Efficacy of Two Commercially Available Adsorbents to Reduce the Combined Toxic Effects of Dietary Aflatoxins, Fumonisins, and Zearalenone and Their Residues in the Tissues of Weaned Pigs.

Mycotoxins present a significant health concern within the animal-feed industry, with profound implications for the pig-farming sector. The objective of this study was to evaluate the efficacy of two commercial adsorbents, an organically modified clinoptilolite (OMC) and a multicomponent mycotoxin detoxifying agent (MMDA), to ameliorate the combined adverse effects of dietary aflatoxins (AFs: sum of AFB1, AFB2, AFG1, and AFG2), fumonisins (FBs), and zearalenone (ZEN) at levels of nearly 0.5, 1.0, and 1.0 mg/kg, on a cohort of cross-bred female pigs (N = 24). Pigs were randomly allocated into six experimental groups (control, mycotoxins (MTX) alone, MTX + OMC 1.5 kg/ton, MTX + OMC 3.0 kg/ton, MTX + MMDA 1.5 kg/ton, and MTX + MMDA 3.0 kg/ton), each consisting of four individuals, and subjected to a dietary regimen spanning 42 days. The administration of combined AFs, FBs, and ZEN reduced the body-weight gain and increased the relative weight of the liver, while there was no negative influence observed on the serum biochemistry of animals. The supplementation of OMC and MMDA ameliorated the toxic effects, as observed in organ histology, and provided a notable reduction in residual AFs, FBs, and ZEN levels in the liver and kidneys. Moreover, the OMC supplementation was able to reduce the initiation of liver carcinogenesis without any hepatotoxic side effects. These findings demonstrate that the use of OMC and MMDA effectively mitigated the adverse effects of dietary AFs, FBs, and ZEN in piglets. Further studies should explore the long-term protective effects of the studied adsorbent supplementation to optimize mycotoxin management strategies in pig-farming operations.

Nicotinamide Nucleotide Transhydrogenase Is Essential for Adrenal Steroidogenesis: Clinical and In Vitro Lessons.

CONTEXT: Nicotinamide nucleotide transhydrogenase (NNT) acts as an antioxidant defense mechanism. NNT mutations cause familial glucocorticoid deficiency (FGD). How impaired oxidative stress disrupts adrenal steroidogenesis remains poorly understood. OBJECTIVE: To ascertain the role played by NNT in adrenal steroidogenesis. METHODS: The genotype-phenotype association of a novel pathogenic NNT variant was evaluated in a boy with FGD. Under basal and oxidative stress (OS) induced conditions, transient cell cultures of the patient's and controls' wild-type (WT) mononuclear blood cells were used to evaluate antioxidant mechanisms and mitochondrial parameters (reactive oxygen species [ROS] production, reduced glutathione [GSH], and mitochondrial mass). Using CRISPR/Cas9, a stable NNT gene knockdown model was built in H295R adrenocortical carcinoma cells to determine the role played by NNT in mitochondrial parameters and steroidogenesis. NNT immunohistochemistry was assessed in fetal and postnatal human adrenals. RESULTS: The homozygous NNT p.G866D variant segregated with the FGD phenotype. Under basal and OS conditions, p.G866D homozygous mononuclear blood cells exhibited increased ROS production, and decreased GSH levels and mitochondrial mass than WT NNT cells. In line H295R, NNT knocked down cells presented impaired NNT protein expression, increased ROS production, decreased the mitochondrial mass, as well as the size and the density of cholesterol lipid droplets. NNT knockdown affected steroidogenic enzyme expression, impairing cortisol and aldosterone secretion. In human adrenals, NNT is abundantly expressed in the transition fetal zone and in zona fasciculata. CONCLUSION: Together, these studies demonstrate the essential role of NNT in adrenal redox homeostasis and steroidogenesis.

Genotoxicity and toxicological evaluations of Brazilian red propolis oral ingestion in a preclinical rodent model.

