Evaluation of potential biomarkers for the diagnosis and monitoring of Systemic Lupus Erythematosus using the Cytometric Beads Array (CBA).

BACKGROUND: Systemic Lupus Erythematosus (SLE) is an autoimmune, multisystemic disease. Currently diagnosis depends on complex criteria developed by the American College of Rheumatology. Moreover, the lack of specific biomarkers also challenges the diagnosis. METHODS: Inflammatory biomarkers such as IL-8, IP-10, MIG, MIP-1α and RANTES were measured in serum samples from SLE patients and subjects in control groups (patients with other autoimmune diseases and healthy individuals). Forty-six SLE patients (22 patients with low activity, SLEDAI-2 K ≤ 4, 24 patients with moderate/high activity, SLEDAI-2 K > 4), 42 patients with other autoimmune diseases (OAD group), and 8 healthy volunteers participated in this study. RESULTS: MIG (p < .001) and RANTES (p < .001) concentrations in SLE patients and healthy controls, and IP-10 concentrations in SLE patients with different disease activities (low activity, p < .01, moderate/high activity, p < .05) differed significantly. IL-8 (p < .001) and MIP-1α (p < .001) concentrations in SLE patients differed from those in patients from the OAD group. IL-8 (p < .05), IP-10 (p < .01), MIG (p < .05), MIP-1α (p < .001), and RANTES (p < .05) were correlated with SLE activity; their concentrations in SLE patients with low and moderate/high activity differed significantly. CONCLUSIONS: Given the findings of this study, one can envision the possibility of future use of some of these cytokines to assist in the screening of SLE patients, or even in monitoring disease activity.

Evaluation of miltefosine for the treatment of dogs naturally infected with L. infantum (=L. chagasi) in Brazil.

Dogs naturally infected with Leishmania Infantum (=L. chagasi) were treated with miltefosine using different therapeutic regimens. The animals were evaluated for clinical evolution, biochemical parameters, parasite load (by real-time PCR), cytokine levels and humoral response. After treatment and during the following 24 months, there was progressive clinical improvement and complete recovery in 50% (7/14) of the treated animals. There was a decrease in the smear positivity of the bone marrow after treatment, and there was also a gradual and constant decrease in positive cultures at the end of the follow-up period. However, the PCR detection of parasite DNA remained positive. In general, all animals presented a significant increase in parasite load 6 months after treatment. The IFN-γ levels in all the groups tended to increase during follow-up period, regardless of the miltefosine dose administered. The IL-4 and IL-10 levels of the animals tended to decrease during follow-up, except after 300 days when only IL-10 increased. The serum antibodies identified antigens that ranged from 116 kDa to less than 29 kDa in the Western blot assay. Furthermore, 300 days after treatment, qualitative and quantitative differences in the antigen profiles were observed. Antigens of 97 and 46 kDa were the most intensely recognized. Higher levels of antigen-specific Leishmania IgG were detected before and 300 days after treatment in all groups. Taking together, the improvement in the clinical symptoms was not followed by parasitological clearance, suggesting that treatment with miltefosine is not recommended, especially in endemic areas like Brazil, where children are the major victims and dogs are involved in the maintenance of the parasite cycle.

Natural killer cell subpopulations in putative resistant individuals and patients with active Mycobacterium tuberculosis infection.

Herein, we intended to perform flow-cytometric analyses of peripheral blood NK-cell subsets in patients with active tuberculosis (TB) and those putative resistant subjects displaying positive tuberculin skin test (TST+) and compared with TST- healthy controls. Our findings demonstrated distinct phenotypic features in TST+ as compared with TB. While lower values of NK-cells with increased frequency of CD3-CD16+ CD56- and CD3-CD16-CD56+ subsets besides lower frequency of CD3-CD16+ CD56+ NK-cells was observed in TST+, unaltered levels of NK-cells with increased levels of CD3-CD16+ CD56- NK-cells with lower frequency of CD3-CD16+ CD56+ NK-cells was found in TB. Additional analysis highlighted a shift towards increased levels of CD3-CD16-/+CD56bright NK-cells as the hallmark of TST+, whereas unaltered frequency was observed in TB. Increased levels of CD3+CD56+ cells were observed in both TST+ and TB. Further focusing on the monocyte/NK-cell network, we have reported that enhanced frequency of CD14+ CD16+ monocytes particularly observed in TST+. Outstanding were the distinct correlation profiles observed between CD3-CD16-CD56+ NK-cells and CD3+ CD56+ cells CD14+ CD16+ monocytes for TST+ and TB. These data suggested that high levels of CD3-CD16-CD56+ NK-cells aside CD14+ CD16+ monocytes as well as non-concurrent increment of CD3+ CD56+ cells, may be involved in protective mechanisms in putative tuberculosis-resistant individuals. On the other hand, the basal levels of macrophage-like monocytes despite its positive correlation with increased levels of CD3+ CD56+ cells may count for the lack of the protective immunity in patients with active tuberculosis. Further studies focusing on the cytokine profiling of peripheral blood innate immunity cells before and after chemotherapeutic treatment are currently under evaluation.

