Assessment of the components of fluid balance in patients with septic shock: a prospective observational study.

BACKGROUND: The optimal amount for initial fluid resuscitation is still controversial in sepsis and the contribution of non-resuscitation fluids in fluid balance is unclear. We aimed to investigate the main components of fluid intake and fluid balance in both survivors and non-survivor patients with septic shock within the first 72 hours. METHODS: In this prospective observational study in two intensive care units, we recorded all fluids administered intravenously, orally, or enterally, and losses during specific time intervals from vasopressor initiation: T1 (up to 24 hours), T2 (24 to 48 hours) and T3 (48 to 72 hours). Logistic regression and a mathematical model assessed the association with mortality and the influence of severity of illness. RESULTS: We included 139 patients. The main components of fluid intake varied across different time intervals, with resuscitation and non-resuscitation fluids such as antimicrobials and maintenance fluids being significant contributors in T1 and nutritional therapy in T2/T3. A positive fluid balance both in T1 and T2 was associated with mortality (p = 0.049; p = 0.003), while nutritional support in T2 was associated with lower mortality (p = 0.040). The association with mortality was not explained by severity of illness scores. CONCLUSIONS: Non-resuscitation fluids are major contributors to a positive fluid balance within the first 48 hours of resuscitation. A positive fluid balance in the first 24 and 48 hours seems to independently increase the risk of death, while higher amount of nutrition seems protective. This data might inform fluid stewardship strategies aiming to improve outcomes and minimize complications in sepsis.

Human B cells infected by Trypanosoma cruzi undergo F-actin disruption and cell death via caspase-7 activation and cleavage of phospholipase Cγ1.

B cells contribute to the immune system in many ways such as antigen presentation to CD4+ T cells, secretion of cytokines and lymphoid tissue organogenesis. Furthermore, they are the only cell type capable of producing immunoglobulins. B cells also account for critical aspects of the resistance against intracellular pathogens. Trypanosoma cruzi is an intracellular parasite that sabotages humoral response by depletion of immature B cells. Polyclonal activation and secretion of non-specific antibodies are also other mechanisms used by T cruzi to evade and subvert the mammalian host immune system, leading to increased parasitemia and susceptibility to Chagas' disease. It remained unclear whether B cell depletion occurs due to direct contact with T. cruzi or results from a global increase in inflammation. Unlike previous reports, we demonstrated in this study that T. cruzi infects human B cells, resulting in parasite-induced activation of caspase-7 followed by proteolytic cleavage of phospholipase Cγ1 and cell death. These data contribute to explain the mechanisms ruling B-cell depletion and evasion of the immune response by T. cruzi.

Processo de implantação e estruturação do Núcleo de Inovação Tecnológica

A implementação dos Núcleo de Inovação Tecnológica (NITs) ainda é apontada como um desafio no Brasil. Assim, este trabalho tem como objetivo avaliar o processo de implantação e estruturação do NIT no Instituto Pasteur (IP), indicando os resultados aferidos neste processo. Foi escolhido o modelo organizacional híbrido, com atividades voltadas para prestação de serviço e Pesquisa & Desenvolvimento. As ações de implantação e estruturação do NIT-IP promoveram alguns resultados positivos na instituição como institucionalização, reconhecimento e sensibilização do NIT-IP frente a comunidade científica; e o acompanhamento e avaliação do potencial inovador dos projetos e resultados científicos da instituição pelo NIT-IP. Devido aos resultados obtidos este modelo pode ser utilizado como uma referência em outros institutos com a estrutura semelhante ou não ao IP. Contudo, o NIT-IP encontra-se em um processo de amadurecimento, devendo transpor gargalos relacionados com a ausência de verba destinada aos NITs e recursos humanos insuficientes.

IL-17 Induction by ArtinM is Due to Stimulation of IL-23 and IL-1 Release and/or Interaction with CD3 in CD4+ T Cells.