ETHNOPHARMACOLOGICAL RELEVANCE: Brazilian red propolis is a natural product known due to its medicinal properties. The efficacy of this natural resin has been proved; however, few studies report the safety of its oral use. Some toxic effects of natural products may not be expressed in traditional use, and preclinical studies are necessary to guarantee their safety. Health regulatory agency currently requires these non-clinical studies to develop drugs and herbal medicines, including genotoxic and oral toxicity tests. AIM OF THE STUDY: Accomplish the preclinical toxicity studies of Brazilian red propolis extract (BRP) in rodents, including genotoxicity, acute and sub-chronic toxicities. MATERIAL AND METHODS: Genotoxicity assays followed the erythrocyte micronucleus test protocol in a range of 500-2000 mg/kg BRP oral treatment on male Swiss mice. After an up-and-down procedure, acute oral toxicity (single dose) was performed on female Wistar Hannover rats, reaching a 2000 mg/kg BRP oral gavage concentration. Animals were monitored periodically until 14 days and euthanized for a macroscopic necropsy analysis. The sub-chronic oral toxicity test (90 days) was achieved with 1000 mg/kg of BRP on Wistar Hannover rats (males/females). Animals were monitored to evaluated behavioral and biometrical changes, then were euthanized to perfomed hematological, biochemical, and histopathological analyses. RESULTS: No genotoxic effect of the BRP was detected. The acute toxicity indicated no toxicity of a single oral dose of 2000 mg/kg of BRP. The long-term oral toxicity performed with 1000 mg/kg of BRP altered water and food intake and the biometrics, hematological and biochemical parameters. Biochemical alterations in hepatic and renal parameters were detected only in the males. Despite the detection of biochemical alterations, no histopathological changes were detected in the organs of any group. CONCLUSIONS: BRP, at a higher dose, showed no signs of immediate toxicity. However, the obtained results suggest that the chemical composition and the intake of higher doses deserve special attention regarding possible toxicity.

Panobinostat (LBH589) increase survival in adult xenografic model of acute lymphoblastic leukemia with t(4;11) but promotes antagonistic effects in combination with MTX and 6MP.

Patients diagnosed with acute lymphoblastic leukemia (ALL) bearing t(4;11)/MLL-AF4 have aggressive clinical features, poor prognosis and there is an urgent need for new therapies to improve outcomes. Panobinostat (LBH589) has been identified as a potential therapeutic agent for ALL with t(4;11) and studies suggest that the antineoplastic effects are associated with reduced MLL-AF4 fusion protein and reduced expression of HOX genes. Here, we evaluated the in vitro effects of the combination of LBH589 with methotrexate (MTX) or 6-mercaptopurine (6MP) by cell proliferation assays and Calcusyn software in ALL cell line (RS4;11); the in vivo effects of LBH589 in xenotransplanted NOD-scid IL2Rgammanull mice measuring human lymphoblasts by flow cytometry; and the expression of HOX genes by qPCR after treatment in an adult model of ALL with t(4;11). LBH589 combination with MTX or 6MP did not promote synergistic effects in RS4;11 cell line. LBH589 treatment leads to increased overall survival and reduction of blasts in xenotransplanted mice but caused no significant changes in HOXA7, HOXA9, HOXA10, and MEIS1 expression. The LBH589, alone, showed promising antineoplastic effects in vivo and may represent a potential agent for chemotherapy in ALL patients with t(4;11).

Vitamin D receptor activation is a feasible therapeutic target to impair adrenocortical tumorigenesis.

OBJECTIVE: To evaluate the therapeutic potential of vitamin D receptor (VDR) signaling in adrenocortical carcinoma (ACC) cells. METHODS: We evaluated VDR's methylation pattern in H295R ACC cells, and investigated the effects of calcitriol and seocalcitol treatments on adrenocortical tumorigenesis. RESULTS: VDR was hypermethylated and underexpressed in basal H295R cells. Treatments with calcitriol and seocalcitol restored VDR signaling, resulted in antiproliferative effects, and impaired Wnt/B-catenin signaling. RNAseq of treated cells demonstrated VDR activation on steroid hormones biosynthesis and Rap1 signaling, among others. In vivo, seocalcitol constrained the growth of H295R xenografts and reduced autonomous tumor steroid secretion without hypercalcemia-associated side effects. CONCLUSIONS: H295R cells present VDR hypermethylation, which can be responsible for its underexpression and signaling inactivation under basal conditions. VDR signaling promoted antiproliferative effects in vitro and in vivo, suggesting that it may be a potential therapeutic target for ACC and a valuable tool for patient's clinical management.

DNA methylation is a comprehensive marker for pediatric adrenocortical tumors.