Acute and chronic toxicological studies of Dimorphandra mollis in experimental animals.

Dimorphandra mollis Benth. (Caesalpiniaceae), known as "faveira" or "fava d'anta" is a common plant in central Brazilian cerrado that is used mainly as a vasoprotector. Its main marker is rutin. The present study aimed to evaluate the security of Dimorphandra mollis dry extract in rodents. The extract presented a rutin content of 76+/-3%. Acute and chronic (180 days) toxicity was evaluated after per os administration. In acute toxicity, 3500 and 5000 mg/kg doses presented reversible effects. In chronic toxicity, 1000 and 2000 mg/kg doses did not provoke significant changes in body weight of the animals and in water and food consumption. Behavioral reversible changes and in blood count parameters (hemoglobin, hematocrit and red cells decrease and platelets increase in male in rats) were observed only in 2000 mg/kg dose. In biochemical evaluation, the results varied a lot considering doses and sex, without a linear profile. Some parameters showed a significant difference but without a clinical correlation. In histopathological examination, lung hemorrhage was observed in 2000 mg/kg dose. In conclusion, the study suggest that the extract is safe in a 1000 mg/kg dose, whereas for 2000 mg/kg dose further studies are needed. In long-term use, caution is required.

Pulmonary tuberculosis: evaluation of interferon-gamma levels as an immunological healing marker based on the response to the Bacillus Calmette-Guerin.

Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis whose interaction with the host may lead to a cell-mediated protective immune response. The presence of interferon gamma (IFN-gamma) is related to this response. With the purpose of understanding the immunological mechanisms involved in this protection, the lymphoproliferative response, IFN-gamma and other cytokines like interleukin (IL-5, IL-10), and tumor necrosis factor alpha (TNF-alpha) were evaluated before and after the use of anti-TB drugs on 30 patients with active TB disease, 24 healthy household contacts of active TB patients, with positive purified protein derivative (PPD) skin tests (induration > 10 mm), and 34 asymptomatic individuals with negative PPD skin test results (induration < 5 mm). The positive lymphoproliferative response among peripheral blood mononuclear cells of patients showed high levels of IFN-gamma, TNF-alpha, and IL-10. No significant levels of IL-5 were detected. After treatment with rifampicina, isoniazida, and pirazinamida, only the levels of IFN-gamma increased significantly (p < 0.01). These results highlight the need for further evaluation of IFN-gamma production as a healing prognostic of patients treated.

Pulmonary tuberculosis: evaluation of interferon-gamma levels as an immunological healing marker based on the response to the Bacillus Calmette-Guerin

Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis whose interaction with the host may lead to a cell-mediated protective immune response. The presence of interferon-gamma is related to this response. With the purpose of understanding the immunological mechanisms involved in this protection, the lymphoproliferative response, IFN-gamma and other cytokines like interleukin (IL-5, IL-10), and tumor necrosis factor alpha (TNF-alfa) were evaluated before and after the use of anti-TB drugs on 30 patients with active TB disease, 24 healthy household contacts of active TB patients, with positive purified protein derivative (PPD) skin tests (induration > 10 mm), and 34 asymptomatic individuals with negative PPD skin test results (induration < 5 mm). The positive lymphoproliferative response among peripheral blood mononuclear cells of patients showed high levels of IFN-gamma, TNF-alfa, and IL-10. No significant levels of IL-5 were detected. After treatment with rifampicina, isoniazida, and pirazinamida, only the levels of IFN-gamma increased significantly (p < 0.01). These results highlight the need for further evaluation of IFN-gamma production as a healing prognostic of patients treated.

Technetium-99m labeling anti-amastigote polyclonal antibodies of Leishmania amazonensis.

