Implications of MTHFD2 expression in renal cell carcinoma aggressiveness.

Renal cell carcinoma (RCC) is the most common type of cancer in kidney and is often diagnosed in advanced stages. Until now, there is no reliable biomarker to assess tumor prognosis during histopathological diagnosis. The Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) overexpression has been suggested as prognostic indicator for RCC, however, its protein profile needs to be clarified. This study investigated the MTHFD2 expression in different RCC cohorts, associating it with tumor characteristics and prognostic factors. Gene expression comparisons between non-neoplastic (NN) and tumor samples, as well as patients' survival analysis, were assessed using KM-Plotter tool. MTHFD2 protein pattern was evaluated in 117 RCC by immunohistochemistry and associations with prognosis, clinical and pathological data were investigated. The tumors exhibited higher MTHFD2 transcript levels than NN, being even higher in the metastatic group. Opposite gene expression patterns were found among clear cell renal cell carcinoma (ccRCC) and pappilary renal cell carcinoma (pRCC) subtypes, showing higher and lower expressions compared to NN samples respectively. Overexpression was associated with shorter overall survival for ccRCC and pRCC subtypes, and shorter recurrence-free survival for pRCC. The immunolabeling profile varied according to tumor subtypes, with lower intensity and expression scores in ccRCC compared to pRCC and to chromophobe renal cell carcinoma (chRCC). MTHFD2 protein expression was associated with larger tumors and higher Fuhrman grades. Although prognostic value of protein immunostaining was not confirmed, patients with higher MTHFD2 tended to have lower survival rates in the pRCC group. The results highlight MTHFD2 different patterns according to RCC histological subtypes, revealing marked variations at both the genetic and protein levels. The mRNA indicated tumor prognosis, and greater expression in the tumor samples. Although MTHFD2 immunolabeling suggests tumor aggressiveness, it needs to be validated in other cohorts as potential prognostic factor.

Interleukin-32γ in the Control of Acute Experimental Chagas Disease.

Chagas disease (CD) is an important parasitic disease caused by Trypanosoma cruzi. Interleukin-32 (IL-32) plays an important role in inflammation and in the development of Th1/Th17 acquired immune responses. We evaluated the influence of IL-32γ on the immune response profile, pathogenesis of myocarditis in acute experimental CD, and control of the disease. For this, C57BL/6 wild-type (WT) and IL-32γTg mice were infected subcutaneously with 1,000 forms of Colombian strain of T. cruzi. In the histopathological analyzes, T. cruzi nests, myocarditis, and collagen were quantified in cardiac tissue. Cytokine productions (IL-32, IFN-γ, TNF-α, IL-10, and IL-17) were measured in cardiac homogenate by ELISA. The IL-32γTg mice showed a better control of parasitemia and T. cruzi nests in the heart than WT mice. Infected-WT and -IL-32γTg mice showed similar levels of IFN-γ, TNF-α, and IL-17, but IL-10 was significantly higher expressed in IL-32γTg than in WT mice. The cytokine profile found in IL-32γTg animals contributed to body weight maintenance, parasitemia control, and survival. Our results indicate that the presence of human IL-32γ in mice infected with the Colombian strain of T. cruzi is important for infection control during the acute phase of Chagas disease.

Catechins as Model Bioactive Compounds for Biomedical Applications.

Research regarding polyphenols has gained prominence over the years because of their potential as pharmacological nutrients. Most polyphenols are flavanols, commonly known as catechins, which are present in high amounts in green tea. Catechins are promising candidates in the field of biomedicine. The health benefits of catechins, notably their antioxidant effects, are related to their chemical structure and the total number of hydroxyl groups. In addition, catechins possess strong activities against several pathogens, including bacteria, viruses, parasites, and fungi. One major limitation of these compounds is low bioavailability. Catechins are poorly absorbed by intestinal barriers. Some protective mechanisms may be required to maintain or even increase the stability and bioavailability of these molecules within living organisms. Moreover, novel delivery systems, such as scaffolds, fibers, sponges, and capsules, have been proposed. This review focuses on the unique structures and bioactive properties of catechins and their role in inflammatory responses as well as provides a perspective on their use in future human health applications.