Children diagnosed with pediatric adrenocortical tumors (pACT) have variable outcomes, and, to date, the disease lacks robust prognostic biomarkers. The prognostic potential of tumor methylation has been demonstrated in several cancers. We aimed to evaluate the pACT methylation profile and its association with disease presentation and survival. In this cross-sectional study, we accessed the DNA methylation (MethylationEPIC Array, Illumina) of 57 primary pACT from Southeastern Brazil and the respective patients' clinicopathological features. We also applied our analysis in an independent 48 pACT methylation dataset. Unsupervised learning whole-methylome analysis showed two groups with distinct methylation signatures: pACT-1 and pACT-2. Compared to pACT-2, pACT-1 tumors were enriched with higher methylation in CpG islands, mainly in gene promoter regions. The topmost hypermethylated gene in these samples was shown to be underexpressed. Patients in the pACT-1 group were older at diagnosis and were more likely to have carcinomas and nonlocalized/advanced and recurrent/metastatic disease. Univariate and bivariate regressions showed that pACT-1 methylation signature confers superior hazard ratio of disease progression and death than known prognostic features. The methylation groups had similar frequencies of germline mutations in the TP53 gene, including the regionally frequent p.R337H. Our analysis replication validated our findings and reproduced those recently described in pACT. We demonstrated the existence of different tumor methylation signatures associated with pACT presentation and clinical evolution, even in the context of germline TP53 mutations. Our data support tumor methylation profiling as a robust and independent prognostic biomarker for pACT and suggest a list of candidate genes for further validation.

Vitamin D receptor hypermethylation as a biomarker for pediatric adrenocortical tumors.

Objective: Pediatric adrenocortical tumors (pACT) display complex genomic backgrounds, lacking robust prognostic markers and targeted therapeutic options. Vitamin D3 receptor (VDR) promoter hypermethylation and underexpression were reported in adrenocortical carcinomas from adult patients. In this study, we aimed to investigate VDR expression levels and methylation status in pACT and their clinical and prognostic significance. Design: Retrospective cross-sectional study enrolling pediatric patients with ACT from two tertiary referral institutions. Methods: We evaluated clinicopathological features, VDR mRNA (qPCR) and protein (immunohistochemistry) expression, and VDR-wide methylation of ACT samples from 108 pediatric patients. Fourteen pediatric and 32 fetal and postnatal normal adrenals were used as controls. Results: Unlike in pre- and post-natal normal adrenals, most pACT lacked nuclear VDR expression and had reduced mRNA levels, especially the carcinomas. Unsupervised analysis of VDR methylation data revealed two groups of pACT with distinct disease features and outcomes. Tumors with high VDR methylation presented lower mRNA levels, and the respective patients presented advanced disease and reduced disease-free and overall survival. Conclusions: VDR has a role in normal adrenocortical development and homeostasis, which is impaired during tumorigenesis. VDR hypermethylation and underexpression may be both predictive and prognostic biomarkers for pACT.

Clinical-like cryotherapy in acute knee arthritis of the knee improves inflammation signs, pain, joint swelling, and motor performance in mice.

To assess the effects of clinical-like cryotherapy on inflammatory signs (in vivo neutrophil migration, cytokines, and joint inflammation), pain, joint swelling, balance, and motor coordination in mice with knee arthritis. Young C57BL/6 mice were randomly divided into three groups (8 to 10 mice per group): Control group: mice with no intervention; antigen-induced arthritis (AIA) group: mice sensitized and immunized with intra-articular (i.a.) injection of methylated bovine serum albumin (mBSA); and AIA + cryotherapy group: mice sensitized, immunized with i.a. injection of mBSA, and submitted to a clinical-like cryotherapy protocol. After 21 days of sensitization, AIA and AIA + cryotherapy groups received i.a. injection of mBSA (100 µg/joint) to induce joint inflammation, and a clinical-like cryotherapy protocol was applied to AIA + cryotherapy group (crushed ice bag, two cryotherapy sessions of 20 min every two hours). Experimental analysis was conducted in the initial (immediately after i.a. injection of mBSA) and final periods (two hours after the second cryotherapy session). The number of synovial fluid neutrophils, cytokine levels, joint histology, pain, joint swelling, and motor performance were also analyzed. Our results showed that clinical-like cryotherapy in mice with acute knee arthritis reduced inflammatory signs, pain, and joint swelling, and improved balance and motor coordination.

Oncogenic and tumor suppressor pathways in subchronic aflatoxicosis in rats: Association with serum and urinary aflatoxin exposure biomarkers.