Anti-amastigote polyclonal antibody (IgG) was incubated with solutions of stannous chloride and sodium borohidride. After that, 3.7 MBq of technetium-99m (99mTc) was added. A labeling yield of the antibody about 84% was obtained. After filtration of 99mTc-IgG, the radiochemical purity increased from 84 to 95%. The labeling of IgG with 99mTc did not modify the immunoreactivity of the antibody, since it was able to identify in vitro and in vivo the specific antigen of Leishmania amazonensis.

A randomized double-blind placebo-controlled trial to evaluate the immunogenicity of a candidate vaccine against American tegumentary leishmaniasis.

This study was aimed at evaluating the immunogenicity of a vaccine composed of killed Leishmania amazonensis promastigotes using several different protocols in a randomized, double-blind and controlled trial design in order to select one of them for further efficacy trials. One hundred and fourteen leishmanin skin test (LST)-negative healthy volunteers were allocated into eight groups that received either two or three deep intramuscular injections of vaccine at doses of 180, 360 and 540 microg or similar injections of placebo. Cell-mediated immune responses were evaluated before and after vaccination by means of LST as well as proliferative responses and cytokine production in Leishmania antigen-stimulated peripheral blood mononuclear cell cultures. The majority of the subjects who actually received vaccine converted to positive LST (89.5%). On the other hand, none of the subjects who received placebo converted to positive LST. Proliferative responses and production of interferon-gamma and interleukin-2 were significantly higher after vaccination than before vaccination in all groups, including those that received placebo. The dose of 360 microg provided the highest LST conversion rate (100%), as well as the greatest increase in interferon-gamma and interleukin-2 production after vaccination.

Immunochemotherapy in American cutaneous leishmaniasis: immunological aspects before and after treatment.

In this study, we evaluated the immune response of patients suffering from cutaneous leishmaniasis treated with two distinct protocols. One group was treated with conventional chemotherapy using pentavalent antimonium salts and the other with immunochemotherapy where a vaccine against cutaneous leishmaniasis was combined with the antimonium salt. Our results show that, although no differences were observed in the necessary time for complete healing of the lesions between the two treatments, peripheral blood mononuclear cells from patients treated by chemotherapy showed smaller lymphoproliferative responses at the end of the treatment than those from patients in the immunochemotherapy group. Furthermore, IFN-gamma production was also different between the two groups. While cells from patients in the chemotherapy group produced more IFN-gamma at the end of treatment, a significant decrease in this cytokine production was associated with healing in the immunochemotherapy group. In addition, IL-10 production was also less intense in this latter group. Finally, an increase in CD8+ -IFN-gamma producing cells was detected in the chemotherapy group. Together these results point to an alternative treatment protocol where healing can be induced with a decreased production of a potentially toxic cytokine.

Immunochemotherapy in American cutaneous leishmaniasis: immunological aspects before and after treatment

In this study, we evaluated the immune response of patients suffering from cutaneous leishmaniasis treated with two distinct protocols. One group was treated with conventional chemotherapy using pentavalent antimonium salts and the other with immunochemotherapy where a vaccine against cutaneous leishmaniasis was combined with the antimonium salt. Our results show that, although no differences were observed in the necessary time for complete healing of the lesions between the two treatments, peripheral blood mononuclear cells from patients treated by chemotherapy showed smaller lymphoproliferative responses at the end of the treatment than those from patients in the immunochemotherapy group. Furthermore, IFN-gamma production was also different between the two groups. While cells from patients in the chemotherapy group produced more IFN-gamma at the end of treatment, a significant decrease in this cytokine production was associated with healing in the immunochemotherapy group. In addition, IL-10 production was also less intense in this latter group. Finally, an increase in CD8+ -IFN-gamma producing cells was detected in the chemotherapy group. Together these results point to an alternative treatment protocol where healing can be induced with a decreased production of a potentially toxic cytokine

Evaluation of the stability and immunogenicity of autoclaved and nonautoclaved preparations of a vaccine against American tegumentary leishmaniasis.