Fish Oil Supplementation Reduces Inflammation but Does Not Restore Renal Function and Klotho Expression in an Adenine-Induced CKD Model.

BACKGROUND: Chronic kidney disease and inflammation promote loss of Klotho expression. Given the well-established anti-inflammatory effects of omega-3 fatty acids, we aimed to investigate the effect of fish oil supplementation in a model of CKD. METHODS: Male C57BL/6 mice received supplementation with an adenine-enriched diet (AD, n = 5) or standard diet (CTL, n = 5) for 10 days. Two other experimental groups were kept under the adenine diet for 10 days. Following adenine withdrawal on the 11th day, the animals returned to a standard diet supplemented with fish oil (Post AD-Fish oil, n = 9) or not (Post AD-CTL, n = 9) for an additional period of 7 days. RESULTS: Adenine mice exhibited significantly higher mean serum urea, creatinine, and renal expression of the pro-inflammatory markers Interleukin-6 (IL-6), C-X-C motif chemokine 10 (CXCL10), and Interleukin-1ß (IL-1ß), in addition to prominent renal fibrosis and reduced renal Klotho gene expression compared to the control. Post AD-Fish oil animals demonstrated a significant reduction of IL-6, C-X-C motif chemokine 9 (CXCL9), and IL-1ß compared to Post AD-CTL animals. However, serum creatinine, renal fibrosis, and Klotho were not significantly different in the fish oil-treated group. Furthermore, renal histomorphological changes such as tubular dilatation and interstitial infiltration persisted despite treatment. CONCLUSIONS: Fish oil supplementation reduced renal pro-inflammatory markers but was not able to restore renal function nor Klotho expression in an adenine-induced CKD model.

TNF-α blockade impairs in vitro tuberculous granuloma formation and down modulate Th1, Th17 and Treg cytokines.

Tuberculosis (TB) is a granulomatous disease that has affected humanity for thousands of years. The production of cytokines, such as IFN-γ and TNF-α, is fundamental in the formation and maintenance of granulomas and in the control of the disease. Recently, the introduction of TNF-α-blocking monoclonal antibodies, such as Infliximab, has brought improvements in the treatment of patients with chronic inflammatory diseases, but this treatment also increases the risk of reactivation of latent tuberculosis. Our objective was to analyze, in an in vitro model, the influence of Infliximab on the granulomatous reactions and on the production of antigen-specific cytokines (TNF-α, IFN-γ, IL-12p40, IL-10 and IL-17) from beads sensitized with soluble Bacillus Calmette-Guérin (BCG) antigens cultured in the presence of peripheral blood mononuclear cells (PBMC) from TB patients. We evaluated 76 individuals, with tuberculosis active, treated and subjects with positive PPD. Granuloma formation was induced in the presence or absence of Infliximab for up to 10 days. The use of Infliximab in cultures significantly blocked TNF-α production (p <0.05), and led to significant changes in granuloma structure, in vitro, only in the treated TB group. On the other hand, there was a significant reduction in the levels of IFN-γ, IL-12p40, IL-10 and IL-17 after TNF-α blockade in the three experimental groups (p <0.05). Taken together, our results demonstrate that TNF-α blockade by Infliximab directly influenced the structure of granuloma only in the treated TB group, but negatively modulated the production of Th1, Th17 and regulatory T cytokines in the three groups analyzed.

Encapsulation of eugenyl acetate in PHBV using SEDS technique and in vitro release evaluation.