In this study, the changes in oncogenic and tumor suppressor signaling pathways in liver and their association with serum and urinary biomarkers of aflatoxin exposure were evaluated in Wistar rats fed diets containing aflatoxin B1 (AFB1) for 90 days. Rats were divided into four groups (n = 15 per group) and assigned to dietary treatments containing 0 (control), 50 (AFB50), 100 (AFB100) and 200 µg AFB1 kg-1 diet (AFB200). Multiple preneoplastic foci of hepatocytes marked with glutathione-S-transferase-placental form (GST-P) were identified in AFB100 and AFB200 groups. Hepatocellular damage induced by AFB1 resulted in overexpression of cyclin D1 and ß-catenin. The liver expression of retinoblastoma (Rb) and p27Kip1 decreased in AFB100 and AFB200 groups, confirming the favorable conditions for neoplastic progression to hepatocellular carcinoma. All samples from rats fed AFB1-contaminated diets had quantifiable AFB1-lysine in serum or urinary AFM1 and AFB1-N7-guanine, with mean levels of 20.42-50.34 ng mL-1, 5.31-37.68 and 39.15-126.37 ng mg-1 creatinine, respectively. Positive correlations were found between AFB1-lysine, AFM1 or AFB1-N7-guanine and GST-P+, ß-catenin+ and cyclin D1+ hepatocytes, while Rb + cells negatively correlated with those AFB1 exposure biomarkers. The pathways evaluated are critical molecular mechanisms of AFB1-induced hepatocarcinogenesis in rats.

Aqueous Pyrostegia venusta (Ker Gawl.) Miers extract attenuates allergen-induced asthma in a mouse model via an antioxidant mechanism.

Objective:Pyrostegia venusta (Ker-Gawl.) Miers (Bignoniaceae) is a perennial invasive vine, distributed worldwide. In folk medicine, its parts are used for the treatment of inflammatory respiratory diseases. Extracts of P. venusta have antioxidant, antimicrobial, and antinociceptive properties. The aim of this study was to evaluate the effects of two extracts (aqueous and hydroethanolic) of P. venusta in the treatment of asthma in an animal model.Methods: Balb/c mice were sensitized twice with ovalbumin (OVA) intraperitoneally (ip), one week apart, and after one week, challenged with OVA intranasally on four alternate days. Mice were treated ip with 300 mg/kg of aqueous or hydroethanolic extracts for seven consecutive days. Control groups received saline on the same days. Bronchial hyperresponsiveness, production of Th1 and Th2 cytokines, lung and airway inflammation, and antioxidant activity in lung tissue were assessed.Results: Treatment with aqueous extract significantly decreased bronchial hyperresponsiveness, measured by total and tissue resistance and elastance. The administration of hydroethanolic extract did not reduce bronchial hyperresponsiveness. In addition, both extracts significantly reduced total cell and eosinophil counts in bronchoalveolar lavage. Both extracts did not change significantly IL-4, IL-5, IL-9, IL-13, IFN-gamma, and TGF-beta levels. Of note, only the aqueous extract significantly increased the total antioxidant activity and reduced lung inflammation.Conclusion: Aqueous extract of P. venusta reduced bronchial hyperresponsiveness, lung and airway inflammation, probably via an antioxidant mechanism. These results demonstrate that P. venusta may have potential for asthma treatment.

Molecular basis of carrageenan-induced cytokines production in macrophages.

BACKGROUND: Low molecular weight carrageenan (Cg) is a seaweed-derived sulfated polysaccharide widely used as inflammatory stimulus in preclinical studies. However, the molecular mechanisms of Cg-induced inflammation are not fully elucidated. The present study aimed to investigate the molecular basis involved in Cg-induced macrophages activation and cytokines production. METHODS: Primary culture of mouse peritoneal macrophages were stimulated with Kappa Cg. The supernatant and cell lysate were used for ELISA, western blotting, immunofluorescence. Cg-induced mouse colitis was also developed. RESULTS: Here we show that Cg activates peritoneal macrophages to produce pro-inflammatory cytokines such as TNF and IL-1ß. While Cg-induced TNF production/secretion depends on TLR4/MyD88 signaling, the production of pro-IL-1ß relies on TLR4/TRIF/SYK/reactive oxygen species (ROS) signaling pathway. The maturation of pro-IL1ß into IL-1ß is dependent on canonical NLRP3 inflammasome activation via Pannexin-1/P2X7/K+ efflux signaling. In vivo, Cg-induced colitis was reduced in mice in the absence of NLRP3 inflammasome components. CONCLUSIONS: In conclusion, we unravel a critical role of the NLRP3 inflammasome in Cg-induced pro-inflammatory cytokines production and colitis, which is an important discovery on the pro-inflammatory properties of this sulfated polysaccharide for pre-clinical studies. Video abstract Carrageenan (Cg) is one the most used flogistic stimulus in preclinical studies. Nevertheless, the molecular basis of Cg-induced inflammation is not totally elucidated. Herein, Lopes et al. unraveled the molecular basis for Cg-induced macrophages production of biological active IL-1ß. The Cg-stimulated macrophages produces pro-IL-1ß depends on TLR4/TRIF/Syk/ROS, whereas its processing into mature IL-1ß is dependent on the canonical NLRP3 inflammasome.

