Effects of selective inhibition of nNOS and iNOS on neuropathic pain in rats.

Various animal models have been employed to understand the pathogenic mechanism of neuropathic pain. Nitric oxide (NO) is an important molecule in nociceptive transmission and is involved in neuropathic pain. However, its mechanistic actions remain unclear. The aim of this study was to better understand the involvement of neuronal and inducible isoforms of nitric oxide synthase (nNOS and iNOS) in neuropathic pain induced by chronic constriction injury (CCI) of the sciatic nerve in rats. We evaluated pain sensitivity (mechanical withdrawal thresholds using Randall and Selitto, and von Frey tests, and thermal withdrawal thresholds using Hargreaves test) prior to CCI surgery, 14 days post CCI and after intrathecal injections of selective nNOS or iNOS inhibitors. We also evaluated the distribution of NOS isozymes in the spinal cord and dorsal root ganglia (DRG) by immunohistochemistry, synthesis of iNOS and nNOS by Western blot, and NO production using fluorescent probe DAF-2 DA (DA). Our results showed higher number of nNOS and iNOS-positive neurons in the spinal cord and DRG of CCI compared to sham rats, and their reduction in CCI rats after treatment with selective inhibitors compared to non-treated groups. Western blot results also indicated reduced expression of nNOS and iNOS after treatment with selective inhibitors. Furthermore, both inhibitors reduced CCI-evoked mechanical and thermal withdrawal thresholds but only nNOS inhibitor was able to efficiently lower mechanical withdrawal thresholds using von Frey test. In addition, we observed higher NO production in the spinal cord and DRG of injured rats compared to control group. Our study innovatively shows that nNOS may strongly modulate nociceptive transmission in rats with neuropathic pain, while iNOS may partially participate in the development of nociceptive responses. Thus, drugs targeting nNOS for neuropathic pain may represent a potential therapeutic strategy.

Antinociceptive effects of treadmill exercise in a rat model of Parkinson's disease: The role of cannabinoid and opioid receptors.

In addition to motor symptoms, Parkinson's disease (PD) presents high prevalence of painful symptoms responsible for worsening quality of life of PD patients. Physical exercise can improve such painful symptoms. This study evaluated the effects of exercise on nociceptive threshold using an unilateral rat model of PD, as well as the role played by cannabinoid and opioid receptors in areas responsible for pain pathways. For PD induction, Wistar rats were injected with 6-OHDA. 15 days after, rats either remained sedentary or were forced to exercise three times a week for 40 min. Motor and nociceptive behaviors were evaluated through cylinder and mechanical hyperalgesia tests, respectively. The animals were euthanized for analysis of cannabinoid receptor type 1 (CB1) and type 2 (CB2), and µ-opioid receptor (MOR) in the anterior cingulate cortex (ACC), periaqueductal gray matter (PAG), and thalamus areas by immunohistochemistry (IHC) and Western blotting. Our data revealed a decrease in the nociceptive threshold in both forepaws after surgery; in contrast, there was improvement in painful symptoms after the exercise protocol. For cannabinoid system there were an increase in CB2 expression in the ACC and PAG, and in CB1 levels in the PAG. And for opioid system there was an increase of MOR expression in the thalamus. Thus, modulation of those receptors by physical exercise can be an important non-pharmacological intervention to reduce painful symptoms in a rat model of PD, contributing to knowledge and promotion of better treatment aimed at improving the quality of life of PD patients.

Defective expression of apoptosis-related molecules in multiple sclerosis patients is normalized early after autologous haematopoietic stem cell transplantation.