Eugenyl acetate obtained via enzymatic esterification using Lipozyme TL IM enzyme was encapsulated in biopolymer poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) through solution-enhanced dispersion by supercritical fluids (SEDS). Produced particles were characterized by SEM and confocal microscopy techniques and in addition in vitro release assays were performed in isopropanol and ethyl acetate. Experimental micronization conditions comprised 8 and 10 MPa, 308 and 313 K and eugenyl acetate concentration ranging from 5 to 20 mg mL-1, keeping PHBV concentration constant (20 mg mL-1 in dichloromethane). The maximum encapsulation efficiency was 58.0 % for 5 mg mL-1of eugenyl acetate at 8 MPa and 308 K. The morphology of the encapsulated particles for most of the trials was spherical, with particle size ranging from 0.061 to 0.276 µm. Regarding the release in ethyl acetate and isopropanol solvents the higher the affinity of the encapsulated ester of these solvents, the faster the release was observed. These results demonstrate the importance of essential clove oil esterification reaction and encapsulation of the ester by SEDS method so that this encapsulated ester can be used in different industrial applications.

Effects of transcutaneous electrical nerve stimulation on fetal and placental development in an experimental model of placental insufficiency.

OBJECTIVE: To elucidate the effects of transcutaneous electrical nerve stimulation (TENS) in pregnancies with placental insufficiency. METHODS: Pregnant rats were subjected to uterine artery ligation and to TENS according to the following groups: ligated stimulated (LS); ligated non-stimulated (LN), control stimulated (CS); and control non-stimulated (CN). Fetal external measurements, such as crown-rump length (CRL), fronto-occipital distance (FOD), thoracic ventral-dorsal (TVDD) and abdominal ventral-dorsal (AVDD) distances were analyzed together with the area occupied by fetal internal organs. Glucose transporter 1 (GLUT-1) expression was evaluated by immunohistochemistry in fetal organs. Thickness of junctional, labyrinth and intermediate placental zones was analyzed by morphometric evaluation in HE-stained slides, and placental hypoxia-inducible factor 1 alfa expression was measured by real-time polymerase chain reaction. RESULTS: In LN and CS groups compared to the CN group, CRL was reduced (27.51/28.95 versus 30.16 mm), as well as FOD (6.63/6.63 versus 7.36 mm), AVDD (7.38/8.00 versus 8.61 mm) and TVDD (6.46/6.87 versus 7.23 mm). Brain GLUT-1 expression was higher in LS (1.3%) and CS (1.8%). The area occupied by placental vessels in the labyrinth zone (29.67 ± 3.51 versus 20.83 ± 7.63) and intermediate zone (26.46 ± 10.21 versus 10.86 ± 8.94) was larger in the LS group than in the LN group. CONCLUSIONS: Our results suggest a negative effect of TENS on placental development, thus compromising the maintenance of adequate blood flow to the fetus.

Expression pattern of transcription factors and intracellular cytokines reveals that clinically cured tuberculosis is accompanied by an increase in Mycobacterium-specific Th1, Th2, and Th17 cells.

Tuberculosis (TB) remains a major global health problem and is the second biggest cause of death by infectious disease worldwide. Here, we investigate in vitro the Th1, Th2, Th17, and Treg cytokines and transcriptional factors produced after Mycobacterium-specific antigen stimulation in patients with active pulmonary tuberculosis, clinically cured pulmonary tuberculosis, and healthy donors with a positive tuberculin skin test (TST+). Together, our data indicate that clinical cure after treatment increases the percentages of Mycobacterium-specific Th1, Th2, and Th17 cells compared with those found in active-TB and TST+ healthy donors. These results show that the host-parasite equilibrium in latent TB breaks in favor of the microorganism and that the subsequent clinical recovery posttreatment does not return the percentage levels of such cells to those observed in latent tuberculosis. Additionally, our results indicate that rather than showing an increase in the percentage of Mycobacterium-specific Tregs, active-TB patients display lower Th1 : Treg and Th17 : Treg ratios. These data, together with lower Th1 : Th2 and Th17 : Th2 ratios, may indicate a mechanism by which the breakdown of the host-parasite equilibrium leads to active-TB and changes in the repertoire of Mycobacterium-specific Th cells that are associated with clinical cure after treatment of pulmonary tuberculosis.