PKM2 promotes Th17 cell differentiation and autoimmune inflammation by fine-tuning STAT3 activation.

Th17 cell differentiation and pathogenicity depend on metabolic reprogramming inducing shifts toward glycolysis. Here, we show that the pyruvate kinase M2 (PKM2), a glycolytic enzyme required for cancer cell proliferation and tumor progression, is a key factor mediating Th17 cell differentiation and autoimmune inflammation. We found that PKM2 is highly expressed throughout the differentiation of Th17 cells in vitro and during experimental autoimmune encephalomyelitis (EAE) development. Strikingly, PKM2 is not required for the metabolic reprogramming and proliferative capacity of Th17 cells. However, T cell-specific PKM2 deletion impairs Th17 cell differentiation and ameliorates symptoms of EAE by decreasing Th17 cell-mediated inflammation and demyelination. Mechanistically, PKM2 translocates into the nucleus and interacts with STAT3, enhancing its activation and thereby increasing Th17 cell differentiation. Thus, PKM2 acts as a critical nonmetabolic regulator that fine-tunes Th17 cell differentiation and function in autoimmune-mediated inflammation.

Wnt/ß-catenin activation cooperates with loss of p53 to cause adrenocortical carcinoma in mice.

Adrenocortical carcinoma (ACC) is a rare and aggressive malignancy with limited therapeutic options. The lack of mouse models that recapitulate the genetics of ACC has hampered progress in the field. We analyzed The Cancer Genome Atlas (TCGA) dataset for ACC and found that patients harboring alterations in both p53/Rb and Wnt/ß-catenin signaling pathways show a worse prognosis compared with patients that harbored alterations in only one. To model this, we utilized the Cyp11b2(AS)Cre mouse line to generate mice with adrenocortical-specific Wnt/ß-catenin activation, Trp53 deletion, or the combination of both. Mice with targeted Wnt/ß-catenin activation or Trp53 deletion showed no changes associated with tumor formation. In contrast, alterations in both pathways led to ACC with pulmonary metastases. Similar to ACCs in humans, these tumors produced increased levels of corticosterone and aldosterone and showed a high proliferation index. Gene expression analysis revealed that mouse tumors exhibited downregulation of Star and Cyp11b1 and upregulation of Ezh2, similar to ACC patients with a poor prognosis. Altogether, these data show that altering both Wnt/ß-catenin and p53/Rb signaling is sufficient to drive ACC in mouse. This autochthonous model of ACC represents a new tool to investigate the biology of ACC and to identify new treatment strategies.

Thirty days after anterior cruciate ligament transection is sufficient to induce signs of knee osteoarthritis in rats: pain, functional impairment, and synovial inflammation.

OBJECTIVE: To compare the unilateral signs of knee osteoarthritis (KOA) 30 and 60 days after anterior cruciate ligament transection (ACLT). Pain, gait function, synovial fluid inflammation, and histopathological changes in the synovial membrane were analyzed, as well as the interaction between the variables. MATERIALS AND METHODS: Male Wistar rats (n = 32; 219.2 ± 18.6 g) were randomly distributed into four groups of eight animals each. Two groups were submitted to unilateral ACLT surgery to induce KOA and analyzed after 30 (KOA30) and 60 days (KOA60). Two control groups (without surgery) were also assessed after the same time periods (C30 and C60). All the groups were evaluated before ACLT from the least to most stressful tests (skin temperature, mechanical response threshold, gait test, thermal response threshold, and joint swelling), as well as 30 and 60 days after surgery. After euthanasia, the synovial fluid and synovial membrane were collected. RESULTS: Thirty days after ACLT, KOA30 showed decrease paw print area and mechanical response threshold, higher joint swelling, skin temperature, leukocyte count, cytokine levels, and synovitis score. No differences were found between KOA30 and KOA60. CONCLUSION: Our data showed that 30 days after ACLT is sufficient to induce signs of KOA in rats, such as pain, functional impairment, and synovial inflammation, suggesting that a shorter time period can be used as an experimental model.