ArtinM is a D-mannose-binding lectin extracted from the seeds of Artocarpus heterophyllus that interacts with TLR2 N-glycans and activates antigen-presenting cells (APCs), as manifested by IL-12 production. In vivo ArtinM administration induces Th1 immunity and confers protection against infection with several intracellular pathogens. In the murine model of Candida albicans infection, it was verified that, in addition to Th1, ArtinM induces Th17 immunity manifested by high IL-17 levels in the treated animals. Herein, we investigated the mechanisms accounting for the ArtinM-induced IL-17 production. We found that ArtinM stimulates the IL-17 production by spleen cells in BALB/c or C57BL/6 mice, a response that was significantly reduced in the absence of IL-23, MyD88, or IL-1R. Furthermore, we showed that ArtinM directly induced the IL-23 mRNA expression and the IL-1 production by macrophages. Consistently, in cell suspensions depleted of macrophages, the IL-17 production stimulated by ArtinM was reduced by 53% and the exogenous IL-23 acted synergistically with ArtinM in promoting IL-17 production by spleen cell suspensions. We verified that the absence of IL-23, IL-1R, or MyD88 inhibited, but did not block, the IL-17 production by ArtinM-stimulated spleen cells. Therefore, we investigated whether ArtinM exerts a direct effect on CD4+ T cells in promoting IL-17 production. Indeed, spleen cell suspensions depleted of CD4+ T cells responded to ArtinM with very low levels of IL-17 release. Likewise, isolated CD4+ T cells under ArtinM stimulus augmented the expression of TGF-ß mRNA and released high levels of IL-17. Considering the observed synergism between IL-23 and ArtinM, we used cells from IL-23 KO mice to assess the direct effect of lectin on CD4+ T cells. We verified that ArtinM increased the IL-17 production significantly, a response that was inhibited when the CD4+ T cells were pre-incubated with anti-CD3 antibody. In conclusion, ArtinM stimulates the production of IL-17 by CD4+ T cells in two major ways: (I) through the induction of IL-23 and IL-1 by APCs and (II) through the direct interaction with CD3 on the CD4+ T cells. This study contributes to elucidation of mechanisms accounting for the property of ArtinM in inducing Th17 immunity and opens new perspectives in designing strategies for modulating immunity by using carbohydrate recognition agents.

[Determinants of maternal near miss in an obstetric intensive care unit]. / Fatores determinantes do near miss materno em uma unidade de terapia intensiva obstétrica.

PURPOSE: To evaluate the risk factors for morbidity and mortality in an obstetric intensive care unit at a university hospital. METHODS: Observational cross-sectional study with 492 pregnant/puerperal women. Patients were admitted to the obstetric intensive care unit over a period of one year, being informed about the proposals of the study and a questionnaire was applied. The analysis was performed using Microsoft Excel 2013 and GraphPad Prism 6. To evaluate risk factors, χ2 tests were used. RESULTS: The main risk factors to near miss were: non-white race (OR=2.5; PR=2.3); marital status (married women) (OR=7.9; PR=7.1), schooling (primary) (OR=3.1; PR=2.8), being from the countryside (OR=4.6; PR=4.0), low income (OR=70; PR=5.5), gestational hypertensive disorders (OR=16.3; PR=13.2), receiving prenatal care (OR=5.0; PR=4.254) and C-section before labor (OR=39.2; PR=31.2). CONCLUSIONS: The prevalence of near miss was associated with socioeconomic/clinical factors and care issues, revealing the importance of interventions to improve these indicators. Additionally, we suggest a better curriculum insertion of this subject in the discipline of the medical course due to the importance of avoiding the near miss using adequate medical education. The importance of correct prenatal care is emphasized in order to identify potential risks, to provide nutritional support to pregnant women, to treat potential diseases and to establish a maternal immunization program, as well as providing better care regarding the clinical features of the patients, in order to reduce obstetrical and neonatal risk.

Would nitric oxide be an effective marker for earlier stages of peri-implant disease? An analysis in human peri-implant sulcular fluid.

Nitric oxide has an important effect on host immune response. However, little has been studied in relation to its potential as a possible diagnostic tool in peri-implant disease. The present study analyzed nitrite levels in the peri-implant sulcular fluid (PISF) of implants with mucositis and the correlation of these nitrite levels with clinical parameters using a simplified fluid collection methodology. Twenty-five partially edentulous patients showing peri-implant mucositis were evaluated, and the peri-implant status was determined based on current clinical parameters: probing depth (PD) and bleeding on probing (BOP). The sulcular fluid (SF) around teeth (control) and implants were collected, and the nitrite levels were evaluated using the Griess method. The mean probing depth (mm) was significantly higher (P < .0001) in implants (2.852 ± 0.6484) than in control teeth (1.585 ± 0.3636). The mean total nitrite level (µM) was statistically higher (P = .0069) in implants with mucositis (14.34 ± 11.83) than in control teeth (9.316 ± 5.534). No correlation was observed between the total nitrite levels and the PD mean in the control group (P = .2558, r = -0.2361) or in the implant group (P = .1160, r = -0.3224), as well as the number of faces showing bleeding on probing (P = .8747, r = 0.0332). These results demonstrated that the nitrite levels were higher in inflamed areas. According to the methodology applied and results obtained, the higher nitrite levels in inflamed areas suggest that, in the future, nitrite could be used as a marker of peri-implant mucositis associated with clinical data to monitor the cure or evolution of the disease.

