Validação de um método analítico rápido por CLAE-UV para determinação de cumarina em guaco (Mikania glomerata Sprengel) confirmado com espectrometria de massas / Validation of a rapid analytical method by HPLC for determining coumarin in guaco (Mikania glomerata Sprengel) confirmed with mass spectrometry

RESUMO A espécie Mikania glomerata Sprengel, popularmente conhecida no Brasil como guaco, é amplamente utilizada como expectorante para tratar doenças respiratórias e tem a sua atividade farmacológica atribuída principalmente a cumarina. Os resultados mostraram que o método apresenta linearidade de 0,05 a 0,8 mg mL-1. Ele foi considerado seletivo, exato e preciso. A proposta de um método rápido para determinação de cumarina em extratos de guaco torna-se interessante para a rotina de controle de qualidade industrial, visando à obtenção de medicamentos fitoterápicos padronizados.

ABSTRACT The species Mikania glomerata Sprengel, popularly known in Brazil as “guaco”, is widely used as an expectorant to treat respiratory diseases. Its pharmacological activity is mainly attributed to coumarin. The results showed that the method for determining coumarin presented linearity from 0.05 to 0.8 mg mL-1. It was considered selective, accurate, and precise according to the specific resolution from ANVISA, the Brazilian regulatory agency. The proposal of a rapid method for the determining coumarin in extracts of guaco is interesting for routine industrial quality control in order to obtain standardized, efficient, and safe phytotherapic medicines.

Effects of triamcinolone acetonide on human trabecular meshwork cells in vitro.

Aim: To study the effects of triamcinolone acetonide (TA) on cultured human trabecular meshwork (HTM) cells. Materials and Methods: HTM cells were cultured and treated with 125, 250, 500 and 1000 μg/mL concentration of TA for 24 h. The cells were treated with both crystalline TA (TA‑C) (commercial preparation) and solubilized TA (TA‑S). Cell viability was measured by a trypan blue dye exclusion test. The activity of caspse‑3/7 was measured by a fluorescence caspase kit and DNA laddering was evaluated by electrophoresis on 3% agarose gel. Levels of lactate dehydrogenase (LDH) were assessed with LDH cytotoxicity assay kit‑II. Results: Mean cell viabilities of HTM cells after 24 h exposure to TA‑C 125, 250, 500, and 1000 μg/mL were 75.4 ±2.45% (P < 0.0001), 49.43 ± 1.85% (P < 0.0001), 17.07 ± 2.39% (P < 0.0001), and 3.7 ± 0.9% (P < 0.0001), respectively, compared with the untreated HTM cells 92.49 ± 1.21%. The mean cell viabilities with 125, 250, 500, and 1000 μg/mL of TA‑S were 94.47 ± 1.60% (P > 0.05), 90.13 ± 0.40% (P < 0.01), 85.57 ± 0.47% (P < 0.001), and 71.67 ± 3.30% (P < 0.0001), respectively, compared to DMSO‑equivalent cultures. Untreated HTM control had a cell viability of 96.57 ± 1.98%. DMSO‑treated controls of 125, 250, 500, and 1000 μg/mL had a cell viability of 94.73 ± 0.57%, 96.97 ± 1.08%, 93.97 ± 1.85%, and 97.27 ± 1.15%, respectively. There was no increase of caspase‑3/7 activity in cultures treated with either TA‑C or TA‑S. DNA laddering showed no bands in the TA‑C or TA‑S treated cultures. There were significantly higher LDH release rates at all concentrations of TA‑C compared to TA‑S. Conclusions: Results show that the effect of TA‑C and TA‑S on HTM cells is due to cell death by necrosis at all concentrations except 125 μg/mL of TA‑S. Elevated levels of LDH confirmed necrotic cell death. Our study also infers the relative safety of TA‑S over TA‑C.