Superior vena cava obstruction after the Glenn procedure.

Acute obstruction of superior vena cava anastomosis right after the Glenn procedure may lead to tragic consequences. We describe the case of a one-year-old child with tricuspid atresia and a previous Blalock-Taussig shunt procedure, who presented severe low cardiac output syndrome right after the Glenn procedure and died forty-four hours after the procedure. The autopsy showed obstruction of the superior vena cava anastomosis. Patients that present superior vena cava syndrome and low cardiac output right after the Glenn procedure should have the surgical anastomosis revised immediately.

Clinical-like cryotherapy improves footprint patterns and reduces synovial inflammation in a rat model of post-traumatic knee osteoarthritis.

Cryotherapy is a non-pharmacological treatment commonly used to control inflammation and improve function after acute traumas. However, there are no definitive findings about its effects on chronic joint diseases such as knee osteoarthritis (KOA). The aim of this study was to investigate the effects of clinical-like cryotherapy on functional impairment and synovial inflammation in a rat model of KOA generated by anterior cruciate ligament transection (ACLT). Thirty-two male Wistar rats were randomly divided into four groups (n = 8/group): Control, KOA, KOA + Cryotherapy and KOA + Placebo. The last two groups were submitted to the relevant interventions twice a day for five days (61 to 65), with each session lasting 20 min. Gait test, skin temperature, thermal response threshold and joint swelling were assessed in all groups before ACLT surgery, and pre (60th day) and post (66th day) intervention protocols. On day 66, the animals were euthanized and exsanguinated to remove the synovial membrane for histopathological examination and synovial fluid to determine the leukocyte count and cytokine concentration. After the intervention period (66th day), footprint area only increased in the KOA + Cryotherapy group (P = 0.004; 14%) when compared to KOA and KOA + Placebo, but did not differ from controls. Cryotherapy lowered the synovial fluid leukocyte count (P < 0.0001; ≥95.0%) and cytokine concentration (P < 0.0001; ≥55%) when compared to the KOA and Placebo groups. Synovial score and synovial fibrosis did not differ in the KOA groups. In conclusion, footprint patterns improved in rats with ACLT-induced KOA as a result of clinical-like cryotherapy, which also lowered the synovial fluid leukocyte count and inflammatory cytokine concentration in these rats.

The NLRP3 inflammasome is involved with the pathogenesis of Mayaro virus.

Mayaro virus (MAYV) is an arbovirus that circulates in Latin America and is emerging as a potential threat to public health. Infected individuals develop Mayaro fever, a severe inflammatory disease characterized by high fever, rash, arthralgia, myalgia and headache. The disease is often associated with a prolonged arthralgia mediated by a chronic inflammation that can last months. Although the immune response against other arboviruses, such as chikungunya virus (CHIKV), dengue virus (DENV) and Zika virus (ZIKV), has been extensively studied, little is known about the pathogenesis of MAYV infection. In this study, we established models of MAYV infection in macrophages and in mice and found that MAYV can replicate in bone marrow-derived macrophages and robustly induce expression of inflammasome proteins, such as NLRP3, ASC, AIM2, and Caspase-1 (CASP1). Infection performed in macrophages derived from Nlrp3-/-, Aim2-/-, Asc-/-and Casp1/11-/-mice indicate that the NLRP3, but not AIM2 inflammasome is essential for production of inflammatory cytokines, such as IL-1ß. We also determined that MAYV triggers NLRP3 inflammasome activation by inducing reactive oxygen species (ROS) and potassium efflux. In vivo infections performed in inflammasome-deficient mice indicate that NLRP3 is involved with footpad swelling, inflammation and pain, establishing a role of the NLRP3 inflammasome in the MAYV pathogenesis. Accordingly, we detected higher levels of caspase1-p20, IL-1ß and IL-18 in the serum of MAYV-infected patients as compared to healthy individuals, supporting the participation of the NLRP3-inflammasome during MAYV infection in humans.