Defective apoptosis might be involved in the pathogenesis of multiple sclerosis (MS). We evaluated apoptosis-related molecules in MS patients before and after autologous haematopoietic stem cell transplantation (AHSCT) using BCNU, Etoposide, AraC and Melphalan (BEAM) or cyclophosphamide (CY)-based conditioning regimens. Patients were followed for clinical and immunological parameters for 2 years after AHSCT. At baseline, MS patients had decreased proapoptotic BAD, BAX and FASL and increased A1 gene expression when compared with healthy counterparts. In the BEAM group, BAK, BIK, BIMEL , FAS, FASL, A1, BCL2, BCLXL , CFLIPL and CIAP2 genes were up-regulated after AHSCT. With the exception of BIK, BIMEL and A1, all genes reached levels similar to controls at day + 720 post-transplantation. Furthermore, in these patients, we observed increased CD8+ Fas+ T cell frequencies after AHSCT when compared to baseline. In the CY group, we observed increased BAX, BCLW, CFLIPL and CIAP1 and decreased BIK and BID gene expressions after transplantation. At day + 720 post-AHSCT, the expression of BAX, FAS, FASL, BCL2, BCLXL and CIAP1 was similar to that of controls. Protein analyses showed increased Bcl-2 expression before transplantation. At 1 year post-AHSCT, expression of Bak, Bim, Bcl-2, Bcl-xL and cFlip-L was decreased when compared to baseline values. In summary, our findings suggest that normalization of apoptosis-related molecules is associated with the early therapeutic effects of AHSCT in MS patients. These mechanisms may be involved in the re-establishment of immune tolerance during the first 2 years post-transplantation.

Different expression patterns of LGALS1 and LGALS3 in polycythemia vera, essential thrombocythemia and primary myelofibrosis.

Despite all the knowledge, the cellular and molecular mechanisms involved in myeloproliferative neoplasm (MPN) pathophysiology remain unclear. Authors have shown galectin-1 (Gal-1) and 3 playing roles in tumour angiogenesis and fibrosis, which were correlated with poor prognosis in patients with MPN. In the present study LGALS1 and LGALS3 were differently expressed between polycythemia vera, essential thrombocythemia (ET) and primary myelofibrosis (PMF) diseases. Increased LGALS3 expression was associated with a negative JAK2 V617F status mutation in leucocytes from PMF but not in patients with ET without this mutation. However, a positive Janus kinase 2 (JAK2) V617F cell line established from patients with ET (SET-2 cells) when treated with JAK inhibitor presented high levels of LGALS3. Additionally, high LGALS1 expression was found in CD34(+) cells but not in leucocytes from patients with PMF, in absence of JAK2 V617F mutation, and also in SET-2 cells treated with JAK inhibitor. Thus, our findings indicate that differential expression of LGALS1 and/or LGALS3 in patients with MPN is linked with JAK2 V617F status mutation in these diseases and state of cell differentiation.

Cyclic adenosine monophosphate protects renal cell lines against amphotericin B toxicity in a PKA-independent manner.

Amphotericin B is the "gold standard" agent in the management of serious systemic fungal infections. However, this drug can cause nephrotoxicity, which contributes up to 25% of all acute kidney injuries in critically ill patients. Cyclic adenosine monophosphate can protect kidney cells from death due to injury or drug exposure in some cases. Hence, the objective of this work was to evaluate if cAMP could prevent cell death that occurs in renal cell lines subjected to AmB treatment and, if so, to assess the involvement of PKA in the transduction of this signal. Two different renal cell lines (LLC-PK1 and MDCK) were used in this study. MTT and flow cytometry assays showed increased cell survival when cells were exposed to cAMP in a PKA-independent manner, which was confirmed by western blot. This finding suggests that cAMP (db-cAMP) may prevent cell death caused by exposure to AmB. This is the first time this effect has been identified when renal cells are exposed to AmB's nephrotoxic potential.

Role of protein kinase A signaling pathway in cyclosporine nephrotoxicity.

Cyclosporine is an important immunosuppressive agent; however, nephrotoxicity is one of the main adverse effects. The purpose of this study was to evaluate the effect of inhibiting the protein kinase A (PKA) signaling pathway in nephrotoxicity caused by cyclosporine from the assessment of cell viability, pro-inflammatory cytokines, and nitric oxide (NO) production in LLC-PK1 and MDCK cell lines. Cyclosporine proved to be cytotoxic for both cell lines, as assessed by the mitochondrial enzyme activity assay (MTT), caused DNA fragmentation, determined by flow cytometry using the propidium iodide dye, and activated the PKA pathway (western blot assay). In MDCK cells, the inhibition of the PKA signaling pathway (H89 inhibitor) caused a significant reduction in DNA fragmentation. In both cell lines, the production of IL-6 proved to be a dependent PKA pathway, while TNF-α was not influenced by the inhibition of the PKA pathway. The NO production was increased when cells were pre-incubated with H89 followed by cyclosporine, and this production was dependent on the PKA pathway in LLC-PK1 and MDCK cells lines. Therefore, considering the present study's results, it can be concluded that the inhibition of PKA signaling pathway can aid in reducing the degree of nephrotoxicity caused by cyclosporine.