This study was designed to evaluate the immunogenicity of autoclaved and nonautoclaved preparations of a vaccine composed of whole antigens from killed promastigotes of Leishmania amazonensis. Leishmanin skin-test (LST)-negative volunteers were immunized with either autoclaved or nonautoclaved vaccine preparations (32 and 36 subjects, respectively) that had been maintained at 4 degrees C for one year before the onset of this trial. Immunological tests were performed two days before and 40 days after vaccination. The LST conversion rates induced by the autoclaved and nonautoclaved vaccines were significantly different: 59% and 83%, respectively. Leishmania antigen-stimulated proliferative responses of peripheral blood mononuclear cells (PBMC) were significantly higher after vaccination than before vaccination in both groups. The CD8+ subset was predominant over the CD4+ subset among the leishmania-reactive cells after vaccination in both groups. The production of IFN-gamma by the leishmania antigen-stimulated PBMC was significantly higher after vaccination than before vaccination in the group receiving the nonautoclaved vaccine but not in the autoclaved vaccine group. IL-2 was found both before and after vaccination with no differences between its levels in these time points in either group. IL-4 was not detected for either group during the study period.

Characterization of human T lymphocyte-mediated immune responses induced by a vaccine against American tegumentary leishmaniasis.

Forty-three Brazilians were immunized against American tegumentary leishmaniasis using a vaccine made of whole antigens from killed promastigotes of five American dermotropic Leishmania strains. None of the immunized subjects had a positive reaction in the Montenegro skin test (leishmanin) before vaccination, and 74% developed positive reactions in the skin test after vaccination. The proliferative responses of peripheral blood mononuclear cells (PBMC) induced by antigens from dermotropic Leishmania species were significantly higher after vaccination than before vaccination. However, with antigens from L. chagasi (a causative agent of American visceral leishmaniasis), there was no significant difference between the proliferative responses obtained before and after vaccination. Interferon-gamma was detected in the supernatants of L. braziliensis antigen-stimulated PBMC cultures after vaccination (but not before vaccination). One year after vaccination, PBMC were obtained from eight of the immunized individuals and stimulated with L. braziliensis antigens in proliferative response assays. In all cases, the majority of the responding cells were CD8+ T cells, in contrast to the results of a group of patients with active lesions of tegumentary leishmaniasis, whose L. braziliensis-reactive cells were mainly of the CD4+ T cell phenotype.

Schistosoma mansoni: identification of a 46KDa antigen of the schistosomular surface by monoclonal antibody

Um anticorpo monoclonal da subclasse IgG2a, designado C6G9, foi obtido pela imunizacao de camundongos BALB/c com antigenos de ovo de Schistosoma mansoni. Esse anticorpo monoclonal possibilitou a identificacao de um antigeno de peso molecular aproximado de 46 quilodaltons (KDa), cuja expressao foi avaliada atraves da reacao de imunofluorescencia indireta. O referido antigeno persistiu no tegumento do esquistossomulo em desenvolvimento pelo menos ate 96 horas pos-transformacao. O anticorpo monoclonal reagiu tambem com a superficie de cercarias, mas nao com a de vermes adultos. O C6G9, em presenca de complemento, foi tambem capaz de mediar niveis significativos de citoxicidade para esquistossomulos recem-transformados.

Schistosoma mansoni: identification of a 46KDa antigen of the schistosomular surface by monoclonal antibody.

An IgG2a subclass monoclonal antibody, C6G9, was obtained by immunization of BALB/c mice with Schistosoma mansoni egg antigens. With this monoclonal antibody, it was possible to identify a schistosomular antigen with a molecular weight of 46 kilodaltons (KDa), and its expression being evaluated by means of indirect immunofluorescence. The antigen persisted in the integument of the developing schistosomulum, for at least 96 hours post-transformation. The monoclonal antibody also reacted with the cercaria surface, but not with that of adult worm. The C6G9 was also able to mediate significant levels of cytotoxicity in the presence of complement for newly transformed schistosomula.

Immunotherapy as a treatment of American cutaneous leishmaniasis: preliminary studies in Brazil.

A prophylactic vaccine composed of killed promastigotes of five stocks of Leishmania was tested as an immunotherapeutic agent against American cutaneous leishmaniasis (ACL). The agent was administered by deep intramuscular injection daily for 10 days, followed by a 10-day interval. Out of 62 patients so treated, 47 (76%) were considered clinically cured; 41 required 2-10 treatment courses and the other six 11-19 courses. None of the patients treated by immunotherapy displayed adverse side-effects. Immunotherapy proved to be effective in the treatment of single cutaneous lesions, multiple cutaneous lesions and in cases of mucocutaneous leishmaniasis. In comparison with chemotherapy (Glucantime), immunotherapy is less efficient and more prolonged but can be safely used when antimonials are contra-indicated or are found to be ineffective. Consideration is given to the treatment of victims of ACL living in rural areas remote from a medical centre.