Superior vena cava obstruction after the Glenn procedure

Acute obstruction of superior vena cava anastomosis right after the Glenn procedure may lead to tragic consequences. We describe the case of a one-year-old child with tricuspid atresia and a previous Blalock-Taussig shunt procedure, who presented severe low cardiac output syndrome right after the Glenn procedure and died forty-four hours after the procedure. The autopsy showed obstruction of the superior vena cava anastomosis. Patients that present superior vena cava syndrome and low cardiac output right after the Glenn procedure should have the surgical anastomosis revised immediately.

Podocin and uPAR are good biomarkers in cases of Focal and segmental glomerulosclerosis in pediatric renal biopsies.

There are controversies whether Minimal Change Disease (MCD) and Focal and Segmental Glomerulosclerosis (FSGS) are distinct glomerular lesions or different manifestations within the same spectrum of diseases. The uPAR (urokinase-type plasminogen activator receptor) and some slit diaphragm proteins may be altered in FSGS glomeruli and may function as biomarkers of the disease in renal biopsies. Thus, this study aims to evaluate the diagnostic potential of uPAR and glomerular proteins for differentiation between MCD and FSGS in renal pediatric biopsy. Renal biopsies from 50 children between 2 and 18 years old were selected, with diagnosis of MCD (n = 29) and FSGS (n = 21). Control group consisted of pediatric autopsies (n = 15) from patients younger than 18 years old, with no evidences of renal dysfunction. In situ expressions of WT1, nephrin, podocin and uPAR were evaluated by immunoperoxidase technique. Renal biopsy of patients with MCD and FSGS expressed fewer WT1 (p≤0.0001, F = 19.35) and nephrin (p<0.0001; H = 21.54) than patients in the control group. FSGS patients expressed fewer podocin than control (p<0.0359, H = 6.655). FSGS cases expressed more uPAR than each of control and MCD (p = 0.0019; H = 12.57) and there was a positive and significant correlation between nephrin and podocin (p = 0.0026, rS = 0.6502) in these cases. Podocin had sensitivity of 73.3% and specificity of 86.7% (p = 0.0068) and uPAR had sensitivity of 78.9% and specificity of 73.3% (p = 0.0040) for diagnosis of FSGS patients. The main limitation of the study is the limited number of cases due to the difficulty in performing biopsy in pediatric patients. Podocin and uPAR are good markers for FSGS and differentiate these cases from MCD, reinforcing the theory of distinct glomerular diseases. These findings suggest that podocin and uPAR can be used as biomarkers in the routine analysis of renal biopsies in cases of podocytopathies when the lesion (sclerosis) is not sampled.

Neutrophil extracellular traps (NETs) exacerbate severity of infant sepsis.

BACKGROUND: Neutrophil extracellular traps (NETs) are innate defense mechanisms that are also implicated in the pathogenesis of organ dysfunction. However, the role of NETs in pediatric sepsis is unknown. METHODS: Infant (2 weeks old) and adult (6 weeks old) mice were submitted to sepsis by intraperitoneal (i.p.) injection of bacteria suspension or lipopolysaccharide (LPS). Neutrophil infiltration, bacteremia, organ injury, and concentrations of cytokine, NETs, and DNase in the plasma were measured. Production of reactive oxygen and nitrogen species and release of NETs by neutrophils were also evaluated. To investigate the functional role of NETs, mice undergoing sepsis were treated with antibiotic plus rhDNase and the survival, organ injury, and levels of inflammatory markers and NETs were determined. Blood samples from pediatric and adult sepsis patients were collected and the concentrations of NETs measured. RESULTS: Infant C57BL/6 mice subjected to sepsis or LPS-induced endotoxemia produced significantly higher levels of NETs than the adult mice. Moreover, compared to that of the adult mice, this outcome was accompanied by increased organ injury and production of inflammatory cytokines. The increased NETs were associated with elevated expression of Padi4 and histone H3 citrullination in the neutrophils. Furthermore, treatment of infant septic mice with rhDNase or a PAD-4 inhibitor markedly attenuated sepsis. Importantly, pediatric septic patients had high levels of NETs, and the severity of pediatric sepsis was positively correlated with the level of NETs. CONCLUSION: This study reveals a hitherto unrecognized mechanism of pediatric sepsis susceptibility and suggests that NETs represents a potential target to improve clinical outcomes of sepsis.