Crotoxin stimulates an M1 activation profile in murine macrophages during Leishmania amazonensis infection.

American tegumentary leishmaniasis is caused by different species of Leishmania. This protozoan employs several mechanisms to subvert the microbicidal activity of macrophages and, given the limited efficacy of current therapies, the development of alternative treatments is essential. Animal venoms are known to exhibit a variety of pharmacological activities, including antiparasitic effects. Crotoxin (CTX) is the main component of Crotalus durissus terrificus venom, and it has several biological effects. Nevertheless, there is no report of CTX activity during macrophage - Leishmania interactions. Thus, the main objective of this study was to evaluate whether CTX has a role in macrophage M1 polarization during Leishmania infection murine macrophages, Leishmania amazonensis promastigotes and L. amazonensis-infected macrophages were challenged with CTX. MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide] toxicity assays were performed on murine macrophages, and no damage was observed in these cells. Promastigotes, however, were affected by treatment with CTX (IC50 = 22·86 µg mL-1) as were intracellular amastigotes. Macrophages treated with CTX also demonstrated increased reactive oxygen species production. After they were infected with Leishmania, macrophages exhibited an increase in nitric oxide production that converged into an M1 activation profile, as suggested by their elevated production of the cytokines interleukin-6 and tumour necrosis factor-α and changes in their morphology. CTX was able to reverse the L. amazonensis-mediated inhibition of macrophage immune responses and is capable of polarizing macrophages to the M1 profile, which is associated with a better prognosis for cutaneous leishmaniasis treatment.

Collagen type IV-related nephropathies in Portugal: pathogenic COL4A3 and COL4A4 mutations and clinical characterization of 25 families.

Pathogenic mutations in genes COL4A3/COL4A4 are responsible for autosomal Alport syndrome (AS) and thin basement membrane nephropathy (TBMN). We used Sanger sequencing to analyze all exons and splice site regions of COL4A3/COL4A4, in 40 unrelated Portuguese probands with clinical suspicion of AS/TBMN. To assess genotype-phenotype correlations, we compared clinically relevant phenotypes/outcomes between homozygous/compound heterozygous and apparently heterozygous patients. Seventeen novel and four reportedly pathogenic COL4A3/COL4A4 mutations were identified in 62.5% (25/40) of the probands. Regardless of the mutated gene, all patients with ARAS manifested chronic renal failure (CRF) and hearing loss, whereas a minority of the apparently heterozygous patients had CRF or extrarenal symptoms. CRF was diagnosed at a significantly younger age in patients with ARAS. In our families, the occurrence of COL4A3/COL4A4 mutations was higher, while the prevalence of XLAS was lower than expected. Overall, a pathogenic COL4A3/COL4A4/COL4A5 mutation was identified in >50% of patients with fewer than three of the standard diagnostic criteria of AS. With such a population background, simultaneous next-generation sequencing of all three genes may be recommended as the most expedite approach to diagnose collagen IV-related glomerular basement membrane nephropathies.

Collagen type IV-related nephropathies in Portugal: pathogenic COL4A5 mutations and clinical characterization of 22 families.

Alport syndrome (AS) is caused by pathogenic mutations in the genes encoding α3, α4 or α5 chains of collagen IV (COL4A3/COL4A4/COL4A5), resulting in hematuria, chronic renal failure (CRF), sensorineural hearing loss (SNHL) and ocular abnormalities. Mutations in the X-linked COL4A5 gene have been identified in 85% of the families (XLAS). In this study, 22 of 60 probands (37%) of unrelated Portuguese families, with clinical diagnosis of AS and no evidence of autosomal inheritance, had pathogenic COL4A5 mutations detected by Sanger sequencing and/or multiplex-ligation probe amplification, of which 12 (57%) are novel. Males had more severe and earlier renal and extrarenal complications, but microscopic hematuria was a constant finding irrespective of gender. Nonsense and splice site mutations, as well as small and large deletions, were associated with younger age of onset of SNHL in males, and with higher risk of CRF and SNHL in females. Pathogenic COL4A3 or COL4A4 mutations were subsequently identified in more than half of the families without a pathogenic mutation in COL4A5. The lower than expected prevalence of XLAS in Portuguese families warrants the use of next-generation sequencing for simultaneous COL4A3/COL4A4/COL4A5 analysis, as first-tier approach to the genetic diagnosis of collagen type IV-related nephropathies.