ApoptomiRs expression modulated by BCR-ABL is linked to CML progression and imatinib resistance.

BACKGROUND: Chronic myeloid leukemia (CML) is a myeloproliferative disease characterized by the presence of Philadelphia chromosome (Ph) leading to expression of a BCR-ABL1 fusion oncogene. The BCR-ABL protein has a constitutive tyrosine kinase activity which is responsible for CML pathogenesis by promoting cell apoptosis resistance; however, the cellular and molecular mechanisms associated with BCR-ABL expression and apoptosis impairment in CML leukemic cells have not been fully elucidated. METHODS: This study evaluated apoptomiRs and their predicted apoptotic genes in BCR-ABL(+) cells from patients in different phases of CML treated with tyrosine kinase inhibitor (TKI) according to their imatinib (IM) response by qPCR. Phosphotyrosine and c-ABL expressions in HL-60.BCR-ABL cells treated with TKI were done by Western blot. RESULTS: We found that dasatinib (DAS) modulated miR-let-7d, miR-let-7e, miR-15a, miR-16, miR-21, miR-130a and miR-142-3p expressions while IM modulated miR-15a and miR-130a levels. miR-16, miR-130a and miR-145 expressions were modulated by nilotinib (NIL). We observed higher miR-15a, miR-130b and miR-145; and lower miR-16, miR-26a and miR-146a expressions in CML-CP in comparison with controls. CML-AP patients showed low miR-let-7d, miR-15a, miR-16, miR-29c, miR-142-3p, miR-145, and miR-146a levels in comparison with CML-CP. We noted that the miR-26a, miR-29c, miR-130b and miR-146a expressions were downregulated in IM resistant patients in comparison with IM responsive patients. CONCLUSIONS: This study showed the modulation of apoptomiRs by BCR-ABL kinase activity and the deregulation of apoptomiRs and their predicted apoptotic target genes in different CML phases and after treatment with TK inhibitors. ApoptomiRs may be involved in the BCR-ABL(+) cell apoptosis regulation.

Alteration in cellular viability, pro-inflammatory cytokines and nitric oxide production in nephrotoxicity generation by Amphotericin B: involvement of PKA pathway signaling.

Amphotericin B is one of the most effective antifungal agents; however, its use is often limited owing to adverse effects, especially nephrotoxicity. The purpose of this study was to evaluate the effect of inhibiting the PKA signaling pathway in nephrotoxicity using Amphotericin B from the assessment of cell viability, pro-inflammatory cytokines and nitric oxide (NO) production in LLC-PK1 and MDCK cell lines. Amphotericin B proved to be cytotoxic for both cell lines, as assessed by the mitochondrial enzyme activity (MTT) assay; caused DNA fragmentation, determined by flow cytometry using the propidium iodide (PI) dye; and activated the PKA pathway (western blot assay). In MDCK cells, the inhibition of the PKA signaling pathway (using the H89 inhibitor) caused a significant reduction in DNA fragmentation. In both cells lines the production of interleukin-6 (IL)-6 proved to be a dependent PKA pathway, whereas tumor necrosis factor-alpha (TNF-α) was not influenced by the inhibition of the PKA pathway. The NO production was increased when cells were pre-incubated with H89 followed by Amphotericin B, and this production produced a dependent PKA pathway in LLC-PK1 and MDCK cells lines. Therefore, considering the present study's results as a whole, it can be concluded that the inhibition of the PKA signaling pathway can aid in reducing the degree of nephrotoxicity caused by Amphotericin B.

