immunologically recognized during ATB and could serve as a biomarker of disease treatment.

Apoptosis of macrophages during pulmonary Mycobacterium bovis infection: correlation with intracellular bacillary load and cytokine levels.

Apoptosis of macrophages infected with pathogenic mycobacteria is an alternative host defence capable of removing the environment supporting bacterial growth. In this work the influence of virulence and bacterial load on apoptosis of alveolar macrophages during the initial phase of infection by Mycobacterium bovis was investigated. BALB/c mice were infected intratracheally with high or low doses of the virulent (ATCC19274) or attenuated (bacillus Calmette-Guérin Moreau) strains of M. bovis. The frequency of macrophage apoptosis, the growth of mycobacteria in macrophages, and the in situ levels of the cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10) and IL-12 and of the anti-apoptotic protein Bcl-2 were measured at day 3 and day 7 post-infection. An increase of macrophage apoptosis was observed after infection with both strains but the virulent strain induced less apoptosis than the attenuated strain. On the 3rd day after infection with the virulent strain macrophage apoptosis was reduced in the high-dose group, while on the 7th day post-infection macrophage apoptosis was reduced in the low-dose group. Inhibition of apoptosis was correlated with increased production of IL-10, reduced production of TNF-alpha and increased production of Bcl-2. In addition, the production of IL-12 was reduced at points where the lowest levels of macrophage apoptosis were observed. Our results indicate that virulent mycobacteria are able to modulate macrophage apoptosis to an extent dependent on the intracellular bacterial burden, which benefits its intracellular growth and dissemination to adjacent cells.

Cellular and humoral immune responses during intrathoracic paracoccidioidomycosis in BALB/c mice.

Paracoccidioidomycosis is a chronic infection that primarily affects the lungs. Here we investigated cellular and humoral immune responses after intrathoracic Paracoccidioidesbrasiliensis infection in BALB/c mice. P. brasiliensis-colony-forming units (CFUs), fungal DNA and granulomas in lungs increased progressively, peaking at day 90 postinfection (p.i.). IFN-gamma production was highest on day 15 p.i., declining thereafter. The kinetics of the NO production was similar to that described for IFN-gamma. In contrast, IL-10 increased from day 45 p.i. reaching a peak at day 90. Levels of serum IgG1 were higher than IgG2a between days 30 and 90 p.i. 30% of mice died by day 90 p.i. These data indicate that infection with P. brasiliensis by the intrathoracic route shows high IFN-gamma and NO production at day 15 p.i., unable to control multiplication of fungi, which appears to be associated with a progressive increase in IL-10 and in the number and complexity of granulomas.

Genistein down-modulates pro-inflammatory cytokines and reverses clinical signs of experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is the most common non-traumatic, disabling neurological human inflammatory demyelinating disease of the central nervous system (CNS). Experimental autoimmune encephalomyelitis (EAE) models MS and is characterized as a CD4+ T-helper type 1 (Th1) cell-mediated autoimmune disease. It is characterized by an influx of activated leukocytes into the CNS. Genistein, occurring abundantly in soy products, has apoptotic, antioxidant, and anti-inflammatory properties. In the present report, we investigated the use of genistein for the treatment of the murine model of MS. After induction of EAE with myelin oligodendrocyte glycoprotein 35-55 peptide (MOG(35-55)), we observed that genistein treatment ameliorated significantly the clinical symptoms, modulating pro- and anti-inflammatory cytokines. Moreover, we analyzed the leukocyte rolling and adherence in the CNS by performing intravital microscopy. Genistein treatment resulted in decreased rolling and adhering of leukocytes as compared to the untreated group. Our data suggest that genistein might be a potential therapy for MS.

Modulatory effects of 6-carboxymethylthiopurine on activated murine macrophages.

The immunological activity of macrophages against pathogens in hosts includes the phagocytosis and the production of nitric oxide. We report herein the investigation of the effect of 6-carboxymethylthiopurine on nitric oxide production by murine macrophages as well as its effect on the cell viability and proliferation after stimulus with Mycobacterium bovis bacille Calmette-Guérin, interferon-gamma or a combination of both. J774A.1 macrophages stimulated or not by bacille Calmette-Guérin (20 microg/mL), interferon-gamma or both, were cultured in the presence of 6-carboxymethylthiopurine (125, 250 and 500 microm). Nitric oxide production was measured by the Griess method and cell viability/proliferation by the diphenyltetrazolium assay [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide]. We observed an increase of J774A.1 cell proliferation after stimulus with bacille Calmette-Guérin at 125, 250 and 500 microm (69.1, 124.0 and 89.7%, respectively) and with interferon-gamma at 125 and 250 microm (64.8% and 61.7%, respectively) (p < 0.05). In all cultures treated with 6-carboxymethylthiopurine, interferon-gamma-activated nitric oxide production by J774A.1 cells decreased as well as when subjected to interferon-gamma plus bacille Calmette-Guérin stimuli at 500 microm (p < 0.05). Altogether these data point to an anti-inflammatory effect of 6-carboxymethylthiopurine on stimulated macrophages.

DNA vaccine using Mycobacterium bovis Ag85B antigen induces partial protection against experimental infection in BALB/c mice.

Bovine tuberculosis is a major cause of economic loss in countries where it is endemic, and in some countries, it may be a significant zoonotic disease problem. Therefore, new strategies for vaccine development are required, and among them, genetic immunization has potential value. The main goal of this study was to test the Mycobacterium bovis Ag85B gene as a DNA vaccine following challenge with an M. bovis virulent strain (ATCC 19274). Groups of BALB/c mice (n = 10) were immunized four times intramuscularly with the pCI-Ag85B construct or the pCI vector alone as the control. High titers of total immunoglobulin G (IgG), IgG1, and IgG2a anti-Ag85B were measured in pCI-Ag85B immunized mice when compared to the pCI control group. Regarding cellular immunity, significant levels of gamma interferon (IFN-gamma) (1,100 +/- 157 pg/ml) and tumor necrosis factor alpha (650 +/- 42 pg/ml) but not interleukin-4 were detected in splenocyte culture supernatants of pCI-Ag85B-vaccinated mice following stimulation with recombinant Ag85B. Further, the main source of IFN-gamma is CD8(+) T cells, as demonstrated by intracellular cytokine staining. As far as protection, a significant reduction in bacterial load in spleens (P < 0.05) was detected in pCI-Ag85B-immunized mice compared to the pCI vector control group. The results obtained here suggest that use of the Ag85B DNA vaccine is a promising strategy to control M. bovis infection due to its ability to induce a Th1 type of immune response. However, protective efficacy needs to be improved, since partial protection was achieved in spleens but not in lungs of vaccinated mice.