Classification systems for lower extremity amputation prediction in subjects with active diabetic foot ulcer: a systematic review and meta-analysis.

AIM: We systematically review the available systems used to classify diabetic foot ulcers in order to synthesize their methodological qualitative issues and accuracy to predict lower extremity amputation, as this may represent a critical point in these patients' care. MATERIAL AND METHODS: Two investigators searched, in EBSCO, ISI, PubMed and SCOPUS databases, and independently selected studies published until May 2013 and reporting prognostic accuracy and/or reliability of specific systems for patients with diabetic foot ulcer in order to predict lower extremity amputation. RESULTS: We included 25 studies reporting a prevalence of lower extremity amputation between 6% and 78%. Eight different diabetic foot ulcer descriptions and seven prognostic stratification classification systems were addressed with a variable (1-9) number of factors included, specially peripheral arterial disease (n = 12) or infection at the ulcer site (n = 10) or ulcer depth (n = 10). The Meggitt-Wagner, S(AD)SAD and Texas University Classification systems were the most extensively validated, whereas ten classifications were derived or validated only once. Reliability was reported in a single study, and accuracy measures were reported in five studies with another eight allowing their calculation. Pooled accuracy ranged from 0.65 (for gangrene) to 0.74 (for infection). CONCLUSION: There are numerous classification systems for diabetic foot ulcer outcome prediction, but only few studies evaluated their reliability or external validity. Studies rarely validated several systems simultaneously and only a few reported accuracy measures. Further studies assessing reliability and accuracy of the available systems and their composing variables are needed.

Bacteremia due to Campylobacter in renal transplantation: a case report and review of literature.

Campylobacter species are the leading cause of acute bacterial diarrhea in industrialized countries. However, bacteremia is detected in <1% of patients with Campylobacter enteritis and is most likely to occur in patients who are immunocompromised or of older age. To our knowledge, only 2 cases of Campylobacter jejuni bacteremia have been reported in renal transplant recipients (RTRs). We present a case of an RTR with C. jejuni bacteremia presenting as self-limiting diarrhea followed by fever and cellulitis. The patient was successfully treated with a 2-week course of imipenem and developed no other complications. We review all cases of Campylobacter bacteremia in RTRs, and discuss clinical presentation and treatment of this potentially fatal disease.

Periodontal inflammation in renal transplant recipients receiving everolimus or tacrolimus - preliminary results.

OBJECTIVE: To compare oral health status between renal transplant recipients (RTRs) receiving tacrolimus (Tac) or everolimus (ERL) as immunosuppressive therapy. DESIGN: This study is a cross-sectional study. METHODS: Thirty-six RTRs receiving Tac and 22 RTRs receiving ERL were included in the study. Age, gender, time since transplant and pharmacological data were recorded for both groups. Oral health status was assessed through the evaluation of teeth, periodontal parameters as well as saliva flow rate and pH. RESULTS: RTRs receiving ERL were older than those receiving Tac. No differences were found between groups concerning oral hygiene habits, oral symptoms, smoking habits, unstimulated and stimulated saliva flow rate and pH, clinical attachment level or the number of decayed, missing and filled teeth. However, RTRs receiving ERL presented lower visible plaque index and lower values for bleeding on probing when compared to RTRs receiving Tac. In addition, RTRs receiving ERL presented a gingival index varying from normal to moderate inflammation whereas RTRs receiving Tac presented a gingival index varying from mild to severe inflammation. CONCLUSIONS: RTRs receiving ERL have lower periodontal inflammation when compared to RTRs receiving Tac.