Glomerular damage as a predictor of renal allograft loss

Interstitial fibrosis and tubular atrophy (IF/TA) are the most common cause of renal graft failure. Chronic transplant glomerulopathy (CTG) is present in approximately 1.5-3.0 percent of all renal grafts. We retrospectively studied the contribution of CTG and recurrent post-transplant glomerulopathies (RGN) to graft loss. We analyzed 123 patients with chronic renal allograft dysfunction and divided them into three groups: CTG (N = 37), RGN (N = 21), and IF/TA (N = 65). Demographic data were analyzed and the variables related to graft function identified by statistical methods. CTG had a significantly lower allograft survival than IF/TA. In a multivariate analysis, protective factors for allograft outcomes were: use of angiotensin-converting enzyme inhibitor (ACEI; hazard ratio (HR) = 0.12, P = 0.001), mycophenolate mofetil (MMF; HR = 0.17, P = 0.026), hepatitis C virus (HR = 7.29, P = 0.003), delayed graft function (HR = 5.32, P = 0.016), serum creatinine ≥1.5 mg/dL at the 1st year post-transplant (HR = 0.20, P = 0.011), and proteinuria ≥0.5 g/24 h at the 1st year post-transplant (HR = 0.14, P = 0.004). The presence of glomerular damage is a risk factor for allograft loss (HR = 4.55, P = 0.015). The presence of some degree of chronic glomerular damage in addition to the diagnosis of IF/TA was the most important risk factor associated with allograft loss since it could indicate chronic active antibody-mediated rejection. ACEI and MMF were associated with better outcomes, indicating that they might improve graft survival.

Synergistic effect of mycophenolate mofetil and angiotensin-converting enzyme inhibitor in patients with chronic allograft nephropathy

Experimental data and few clinical non-randomized studies have shown that inhibition of the renin-angiotensin system by angiotensin-converting enzyme (ACE) associated or not with the use of mycophenolate mofetil (MMF) could delay or even halt the progression of chronic allograft nephropathy (CAN). In this retrospective historical study, we investigated whether ACE inhibition (ACEI) associated or not with the use of MMF has the same effect in humans as in experimental studies and what factors are associated with a clinical response. A total of 160 transplant patients with biopsy-proven CAN were enrolled. Eighty-one of them were on ACE therapy (G1) and 80 on ACEI_free therapy (G2). Patients were further stratified for the use of MMF. G1 patients showed a marked decrease in proteinuria and stabilized serum creatinine with time. Five-year graft survival after CAN diagnosis was more frequent in G1 (86.9 vs 67.7 percent; P < 0.05). In patients on ACEI-free therapy, the use of MMF was associated with better graft survival. The use of ACEI therapy protected 79 percent of the patients against graft loss (OR = 0.079, 95 percentCI = 0.015-0.426; P = 0.003). ACEI and MMF or the use of MMF alone after CAN diagnosis conferred protection against graft loss. This finding is well correlated with experimental studies in which ACEI and MMF interrupt the progression of chronic allograft dysfunction and injury. The use of ACEI alone or in combination with MMF significantly reduced proteinuria and stabilized serum creatinine, consequently improving renal allograft survival.

Are the current chronic allograft nephropathy grading systems sufficient to predict renal allograft survival?

A major problem in renal transplantation is identifying a grading system that can predict long-term graft survival. The present study determined the extent to which the two existing grading systems (Banff 97 and chronic allograft damage index, CADI) correlate with each other and with graft loss. A total of 161 transplant patient biopsies with chronic allograft nephropathy (CAN) were studied. The samples were coded and evaluated blindly by two pathologists using the two grading systems. Logistic regression analyses were used to evaluate the best predictor index for renal allograft loss. Patients with higher Banff 97 and CADI scores had higher rates of graft loss. Moreover, these measures also correlated with worse renal function and higher proteinuria levels at the time of CAN diagnosis. Logistic regression analyses showed that the use of angiotensin-converting enzyme inhibitor (ACEI), hepatitis C virus (HCV), tubular atrophy, and the use of mycophenolate mofetil (MMF) were associated with graft loss in the CADI, while the use of ACEI, HCV, moderate interstitial fibrosis and tubular atrophy and the use of MMF were associated in the Banff 97 index. Although Banff 97 and CADI analyze different parameters in different renal compartments, only some isolated parameters correlated with graft loss. This suggests that we need to review the CAN grading systems in order to devise a system that includes all parameters able to predict long-term graft survival, including chronic glomerulopathy, glomerular sclerosis, vascular changes, and severity of chronic interstitial fibrosis and tubular atrophy.