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.

In-vitro study of cellular viability and nitric oxide production by J774 macrophages stimulated by interferon gamma with ceramic, polycarbonate, and polyoxymethylene brackets.

INTRODUCTION: Ceramic brackets are chemically inert in the oral cavity, whereas polycarbonate and polyoxymethylene brackets can degrade and release bisphenol-A and formaldehyde, respectively. More reliable tests are needed to assess the potential toxicity of these materials. In addition to traditional cytotoxicity tests, the study of nitric oxide (NO) cellular production stimulated by a specific material has been shown to be a reliable tool for evaluating cytotoxic potential. The purpose of this study was to assess, with esthetic brackets, cellular viability by 3,(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide assay (Sigma, St. Louis, Mo) in the macrophage cell line J774 stimulated with interferon gamma. Interferon gamma is a key cytokine in the activation of macrophages, plays an important role in immunologic processes, and also quantifies NO production by these macrophages. METHODS: Well plates were seeded with 2 x 104 J774 cells per well, in a volume of 100 microL, resuspended in Roswell Park Memorial Institute Supplemented Medium 1640. The macrophage cell line J774 was stimulated with interferon gamma. Ceramic, polycarbonate, and polyoxymethylene brackets were added and kept in the culture for 24, 48, or 72 hours in 5% carbon dioxide at 37 degrees C; the control samples did not include brackets. At the end of each incubation period, the supernatant was collected for posterior NO quantification, and the cells were evaluated for cytotoxicity. RESULTS: Cellular viability in all groups was higher at 72 hours than at 24 hours. The final means in the bracket groups did not show significant differences compared with the control group. NO production was significantly greater in all groups at the final time than at the initial time. However, the brackets with the interferon gamma stimulation did not result in greater NO production than did the cells in the control group.

In-vitro study of the cellular viability and nitric oxide production by J774 macrophages with ceramic, polycarbonate, and polyoxymethylene brackets.

INTRODUCTION: Studies show that ceramic brackets are chemically inert in the oral cavity, whereas polycarbonate and polyoxymethylene brackets can degrade, releasing bisphenol-A and formaldehyde, respectively. In addition to the traditional cytotoxicity tests, the study of nitric oxide cellular production stimulated by a specific material has been shown to be a reliable tool for evaluating its cytotoxic potential. METHODS: We aimed to assess cellular viability by MTT (Sigma, St. Louis, Mo): 3,(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide assay in a murine macrophage cell line J774 with esthetic brackets and quantify nitric oxide production by these macrophages. Cell cultures were evaluated at 3 times: 24, 48, and 72 hours. RESULTS: Cellular viability in all groups was higher at 72 hours compared with 24 hours. This increase was significant in the control and ceramic brackets groups. Final means in the bracket groups showed no significant differences compared with the control group. Nitric oxide production was significantly greater in all groups at final time. There was no significant difference between the final means of the bracket groups and the control group, although polyoxymethylene brackets showed significantly greater means at 24 and 48 hours. CONCLUSIONS: Final means in the bracket groups showed no significant differences compared with the control group.

Leishmania amazonensis: xylitol as inhibitor of macrophage infection and stimulator of macrophage nitric oxide production.