Early-onset of disseminated cryptococcal infection in two renal transplant recipients.

Cryptococcosis is the third most common invasive fungal infection in organ transplant recipients after candidiasis and aspergillosis. Newly acquired and reactivation of latent infection are the major causes of infection, with typical later-onset and mainly as disseminated infection. The type and intensity of immunosuppression, diabetes mellitus and other co-morbidities as well as uremia seem to be important determinants on clinical presentation and outcome. Moreover, the diagnosis is not always apparent since it usually presents subacutely, as well as mimicking bacterial infections, which may be responsible for a delay in the diagnosis. Thus, a high degree of suspicion and need of invasive procedures for microbiological and histological evaluation are critical for definitive diagnosis and prompt institution of adequate treatment. We report two cases of disseminated cryptococcosis with different presentations and with an early-onset after renal transplantation.

Cerebral coccidioidomycosis after renal transplantation in a non-endemic area.

Coccidioidomycosis is a fungal infection caused by Coccidioides species endemic to the desert south western United States. In healthy people, manifestations range mainly from asymptomatic to mild influenza-like syndrome. In immunosuppressed patients, extrapulmonary dissemination is common. We report the case of a patient with a parenchymal central nervous system lesion. This presentation is considered very rare. The highest risk of infection after solid organ transplantation occurs during the first year. Risk factors include treatment of acute rejection. In the reported case, the infection was probably donor-derived, as our patient had no history of traveling to endemic areas. Mortality is high among transplanted recipients with coccidioidomycosis, particularly those with disseminated infection.

Rituximab for steroid-dependent nephrotic syndrome.

Minimal change disease (MCD) is characterized by marked sensitivity to glucocorticoid therapy. However, in 40 - 50% of all cases the disease presents with frequent relapses and needs repeated courses of steroids as well as additional immunosuppressive therapy including azathioprine, cyclophosphamide or cyclosporine. Because such regimens are associated with significant toxicity, the therapeutic challenge of this disease is to identify the treatment with the highest probability of producing a sustained remission with the lowest risk of toxicity. There is increasing evidence that rituximab may play an important role in the treatment of idiopathic nephrotic syndrome. Here, we present an adult patient with steroid-sensitive but high-dose steroid-dependent MCD beginning in childhood with a heavy history of multiple immunosuppressive therapy that was brought into 1 year sustained remission of proteinuria with two infusions of rituximab (375 mg/m2).

[Endovascular aneurysms repair: 100 consecutive cases outcome analysis]. / Cirurgia endovascular de aneurismas daaorta abdominal: análise de 100 casos consecutivos.

OBJECTIVE: To evaluate the effectiveness and clinical outcomes of endovascular aneurysm repair in a single regional centre Methods: The medical records one hundred consecutive patients who underwent elective endovascular repair of nonruptured infra-renal abdominal aortic aneurysm were retrospectively reviewed. The assessed outcomes were all-cause mortality, aneurysm-related mortality, incidence of perioperative complications and reinterventions. Patient demographics and procedure characteristics were also analysed. RESULTS: The patient's mean age was 74.4 years-old. There was a male preponderance, with only 5 women treated. Two-thirds were American Society of Anesthesiologists (ASA) class ≥3. Loco-regional blockade was the anaesthetic technique most commonly used (65%). There were no perioperative deaths. Medical complications occurred in 10.3% of cases, pulmonary and cardiac being the most frequent. The 30-day reintervention rate was 6.1% (SE: 2.4%). The overall median hospital length of stay was 5 days. At 8 years, all cause mortality was 28.5%(SE: 8.5%) and aneurysm-related death was 1.3% (SE: 1.3%). During the follow-up period, 87.9% (SE: 3.7%) of patients remained free from reintervention CONCLUSIONS: In our institution, EVAR is associated with no early mortality and significantly good perioperative outcomes such as low rate of systemic complications, minimal blood loss, low rate of postoperative mechanical ventilation use and short hospital stay. Although the high reintervention rate for EVAR has been confirmed in several studies, our study did not find such a high need for secondary procedures. We found a durable benefit since aneurysm-related mortality is very low and late overall survival is similar to other reports. In this study's setting, our findings support endovascular management of large AAAs.