Acute kidney injury and progression of renal failure after fetal programming in the offspring of diabetic rats.

BACKGROUND: Diseases of adulthood, such as diabetes and hypertension, may be related to changes during pregnancy, particularly in kidney. We hypothesized that acute kidney injury progresses more rapidly in cases of fetal programming. METHODS: Diabetic dams' offspring were divided into: CC (controls, receiving vehicle); DC (diabetics, receiving vehicle); CA (controls receiving folic Acid solution, 250 mg/kg); and DA (diabetics receiving folic acid solution). Renal function tests, morphometry, gene, and protein expression of epithelial-mesenchymal transition (EMT) markers were analyzed by qPCR and immunohistochemistry, respectively. RESULTS: Creatinine, urea, Bowman's space, and EMT markers were increased in CA and DA groups. TGF-ß3, actin, and fibronectin expression was higher in CA and DA, with significant increase in DA compared to CA 2-mo offspring. There was higher expression level of TGF-ß1, TGF-ß3, fibronectin, and vimentin in the offspring of diabetic dams at 5 mo. Increases in TGF-ß1 and TGF-ß3 were more evident in the offspring of diabetic dams. CONCLUSION: Fetal programming promotes remarkable changes in kidney morphology, and function in offspring and renal failure progression may be faster in younger offspring of diabetic dams subjected to an additional injury.

Interleukin-6 and C-reactive protein are overexpressed in the liver of perinatal deaths diagnosed with fetal inflammatory response syndrome.

Anatomopathologic studies have failed to define the fetal inflammatory response syndrome (FIRS) as a cause of fetal death. Here, liver fragments of perinatal autopsies were collected at a university hospital from 1990 to 2009 and classified according to the cause of death, perinatal stress, and gestational age (GA) of the fetus. IL-6, TNF-α, and C-reactive protein (CRP) expression were immunostained, respectively, with primary antibody. Cases with congenital malformation, ascending infection, and perinatal anoxia showed increased IL-6, CRP, and TNF-α, respectively. Prematures presented higher expression of IL-6 whereas term births showed higher expression of CRP. Cases classified as acute stress presented higher expression of IL-6 and TNF-α and cases with chronic stress presented higher expression of CRP. GA correlated negatively with IL-6 and positively with CRP and TNF-α. Body weight correlated negatively with IL-6 and positively with CRP and TNF-α. Despite the diagnosis of FIRS being clinical and based on serum parameters, the findings in the current study allow the inference of FIRS diagnosis in the autopsied infants, based on an in situ liver analysis of these markers.

T Cell Activation and Proinflammatory Cytokine Production in Clinically Cured Tuberculosis Are Time-Dependent and Accompanied by Upregulation of IL-10.

BACKGROUND: Th1 cytokines are essential for the control of M. tuberculosis infection. The role of IL-10 in tuberculosis is controversial and there is an increasing body of evidence suggesting that the relationship between Th1 cytokines and IL-10 is not as antagonistic as it was first believed, and that these cytokines may complement each other in infectious diseases. METHODS: The present study evaluated the activating capacity of CD4+ and CD8+ T cell repertoire in response to antigen stimulation through the expression of CD69 using Flow Cytometry, as well as the functionality of PBMCs by determining the cytokine profile in patients with active tuberculosis and in clinically cured patients after in vitro stimulation using ELISA. Treated patients were subdivided according to time after clinical cure (<12 months or >12 months post-treatment). RESULTS: We observed that T cell activation was higher in TB-treated patients, especially CD8+ T cell activation in TB-Treated >1 year. Th1 cytokines were significantly higher in TB-Treated, and the levels of IFN-γ and TNF-α increased continuously after clinical cure. Moreover, IL-10 production was significantly higher in cured patients and it was also enhanced in cured patients over time after treatment. Th17, Th2 and Th22 cytokines showed no statistically significant differences between Healthy Donors, Active-TB and TB-Treated. CONCLUSIONS: This study describes a scenario in which potentiation of CD4+ and CD8+ T cell activation and increased Th1 cytokine production are associated with the clinical cure of tuberculosis in the absence of significant changes in Th2 cytokine production and is accompanied by increased production of IL-10. In contrast to other infections with intracellular microorganisms, this response occurs later after the end of treatment.