Contribution of CD4+ T cells to the early mechanisms of ischemia- reperfusion injury in a mouse model of acute renal failure

Renal ischemia-reperfusion (IR) injury is the major cause of acute renal failure in native and transplanted kidneys. Mononuclear leukocytes have been reported in renal tissue as part of the innate and adaptive responses triggered by IR. We investigated the participation of CD4+ T lymphocytes in the pathogenesis of renal IR injury. Male mice (C57BL/6, 8 to 12 weeks old) were submitted to 45 min of ischemia by renal pedicle clamping followed by reperfusion. We evaluated the role of CD4+ T cells using a monoclonal depleting antibody against CD4 (GK1.5, 50 æ, ip), and class II-major histocompatibility complex molecule knockout mice. Both CD4-depleted groups showed a marked improvement in renal function compared to the ischemic group, despite the fact that GK1.5 mAb treatment promoted a profound CD4 depletion (to less than 5 percent compared to normal controls) only within the first 24 h after IR. CD4-depleted groups presented a significant improvement in 5-day survival (84 vs 80 vs 39 percent; antibody treated, knockout mice and non-depleted groups, respectively) and also a significant reduction in the tubular necrosis area with an early tubular regeneration pattern. The peak of CD4-positive cell infiltration occurred on day 2, coinciding with the high expression of ßC mRNA and increased urea levels. CD4 depletion did not alter the CD11b infiltrate or the IFN-g and granzyme-B mRNA expression in renal tissue. These data indicate that a CD4+ subset of T lymphocytes may be implicated as key mediators of very early inflammatory responses after renal IR injury and that targeting CD4+ T lymphocytes may yield novel therapies.

Exploratory calcineurin inhibitor-free regimens in living-related kidney transplant recipients

Chronic allograft nephropathy is among the major causes of graft loss even in low-risk kidney transplant recipients and correlates with acute nephrotoxic events during the first year post-transplant. Therefore, calcineurin inhibitor-free regimens may improve patient and graft survival among recipients of living-related kidney transplants. To confirm this hypothesis, we evaluated the efficacy and safety of two calcineurin inhibitor-free regimens in 92 low-risk recipients of one-haplotype living-related kidney transplants. Immunosuppression consisted of tacrolimus, azathioprine and prednisone (group I, GI, N = 38), 2 doses of daclizumab, mycophenolate mofetil (MMF), and prednisone (GII, N = 33) and 2 doses of daclizumab, MMF, sirolimus and prednisone (GIII, N = 21). At 12 months, treatment failure (biopsy-confirmed acute rejection, graft loss or death) was higher in GII compared to GIII and GI (54.5 vs 24.0 vs 13.1 percent, P < 0.01, respectively). In patients of black ethnicity the incidence of acute rejection was 25 vs 83.3 vs 20 percent (P = 0.055), respectively. Patient and graft survival was comparable. There were no differences in mean creatinine or calculated creatinine clearance at 12 months. Overall incidence of post-transplant diabetes mellitus (3.3 percent) and cytomegalovirus disease (4.3 percent) was similar in all groups. Further development of effective calcineurin inhibitor-free regimens should exclude patients of black ethnicity and may need full-induction therapy, perhaps with depleting agents, and concentration-controlled use of sirolimus and MMF.

Effect of antibodies to intercellular adhesion molecule type 1 on the protection of distant organs during reperfusion syndrome in rats

We investigated kidney and lung alterations caused by intercellular adhesion molecule type 1 (ICAM-1) blockade after ischemia and reperfusion of hind limb skeletal muscles. Rats were submitted to ligature of the infrarenal aorta for 6 h. The animals were randomized into three groups of 6 rats each: group I, sacrificed after ischemia; group II, reperfusion for 24 h, and group III, reperfusion for 24 h after receiving monoclonal anti-ICAM-1 antibodies. At the end of the experiment, blood samples were collected for creatinine, lactate dehydrogenase, creatine phosphokinase, potassium, pH and leukocyte counts. Samples were taken from the muscles of the hind limbs and from the kidneys and lungs for histological analysis and measurement of the neutrophil infiltrate by myeloperoxidase staining. The groups did not differ significantly with regard to the laboratory tests. There were no major histological alterations in the kidneys. An intense neutrophil infiltrate in the lungs, similar in all groups, was detected. Myeloperoxidase determination showed that after reperfusion there was significantly less retention of polymorphonuclear neutrophils in the muscles (352 ± 70 vs 1451 ± 235 I 10² neutrophils/mg; P<0.01) and in the kidneys (526 ± 89 vs 852 ± 73 I 10² neutrophils/mg; P<0.01) of the animals that received anti-ICAM-1 before perfusion compared to the group that did not. The use of anti-ICAM-1 antibodies in this experimental model minimized neutrophil influx, thus reducing the inflammatory process, in the muscles and kidneys after ischemia and reperfusion of the hind limbs

Clinical and serologic features of 47 patients with paracoccidioidomycosis treated by amphotericin B.