[Hybrid treatment of an aortic arch aneurysm]. / Aneurisma do arco aórtico: cirurgia híbrida.

Traditional open repair of aortic arch aneurysms is a complex and demanding surgery that requires cardiopulmonary bypass, hypothermia and circulatory arrest, being associated with considerable morbidity and mortality. Even with significant advances in pre-operative assessment and optimization, advanced anaesthetic techniques and intensive care, conventional aortic arch repairs remain amongst the highest risk of across all surgical specialties. Therefore, a hybrid approach with sequential transposition of the supra-aortic branches and endovascular stent-graft placement is a less invasive alternative treatment that opens the indication for repair to more critical patients. We report our institution's first experience in the hybrid surgery of aortic arch aneurysms.

[Supra-aortic trunks occlusive disease: three different treatment approaches]. / Doença oclusiva dos troncos supra-aórticos: Três opções de revascularização.

Unlike carotid bifurcation atherosclerotic stenosis, supra-aortic trunks (SAT) occlusive disease is rare and its revascularization uncommon, accouting for less than 10% of the operations performed on the extracranial brain-irrigating arteries. There are three different treatment approaches: transthoracic, extra-anatomic cervical and endovascular. Endovascular repair is gaining popularity as first-line therapy for proximal lesions with favorable anatomy because of its low morbidity and rare mortality. Extra-anatomic bypass is a safe and durable reconstruction and should be considered in patients with single vessel disease, with cardiopulmonary high-risk or with limited life expectancy. If cardiac surgery is needed, central transthoracic reconstruction is preferable, and the two procedures should be combined. The long-term patency of bypasses with aortic origin, specially when multiple vessels are involved, is superior to other repair techniques. We present three clinical cases that illustrate each of these therapeutic strategies: central brachiocephalic revascularization and synchronous cardiac surgery in a patient with complex SAT atherosclerosis disease; subclavian-carotid transposition for disabling upper limb claudication; and subclavian artery stenting for subclavian-steal syndrome. Surgical approach selection should be based on the individual patient's anatomy and operative risk.

Inflammation and coagulation crosstalk induced by BJcuL, a galactose-binding lectin isolated from Bothrops jararacussu snake venom.

Inflammation and coagulopathies are important systemic events following snakebite. Snake venom galactoside-binding lectins (SVgalLs) are known modulators of the immune response with no direct effect on hemostasis. Considering the crosstalk between inflammation and coagulation, the present study investigated how BJcuL, a proinflammatory SVgalL isolated from Bothrops jararacussu venom, mediated the inflammation-induced procoagulant activity. We examined the proinflammatory cytokine production and procoagulant tissue factor (TF) activity in human whole blood and monocyte-rich cell suspension (MR-PBMC) treated with BJcuL. This lectin increased production of the cytokines TNF-α, IL-1ß and IL-6, upregulated TF expression on the cell surface, and induced procoagulant activity. The proinflammatory behavior was mediated by the direct interaction between the lectin and toll-like receptor 4, via binding to ß-galactoside-containing glycoconjugates on the cell surface, and activation of NFκ-B signaling. Interestingly, the BJcuL-induced inflammation was directly associated with the procoagulant activity of MR-PBMC cells. In whole blood culture, the lectin exhibited similar behavior, i.e. it induced cytokine production and MR-PBMC TF-mediated procoagulant activity. Therefore, the present study is the first report on the inflammation-induced procoagulant activity of SVgalLs, and it indicates that BJcuL is an important factor associated with coagulopathy in patients with snake envenomation.

Physicochemical quality of and Escherichia coli resistance profiles in urban surface waters.