Evaluation of human polymorphonuclear behavior on textured titanium and calcium-phosphate coated surfaces.

Few studies have evaluated the effects of titanium (Ti) surface modifications on polymorphonuclear neutrophils (PMNs). Human PMNs' viability and release of key mediators-such as IL1ß, IL6, TNFα, IL12, IL10, IL4, TGFß1, IL8, IP-10, and Mig-were evaluated on three different Ti surface treatments: (1) machined Ti; (2) alumina-blasted and acid-etched Ti (AB/AE); and (3) calcium phosphate coating of 300-500 nm by ion beam onto the AB/AE Ti surface (CaP). A polystyrene surface was used as a negative control. The PMNs were purified from whole human blood and cultured for 6 h. Cell viability was determined by flow cytometry, and the supernatant was evaluated to determine the levels of cytokines and chemokines. Results showed that the percentage of viable cells was significantly lower on the CaP surface compared to the control (p < 0.05) relative to the other groups. No differences in the levels of IL8, MIG, and IP10 were detected between groups. Significantly higher levels of IL1ß (p = 0.046) and TNFα (p = 0.016) were detected for the CaP surfaces compared to AB/AE surface only. The levels of IL4, IL10, and TGFß1 secreted from the PMNs in the CaP group were significantly lower than in the control and machined groups (p < 0.05) that were statistically comparable to AB/AE. Overall, the addition of a thin CaP coating to the AB/AE Ti surface influenced the secretion profile of pro-inflammatory cytokines due to the higher release of pro-inflammatory cytokines (IL1ß and TNFα) on these surfaces.

CD28 family and chronic rejection: "to belatacept...And beyond!".

Kidneys are one of the most frequently transplanted human organs. Immunosuppressive agents may prevent or reverse most acute rejection episodes; however, the graft may still succumb to chronic rejection. The immunological response involved in the chronic rejection process depends on both innate and adaptive immune response. T lymphocytes have a pivotal role in chronic rejection in adaptive immune response. Meanwhile, we aim to present a general overview on the state-of-the-art knowledge of the strategies used for manipulating the lymphocyte activation mechanisms involved in allografts, with emphasis on T-lymphocyte costimulatory and coinhibitory molecules of the B7-CD28 superfamily. A deeper understanding of the structure and function of these molecules improves both the knowledge of the immune system itself and their potential action as rejection inducers or tolerance promoters. In this context, the central role played by CD28 family, especially the relationship between CD28 and CTLA-4, becomes an interesting target for the development of immune-based therapies aiming to increase the survival rate of allografts and to decrease autoimmune phenomena. Good results obtained by the recent development of abatacept and belatacept with potential clinical use aroused better expectations concerning the outcome of transplanted patients.