Incidência de endometrite citológica e desempenho reprodutivo em vacas de corte Nelore / Incidence of cytological endometritis and its effects on reproductive performance in Nelore beef cows

Avaliaram-se a incidência de endometrite citológica dos 29 aos 90 dias pós-parto e seus efeitos sobre o desempenho reprodutivo de vacas de corte Nelore submetidas a uma estação de monta (EM) de 90 dias. Foram utilizadas 49 matrizes Nelores, sem histórico de retenção de placenta, sem a presença de uma infecção uterina clínica, e com escore de condição corporal acima de 2,5. Realizou-se exame ultrassonográfico para avaliar a parede uterina e a atividade ovariana. O diagnóstico de endometrite citológica foi feito pela técnica de lavagem uterina, considerando-se caso de endometrite ≥5% de neutrófilos em cada lâmina. A incidência de endometrite citológica do rebanho foi de 22%, não diferindo entre as categorias analisadas (primíparas versus multíparas) (P>0,05), a taxa de concepção à primeira inseminação também foi semelhante entre primíparas versus multíparas (P>0,05), porém a taxa de gestação ao final da EM foi maior nas vacas multíparas (83,8%) quando comparadas às primíparas (50,0%) (P<0,05). A presença ou ausência da endometrite citológica não influenciou a taxa de concepção (P>0,05), tampouco a taxa de gestação ao final da EM (P>0,05). Conclui-se que o uso da citologia endometrial não se justifica como ferramenta de diagnóstico em vacas de corte Nelore.

Were evaluated the incidence of cytological endometritis from 29 to 90 days postpartum and its effect on the reproductive performance of Nelore beef cows submitted to a breeding season (BS) for 90 days. A total of 49 cows, with no history of retained placenta, without the presence of a clinic uterine infection, and with a body condition score above 2.5 were used. Ultrasound examination was performed to evaluate the uterine wall and ovarian activity. The cytological diagnosis of endometritis was done by uterine lavage, and endometritis was considering cases of ≥5% neutrophils in each blade. The incidence of cytological endometritis in the herd was 22%, and did not differ between the categories analyzed (primiparous versus multiparous) (P>0.05), and the conception rate for first insemination was also similar between primiparous versus multiparous (P>0.05). However, the pregnancy rate at the end of BS was higher in multiparous cows (83.8%) when compared to primiparous (50.0%) cows (P<0.05). The presence or absence of cytological endometritis did not influence the conception rate (P>0.05) nor pregnancy rate at the end of the BS (P>0.05). Therefore, it can be concluded that the use of endometrial cytological cannot be justified as a diagnostic tool in Nelore beef cows.

Up-regulation of fas and fasL pro-apoptotic genes expression in type 1 diabetes patients after autologous haematopoietic stem cell transplantation.

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by T cell-mediated destruction of pancreatic ß cells, resulting in insulin deficiency and hyperglycaemia. Recent studies have described that apoptosis impairment during central and peripheral tolerance is involved in T1D pathogenesis. In this study, the apoptosis-related gene expression in T1D patients was evaluated before and after treatment with high-dose immunosuppression followed by autologous haematopoietic stem cell transplantation (HDI-AHSCT). We also correlated gene expression results with clinical response to HDI-AHSCT. We observed a decreased expression of bad, bax and fasL pro-apoptotic genes and an increased expression of a1, bcl-x(L) and cIAP-2 anti-apoptotic genes in patients' peripheral blood mononuclear cells (PBMCs) compared to controls. After HDI-AHSCT, we found an up-regulation of fas and fasL and a down-regulation of anti-apoptotic bcl-x(L) genes expression in post-HDI-AHSCT periods compared to pre-transplantation. Additionally, the levels of bad, bax, bok, fasL, bcl-x(L) and cIAP-1 genes expression were found similar to controls 2 years after HDI-AHSCT. Furthermore, over-expression of pro-apoptotic noxa at 540 days post-HDI-AHSCT correlated positively with insulin-free patients and conversely with glutamic acid decarboxylase autoantibodies (GAD65) autoantibody levels. Taken together, the results suggest that apoptosis-related genes deregulation in patients' PBMCs might be involved in breakdown of immune tolerance and consequently contribute to T1D pathogenesis. Furthermore, HDI-AHSCT modulated the expression of some apoptotic genes towards the levels similar to controls. Possibly, the expression of these apoptotic molecules could be applied as biomarkers of clinical remission of T1D patients treated with HDI-AHSCT therapy.

Fractions composition study of the pyrolysis oil obtained from sewage sludge treatment plant.