Aquatic ecosystems of urban rivers are contaminated through waste disposal, which poses a public health problem. The objective of this research was to evaluate the quality of water used for recreation and public supply of six rivers in the city of Cascavel - Paraná, including Cascavel, Quati, Bezerra, Antas, Clarito and Amambay. Samples were collected every 4 months in 2017, and their physicochemical and microbiological parameters, as well as resistance profiles of strains of Escherichia coli to antimicrobials distributed by pharmacies of the primary healthcare network, were evaluated. Parameters such as water temperature, turbidity, total nitrogen, total phosphorus, total coliforms and thermotolerant coliforms showed significant differences. The allowed limit for thermotolerant coliforms, which was set by National Environment Council, Resolution 357/2005, was exceeded in all of the six analyzed rivers. It was determined that 48.1% of E. coli strains showed resistance to nine antimicrobial tested. The highest levels of resistance were found for ampicillin (27.7%), tetracycline (27.7%) and amoxicillin (24.0%). The results of this study contribute to the understanding of the hazards associated with the contamination of springs in urban centers with wastewater containing resistant bacteria. Therefore, recovery work is necessary in these areas because of the importance of these water sources for the entire western region of Paraná state.

QSPR modelling of the soil sorption coefficient from training sets of different sizes.

Quantitative structure-property relationship (QSPR) modelling has been used in many scientific fields. This approach has been extensively applied in environmental research to predict physicochemical properties of compounds with potential environmental impact. The soil sorption coefficient is an important parameter for the evaluation of environmental risks, and it helps to determine the final fate of substances in the environment. In the last few years, different QSPR models have been developed for the determination of the sorption coefficient. In this study, several QSPR models were generated and evaluated for the prediction of log Koc from the relationship with log P. These models were obtained from an extensive and diverse training set (n = 639) and from subsets of this initial set (i.e. halves, fourths and eighths). The aim of this study was to investigate whether the size of the training set affects the statistical quality of the obtained models. Furthermore, statistical equivalence was verified between the models obtained from smaller sets and the model obtained from the total training set. The results confirmed the equivalence between the models, thus indicating the possibility of using smaller training sets without compromising the statistical quality and predictive capability, as long as most chemical classes in the test set are represented in the training set.

Elevated plasma levels of hepatocyte growth factor in rats experimentally envenomated with Bothrops jararaca venom: Role of snake venom metalloproteases.

The hepatocyte growth factor (HGF)/c-met pathway, which mainly consists of HGF activator (HGFA) and its substrate HGF, protects various types of cells via anti-apoptotic and anti-inflammatory signals. Thrombin is the main physiological activator of such plasmatic pathway, and increased plasma concentrations of HGF have been considered as a molecular marker for some pathological conditions, such as disseminated intravascular coagulation. Since thrombin generation is often linked to tissue injury, and these events are common during snake venom-induced consumption coagulopathies (VICC), our goals were to examine whether Bothrops jararaca venom (Bjv), which induces VICC in vivo: (i) activates the HGF/c-met pathway in vivo and (ii) cleaves zymogen forms of HGFA and HGF (proHGFA and proHGF, respectively) in vitro. Two experimental groups (n = 6, each) of male adult Wistar rats were subcutaneously injected with 500 µL of 0.9% NaCl solution (control) or sub-lethal doses (1.6 mg/kg) of Bjv. Three hours after envenomation, whole blood samples were collected from the carotid arteries to evaluate relevant coagulation parameters using rotational thromboelastometry and fibrinogen level (colorimetric assay). Additionally, the plasma concentration of HGF was assayed (ELISA). Thromboelastometric assays showed that blood clotting and fibrin polymerization were severely impaired 3 h after Bjv injection. Total plasma HGF concentrations were almost 6-fold higher in the Bjv-injected group (410.0 ±â€¯91) compared with control values (68 ±â€¯18 pg/mL, p < 0.05). Western blotting assay showed that Bjv processed proHGFA and proHGF, generating bands resembling those generated by thrombin and kallikrein, respectively. In contrast to the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), the metalloprotease inhibitor ethylenediaminetetraacetic acid disodium salt (Na2-EDTA) strongly reduced the ability of Bjv to process proHGFA and generated one active band similar to that of thrombin. Since Bjv contains prothrombin and factor X activators, increased intravascular thrombin formation might partly explain the increased HGF levels after bothropic envenomation. In conclusion, these findings suggest that snake venom metalloproteases may be determinant for elevation of plasma levels of HGF in rats experimentally envenomated with Bjv.