Sepse neonatal precoce: níveis de citocinas no sangue de cordão umbilical no diagnóstico e durante o tratamento / Early-onset neonatal sepsis: cord blood cytokine levels at diagnosis and during treatment

OBJETIVO: Avaliar parâmetros clínicos, laboratoriais e citocinas séricas em 55 neonatos que desenvolveram sepse precoce. MÉTODOS: Avaliamos os parâmetros clínicos dos neonatos relacionados com sepse precoce. No dia do diagnóstico de sepse e 48 horas após, foram realizados o leucograma diferencial e a dosagem de proteína C reativa e glicemia. As citocinas IL-1β, IL-10, IL-6 e TNF-α foram determinadas no sangue do cordão, no dia do diagnóstico de sepse, 48 e 96 horas após o início do tratamento. RESULTADOS: O tempo de internação dos neonatos foi inversamente proporcional ao peso no nascimento. Os parâmetros clínicos foram variados, especialmente a temperatura corpórea. Alterações de glicemia foram frequentes, principalmente a hipoglicemia. A alteração de hemograma mais prevalente foi a leucopenia, devido principalmente à neutropenia. Os níveis de proteína C reativa se mostraram correlacionados com o índice neutrofílico. Observamos uma correlação positiva entre os níveis de TNF-α e IL-10 entre o curso da sepse precoce e os níveis observados no cordão umbilical. CONCLUSÕES: As alterações clínicas e laboratoriais entre os neonatos com sepse são variadas. Neonatos que apresentam elevações no padrão de citocinas no momento do parto permanecem com seus níveis elevados durante o processo infeccioso.

OBJECTIVE: To assess clinical and laboratory parameters and serum cytokine levels in 55 neonates who developed early-onset sepsis. METHODS: Clinical parameters associated with early-onset neonatal sepsis were assessed. White blood cell differential and serum C-reactive protein and glucose levels were measured upon diagnosis of sepsis and 48 hours later. IL-1β, IL-10, IL-6, and TNF-α levels were measured in cord blood samples obtained on the day of diagnosis and from samples collected 48 and 96 hours after treatment onset. RESULTS: Among newborns with early-onset sepsis, the length of hospital stay was inversely correlated with birth weight. Clinical parameters varied widely, especially body temperature. Blood glucose changes - particularly hypoglycemia - were common. Leukopenia, usually due to neutropenia, was the most prevalent change in blood cell count. C-reactive protein levels correlated with the immature-to-total neutrophil ratio. Serum TNF-α and IL-10 levels measured early in the course of sepsis were positively correlated with those detected in cord blood. CONCLUSIONS: Clinical and laboratory parameters varied widely among neonates with sepsis in this sample. In neonates who presented with increased cytokine levels at birth, this abnormality persisted throughout the infectious process.

Early-onset neonatal sepsis: cord blood cytokine levels at diagnosis and during treatment.

OBJECTIVE: To assess clinical and laboratory parameters and serum cytokine levels in 55 neonates who developed early-onset sepsis. METHODS: Clinical parameters associated with early-onset neonatal sepsis were assessed. White blood cell differential and serum C-reactive protein and glucose levels were measured upon diagnosis of sepsis and 48 hours later. IL-beta, IL-10, IL-6, and TNF-α levels were measured in cord blood samples obtained on the day of diagnosis and from samples collected 48 and 96 hours after treatment onset. RESULTS: Among newborns with early-onset sepsis, the length of hospital stay was inversely correlated with birth weight. Clinical parameters varied widely, especially body temperature. Blood glucose changes - particularly hypoglycemia - were common. Leukopenia, usually due to neutropenia, was the most prevalent change in blood cell count. C-reactive protein levels correlated with the immature-to-total neutrophil ratio. Serum TNF-α and IL-10 levels measured early in the course of sepsis were positively correlated with those detected in cord blood. CONCLUSIONS: Clinical and laboratory parameters varied widely among neonates with sepsis in this sample. In neonates who presented with increased cytokine levels at birth, this abnormality persisted throughout the infectious process.

Evaluation of a modified microscopic direct diagnosis of dermatophytosis.

Here we present a modified protocol for dematophyte diagnosis, utilizing a simple centrifugation step to significantly decrease false-negative results of the original KOH direct microscopy-based technique. Although culture constitutes the gold-standard diagnosis, the time spent for results is a limit. Fast and low-cost techniques are important for infection screening in underdeveloped countries.