No presente trabalho, foram avaliados 47 doentes com paracoccidioidomicose. Utilizou-se ficha padronizada que continha todas as informacoes clinicas, radiologicas e laboratoriais dos doentes antes, durante e apos terapia pela anfotericina B. Os resultados mostraram que a faixa etaria oscilou entre 13 a 50 anos com predominio do sexo masculino (10:1). O tabagismo e o etilismo foram os antecedentes mais referidos. Os doentes provieram de municipios vizinhos de Botucatu. Tosse expectorativa, dispneia, emagrecimento e anorexia foram as manifestacoes clinicas mais assinaladas. A forma mista predominou. Cinco pacientes nao apresentaram lesoes pulmonares.Nos demais 42 pacientes, a radiologia pulmonar revelou exsudatos intersticiais dos tipos nodular e fibrocicatricial em 85,7%, enfisema pulmonar em 37,7% e associacao com tuberculose em 14,2% dos casos. A elevacao da ureia, creatinina e do potassio foram as alteracoes laboratoriais mais frequentes decorrentes do uso de anfotericina B . A avaliacao clinico-laboratorial tardia mostrou: alteracoes eletrocardiograficas em 13; hipertensao arterial em 9 e resposta hiporreatora ao teste de Thorn em 1 doente. A reacao de fixacao do complemento foi o teste sorologico que melhor se prestou para estabelecer o criterio de cura. O insucesso terapeutico coincidiu com o emprego da dose incompleta da anfotericina B

Aspectos epidemiologicos da paracoccidioidomicose na area endemica de Botucatu (Sao Paulo - Brasil). / Epidemiological aspects of paracoccidioidomycosis in the endemic area of Botucatu (Sao Paulo - Brazil)

Estudamos o perfil epidemiologico de 176 pacientes portadores de paracoccidioidomicose atendidos no Hospital das Clinicas de Botucatu, tendo sido delimitada a area endemica correspondente e investigadas as caracteristicas de seu ecosistema. Como controle, utilizou-se grupo de 29063 pacientes com outras afeccoes, atendidos no mesmo Hospital, durante o mesmo periodo.Observou-se: 1) A relacao de pacientes masculino/feminino foi de 10,7/1; 2) 54% dos pacientes tinham entre 35 e 54 anos; 3) A frequencia de individuos pardos com paracoccidioidomicose (9,7%) foi maior quando comparada com a do grupo controle (2,7%); 4) Lavradores e pedreiros forneceram o principal grupo profissional entre os doentes com paracoccidioidomicose; 5) 80% dos pacientes originaram-se da regiao centro-sudoeste do Estado de Sao Paulo, especialmente de Botucatu, Sao Manuel e Avare (area endemica de Botucatu); 6) Esta area endemica localiza-se entre os 22 graus01' e 23 graus37' de latitude sul, possuindo temperatura anual entre 18 a 24 graus C; pluviosidade anual entre 1187 a 1463 mm; altitude entre 400 a 873 m; solos acidos e predominantemente pobre

Correlacao anatomo-clinica em glomerulopatias. Estudo retrospectivo de 65 casos em adultos. / Anatomo-clinical correlation in glomerulopathies. Retrospective study of 65 cases in adults