Cytotoxic, genotoxic, and oxidative stress-inducing effect of an l-amino acid oxidase isolated from Bothrops jararacussu venom in a co-culture model of HepG2 and HUVEC cells.

Hepatocellular carcinoma incidence rates have increased worldwide, which encouraged the development of new chemotherapeutic drugs. l-Amino acid oxidases from snake venoms are cytotoxic towards human tumor cells in in vitro monoculture systems, which do not simulate the tumor microenvironment. We examined the antitumor potential of BjussuLAAO-II, an l-amino acid oxidase from Bothrops jararacussu venom, in hepatocarcinoma cells (HepG2) in monoculture and co-culture with human umbilical vein endothelial cells (HUVEC) in vitro. All the concentrations tested (0.25-5.00 µg/mL) were cytotoxic (MTT and clonogenic survival assays) towards HepG2 and HUVEC cells in monoculture, and increased oxidative stress by 2',7'-dichlorofluorescin diacetate fluorescence assay. Only 1.00 and 5.00 µg/mL exerted these effects in HepG2 cells co-cultured with HUVEC cells, and were genotoxic (comet assay) to HUVEC cells in monoculture. BjussuLAAO-II at 5.00 µg/mL induced DNA, but not chromosomal damage (micronucleus assay) in HepG2 cells in mono- and co-culture. The cytotoxicity and genotoxicity was more pronounced in monoculture, indicating that the tumor microenvironment influences the cellular response. BjussuLAAO-II caused cell death and DNA damage in HepG2 cells in vitro by inducing oxidative stress. Therefore, BjussuLAAO-II is a promising molecule for the development of new antitumor drugs.

Antineurotoxic activity of Galactia glaucescens against Crotalus durissus terrificus venom.

Ethanolic extract of leaves of Galactia glauscescens (GGE) at concentration of 100 and 500 microg/ml prevented the neuromuscular paralysis induced by Crotalus durissus terrificus venom on mouse phrenic nerve-diaphragm preparation.

Crotoxin Isolated from Crotalus durissus terrificus Venom Modulates the Functional Activity of Dendritic Cells via Formyl Peptide Receptors.

The Crotalus durissus terrificus rattlesnake venom, its main toxin, crotoxin (CTX), and its crotapotin (CA) and phospholipase A2 (CB) subunits modulate the immune system. Formyl peptide receptors (FPRs) and lipoxin A4 (LXA4) are involved in CTX's effect on macrophages and neutrophils. Dendritic cells (DCs) are plasticity cells involved in the induction of adaptive immunity and tolerance maintenance. Therefore, we evaluated the effect of CTX, CA or CB on the maturation of DCs derived from murine bone marrow (BM). According to data, CTX and CB-but not CA-induced an increase of MHC-II, but not costimulatory molecules on DCs. Furthermore, CTX and CB inhibited the expression of costimulatory and MHC-II molecules, secretion of proinflammatory cytokines and NF-κBp65 and p38/ERK1/2-MAPK signaling pathways by LPS-incubated DCs. Differently, CTX and CB induced IL-10, PGE2 and LXA4 secretion in LPS-incubated DCs. Lower proliferation and IL-2 secretion were verified in coculture of CD3+ cells and DCs incubated with LPS plus CTX or CB compared with LPS-incubated DCs. The effect of CTX and CB on DCs was abolished in cultures incubated with a FPRs antagonist. Hence, CTX and CB exert a modulation on functional activity of DCs; we also checked the involvement the FPR family on cell activities.