Xylitol is a sugar alcohol being explored for clinical uses. The aim was to evaluate the effects of xylitol on Leishmania amazonensis-infected J774A.1 macrophages. Macrophages were infected with L. amazonensis for 3h, washed and incubated with 2.5 or 5.0% xylitol for 24, 48, and 72 h at 37 degrees C. Infection indexes for macrophages incubated only in medium were compared to those treated with xylitol. Cell viability and nitric oxide production were determined each time. Xylitol did not affect L. amazonensis or J774A.1 cell viabilities. Xylitol at 5.0% stimulated nitric oxide production by macrophages at 72 h (p<0.01). At 2.5 and 5.0%, xylitol inhibited nitric oxide production by L. amazonensis at 48 h (p<0.05) when compared to control. Infection indexes were significantly lower at 72 h (p<0.05), (16.9% and 9.6%) in cells cultivated with 2.5 and 5.0% xylitol, respectively, compared to control (38.4%). Results suggest a potential leishmanicidal action of the xylitol on infected macrophages.

Preparation and antitubercular activities of alkylated amino alcohols and their glycosylated derivatives.

A series of N- and C-alkylated amino alcohols and of their protected galactopyranosyl derivatives was synthesized and evaluated for antitubercular activity. Five of these compounds displayed good activity, with a MIC below 12.5mug/mL. The presence of the carbohydrate slightly affected the antibacterial activity.

[Immunoglobulin isotype and IgG subclass profiles in American tegumentary leishmaniasis]. / Perfil de isotipos de imunoglobulinas e subclasses de IgG na leishmaniose tegumentar americana.

The present work investigated the serum antibody profiles in 37 patients with American tegumentary leishmaniasis, who were attended at Hospital de Clinicas - Universidade Federal de Uberlandia, MG, Brazil. The immunoglobulin class and IgG subclass profiles were analyzed by indirect ELISA using Leishmania (Leishmania) amazonensis soluble antigen. The antibody avidity was determined by 6 M urea treatment after incubation with immunoenzymatic conjugate. It was observed that 97% of the serum samples presented anti-Leishmania antibodies for IgE class, 94.6% IgG, 57.5% IgA and 21.5% IgM class. For IgG subclasses the profiles were in the following order of frequency: IgG1>IgG3>IgG2>IgG4. High avidity of anti-Leishmania IgE antibodies was found in 44.4% of the samples. On the other hand, moderate avidity of specific IgG and IgA was observed in 62.8% and 47.8% of samples, respectively. These results indicate a very complex antibody response profile against American tegumentary leishmaniasis.

Immunoblotting analyses using two-dimensional gel electrophoresis of Trypanosoma cruzi excreted-secreted antigens.

Trypanosoma cruzi trypomastigotes excrete-secrete a complex mixture of antigenic molecules. This antigenic mixture denominated trypomastigote excreted-secreted antigens contains a 150-160 kDa band that shows excellent performance in Chagas' disease diagnosis by immunoblotting. The present study partially characterized by two-dimensional gel electrophoresis the immunoreactivity against the 150-160 kDa protein using sera samples from chagasic patients in different phases of the disease. Trypomastigote excreted-secreted antigen preparations were subjected to high-resolution two-dimensional (2D) gel electrophoresis followed by immunoblotting with sera from chagasic and non-chagasic patients. The 150-160 kDa protein presented four isoforms with isoelectric focusing ranging from 6.2 to 6.7. The four isoforms were recognized by IgM from acute phase and IgG from chronic phase sera of chagasic patients. The 150-160 kDa isoform with IF of approximately 6.4 became the immunodominant spot with the progression of the disease. No cross-reactivity was observed with non-chagasic or patients infected with Leishmania sp. In this study we provide basic knowledge that supports the validation of trypomastigote excreted-secreted antigens for serological diagnosis of Chagas' disease.

Detection of antibodies to the 97 kDa component of Toxoplasma gondii in samples of human serum.

This study was carried out to investigate the immune response against 97 kDa (p97) molecular marker of Toxoplasma gondii that has been characterized as a cytosolic protein and a component of the excreted-secreted antigens from this parasite. A total of 60 serum samples from patients were analyzed by enzime-linked immunosorbent assay and Western blot for toxoplasmosis. These samples were organized in three groups, based on clinical symptoms and results of serological tests. Group I: 20 samples reactive to IgG and IgM (acute phase); group II: 20 non-reactive samples (control group); and group III: 20 samples reactive only to IgG (chronic phase). Western blot was performed with total antigenic extracts or with excreted and secreted antigen from T. gondii to identify the fraction correspondent to p97. It was observed that this cytosolic component from T. gondii stimulates the immunologic system to produce both IgM and IgG antibodies in the beginning of the acute infection and IgG throughout the chronic stage of the asymptomatic toxoplasmosis.