O presente trabalho compreende estudo anatomo-clinico retrospectivo de 65 casos de glomerulopatias em adultos. Os casos foram classificados em tres sindromes clinicas: nefritica, nefrotica, nao nefritica - nao nefrotica. A intensidade das lesoes histologicas renais foi avaliada no material de biopsia e/ou autopsia atraves de metodo semiquantitativo (0 a 4 +) obtendo-se indices de lesao glomerular, tubular, intersticio-vascular e renal total. Para cada paciente, foram estabelecidas correlacoes entre estes indices com a apresentacao clinica, niveis pressoricos, funcao renal (creatinina serica) e evolucao. No conjunto, observou-se que lesoes historicas mais graves associam-se a agravamento da funcao renal e piora da evolucao dos pacientes.A analise estatistica de regressao linear simples demonstrou correlacao significante entre os diferentes indice de lesao com os niveis de creatinina serica, porem o indice de lesao intersticio-vascular foi o que melhor se correlacionou com a funcao renal (r2 = 0,53)

Significado clinico do anticorpo antiDNA nativo pesquisado pelo metodo "Crithidia luciliae". / Clinical significance of anti-DNA antibodies using the \"Crithidia luciliae\" method

Os autores utilizaram a tecnica da Crithidia luciliae para estudar: 1) a presenca de anticorpo IgG, IgM e IgA anti-DNA nativo em 213 soros (54 pacientes) positivos para anticorpo antinucleo; 2) a correlacao entre a presenca destes anticorpos e a doenca basica dos pacientes; 3)a presenca e os titulos destes anticorpos em pacientes com LES ativo, com e sem nefrite. Observaram: 1) anticorpos anti-DNA nativo foram notados em 65,8% dos soros dos pacientes com LES; 2) anticorpos anti-DNA nativo das tres classes de imunoglobulinas foram encontrados com frequencia variavel em outras doencas auto-imunes; 3) nao houve diferenca entre a incidencia de anticorpos IgG, IgM e IgA anti-DNA nativo nos pacientes com LES ativo, com e sem nefrite; 4) os titulos de anticorpos anti-DNA nativo foram maiores nos pacientes com LES ativo com nefrite

Correlacao anatomo-cintilografica em 78 casos cirurgicos de nodulos tireoidianos foliculares unicos benignos (adenomas). / Anatomo-scintigraphic correlation in 78 surgical cases of benign follicular thyroid nodule (adenomas)

No presente trabalho, sao apresentados dados referentes a incidencia e correlacao entre achados cintilograficos e anatomo-patologicos de 78 nodulos tireiodianos foliculares unicos benignos (adenomas), encontrados em 249 tireoidectomias consecutivas realizadas no Hospital das Clinicas da Faculdade de Medicina de Botucatu, em seis anos (1973 a 1978). A maioria dos pacientes foi do sexo feminimo, com idade entre 20 e 59 anos. Em 75% dos 48 adenomas frios, observou-se concordancia entre a cintilografia e os dados morfologicos: adenoma cistico ou de tipo histologico nao-funcionante (embrionario, fetal ou macrofolicular).Nos 12 nodulos mornos, houve concordancia anatomo-cintilografica em 50% dos casos (normocaptacao e histologia de adenoma folicular simples) e discordancia em 50% (normocaptacao e adenoma cistico ou histologicamente nao-funcionante); este achado indica a importancia do exame cintilografico de perfil dos nodulos tireoidianos. Dezessete dos 18 adenomas quentes apresentaram histologia hiperplasica.O tecido tireoidiano circunvizinho aos adenomas mostrou focos histologicos de hiperplasia folicular em 8,9% e de tiroidite linfocitaria focal em 33,3% dos casos

Tirotoxicose endogena no decurso de terapeutica de bocio difuso atoxico com hormonio tiroidiano. Relato de caso e revisao da literatura. / Endogenous thyrotoxicosis on the course of nontoxic diffuse goiter treatment with thyroid hormone. A case report and review of the literature

Os autores descrevem paciente de 49 anos, branca, com bocio atoxico de longa duracao, que na vigencia de terapeutica com hormonio tiroidiano desenvolveu hipertiroidismo, com aumento do bocio. O hormonio tiroidiano foi tratada com 10mCi de 131 I. Como a melhora clinica foi discreta, foi introduzido propiltiouracil, com o que a paciente alcancou eutiroidismo. Apos 10 meses o PTU foi interrompido e a paciente permaneceu eutiroidea. A biopsia percutanea da tiroide mostrou bocio difuso atoxico, sem evidencias de tiroidite de Hashimoto. Os autores, apos discutirem varias hipoteses, concluem que, no presente caso, o bocio difuso atoxico provavelmente foi manifestacao inicial da doenca de Graves