In this work the parameters of Low Temperature Conversion--LTC were applied in a centrifuged sludge from a sewage treatment plant located in Rio de Janeiro, Brazil. Before the conversion, the sludge was dried and analyzed by TGA to observe its behavior with increasing temperature. The chemical composition of the crude pyrolysis oil was analyzed by FTIR, 1H NMR and GC-MS. The results showed that the oil is a mixture of hydrocarbons, oxygenated and nitrogenated compounds. Using a catalytic treatment it was possible to fractionate the oil where the predominant constituents were hydrocarbons showing that the cracking was effective. An important result was the difference between the calorific value of dry sludge (10 MJ kg(-1)), the pyrolysis oil (36 MJ kg(-1)) and one of the fractions separated by catalytic cracking (40 MJ kg(-1)) when compared with commercial diesel (45 MJ kg(-1)).

In vitro photodynamic inactivation of Candida spp. by different doses of low power laser light.

This study evaluated the efficacy of PDT in photoinactivation of Candida species using methylene blue (MB) and irradiation with a diode laser (660nm, 40mW). Suspensions of Candida species were obtained containing 10(6)cfu/ml, transferred to 96-holes plates and exposed to 03 doses of laser light (60J/cm(2), 120J/cm(2), 180J/cm(2)) in the presence of MB. Additional suspensions were treated with only the MB, the laser light or with 0.85% saline (control groups). After the treatments, 1µl aliquot of the suspensions was plated in duplicate on SDA. The plates were incubated at 37°C for 24-48h and after this period there was the counting of colonies (cfu/ml). The three evaluated doses determined meaningful inactivation of Candida spp. (p<0.05). The 180J/cm(2) dose was the most effective, inactivating 78% of cfu/ml. At a dose of 180J/cm(2)C. albicans was the most susceptible specie. PDT has demonstrated effectiveness in the inactivation of Candida spp.

[Thermal colorectal lesions]. / Lesões térmicas colo-rectais.

A case report of thermal burns of the rectum and colon in a 94-year-old woman. The thermal lesions were caused by a scalding water enema. Flexible sigmoidoscopy was useful in documenting the extension of the lesions and histology was helpful in documenting the diagnosis. The authors review the enema's complications.

[Acute pancreatitis of biliary etiology. The case histories of the Department of General Surgery of S. Francisco Xavier Hospital (1990-1993)]. / Pancreatite aguda de etiologia biliar. Casuística do Serviço de Cirurgia Geral do H. S. Francisco Xavier (1990-1993).

Gallstones and alcohol are the most important causes of acute pancreatitis, accounting for 80% of cases. One hundred and four cases of Acute Gallstone Pancreatitis were retrospectively studied, representing 39.5% of all cases of Acute Pancreatitis that have been treated between 1990-93. Abdominal ultrasound, demonstrating gallstones in 95% of the cases, was a very useful examination in the initial study of these patients. ERCP with sphincterotomy was performed in 25 patients: 6 in a urgent basis and the others as elective procedure. Gallstones have been treated during the initial admission in 80.6% of the cases and the others at a second admission: ERCP with sphincterotomy in 14 patients as the only etiologic treatment, open cholecystectomy in 50 cases and laparoscopic cholecystectomy in 29 cases. The overall mortality rate was 3.8%--four cases.

Enhancement of Streptococcus faecalis infection and complement depletion in yeast-treated mice.

Enhancement of Streptococcus faecalis infection and lowering of the complement level have been demonstrated in mice injected with a heat-treated suspension of baker's yeast (Saccharomyces cerevisiae). The leucocyte response to the infection was not affected. The yeast preparation showed, in vitro, an intense anti-complementary activity on mouse serum and interfered with the microbial killing function of the mouse peritoneal macrophages. No significant stimulation of the growth of S. faecalis in vitro in the presence of the yeast was observed. The enhancement of the infection in mice treated with the yeast seems to be mediated, mainly, by complement depletion.

Aspergilose pulmonar, aspectos anatomo-patologicos de um paciente portador de leucemia linfoide aguda. / Pulmonary aspergillosis, anatomo-pathological aspects of a patient with acute lymphoid leukemia

Este e o relato de um caso de um paciente com leucemia linfoide aguda em tratamento que desenvolveu uma pneumonia necrotizante bilateral por Aspergillus sp. Os autores comentam o aumento da incidencia de infeccoes oportunistas principalmente em pacientes portadores de neoplasias malignas particularmente linfomas e leucemias