Translation into Brazilian Portuguese, cultural adaptation and evaluation of the reliability of the Disabilities of the Arm, Shoulder and Hand Questionnaire

The objective of the present study was to translate, adapt and validate a Brazilian Portuguese version of the Disabilities of the Arm, Shoulder and Hand (DASH) Questionnaire. The study was carried out in two steps. The first was to translate the DASH into Portuguese and to perform cultural adaptation and the second involved the determination of the reliability and validity of the DASH for the Brazilian population. For this purpose, 65 rheumatoid arthritis patients of either sex (according to the classification criteria of the American College of Rheumatology), ranging in age from 18 to 60 years and presenting no other diseases involving the upper limbs, were interviewed. The patients were selected consecutively at the rheumatology outpatient clinic of UNIFESP. The following results were obtained: in the first step (translation and cultural adaptation), all patients answered the questions. In the second step, Spearman's correlation coefficients for interobserver evaluation ranged from 0.762 to 0.995, values considered to be highly reliable. In addition, intraclass correlation coefficients ranged from 0.97 to 0.99, also highly reliable values. Spearman's correlation coefficients and the intraclass correlation coefficients obtained during intra-observer evaluation ranged from 0.731 to 0.937 and from 0.90 to 0.96, respectively, being highly reliable values. The Ritchie Index showed a weak correlation with Brazilian DASH scores, while the visual analog scale of pain showed a good correlation with DASH score. We conclude that the Portuguese version of the DASH is a reliable instrument.

Preclinical evaluation of the antidiabetic effect of Eugenia jambolana seed powder in streptozotocin-diabetic rats

The world is facing an explosive increase in the incidence of diabetes mellitus and cost-effective complementary therapies are needed. The effects of Eugenia jambolana, a household remedy for diabetes, were studied. Streptozotocin diabetic female albino Wistar rats weighing 150-200 g (N = 6) were fed E. jambolana seed powder (250, 500 or 1000 mg/kg) for 15 days. Diabetic rats fed 500 and 1000 mg/kg seed powder showed an increase in body weight on day 20 in relation to day 5 (6 ± 4.7, 9 ± 7.8 vs diabetic control -16 ± 7.1 g, P < 0.001), a decrease in fasting blood glucose (75 ± 11.9, 123 ± 14.4 vs diabetic control -34 ± 12.1 mg/dl, P < 0.001), a difference in post-treatment fasting and peak blood glucose (38 ± 11.9, 36 ± 14.2 vs diabetic control 78 ± 11.9 mg/dl, P < 0.001), and a difference in liver glycogen (50 ± 6.8, 52 ± 7.5 vs normal control 90 ± 6.6 µg/g of liver tissue, P < 0.001). Tri-terpenoids, tannins, gallic acid, and oxalic acid were the chemical constituents detected in E. jambolana seed. The best results were obtained with an oral dose of 500 mg/kg. Subacute toxicity studies with a single administration of 2.5 and 5.0 g/kg seed powder showed no mortality or abnormality. These data on the antidiabetic effect of E. jambolana seed are adequate for approval of phase 2 clinical trials to evaluate this seed powder as complementary therapy in type 2 and type 1 diabetes.

Thermodynamic evaluation and modeling of proton and water exchange associated with benzamidine and berenil binding to ß-trypsin

Serine-proteases are involved in vital processes in virtually all species. They are important targets for researchers studying the relationships between protein structure and activity, for the rational design of new pharmaceuticals. Trypsin was used as a model to assess a possible differential contribution of hydration water to the binding of two synthetic inhibitors. Thermodynamic parameters for the association of bovine ß-trypsin (homogeneous material, observed 23,294.4 ± 0.2 Da, theoretical 23,292.5 Da) with the inhibitors benzamidine and berenil at pH 8.0, 25ºC and with 25 mM CaCl2, were determined using isothermal titration calorimetry and the osmotic stress method. The association constant for berenil was about 12 times higher compared to the one for benzamidine (binding constants are K = 596,599 ± 25,057 and 49,513 ± 2,732 M-1, respectively; the number of binding sites is the same for both ligands, N = 0.99 ± 0.05). Apparently the driving force responsible for this large difference of affinity is not due to hydrophobic interactions because the variation in heat capacity (DCp), a characteristic signature of these interactions, was similar in both systems tested (-464.7 ± 23.9 and -477.1 ± 86.8 J K-1 mol-1 for berenil and benzamidine, respectively). The results also indicated that the enzyme has a net gain of about 21 water molecules regardless of the inhibitor tested. It was shown that the difference in affinity could be due to a larger number of interactions between berenil and the enzyme based on computational modeling. The data support the view that pharmaceuticals derived from benzamidine that enable hydrogen bond formation outside the catalytic binding pocket of ß-trypsin may result in more effective inhibitors.

Pharmacokinetic and parasitological evaluation of the bone marrow of dogs with visceral leishmaniasis submitted to multiple dose treatment with liposome-encapsulated meglumine antimoniate

The aim of the present study was to evaluate the impact of a multiple dose regimen of a liposomal formulation of meglumine antimoniate (LMA) on the pharmacokinetics of antimony in the bone marrow of dogs with visceral leishmaniasis and on the ability of LMA to eliminate parasites from this tissue. Dogs naturally infected with Leishmania chagasi received 4 intravenous doses of either LMA (6.5 mg antimony/kg body weight, N = 9), or empty liposomes (at the same lipid dose as LMA, N = 9) at 4-day intervals. A third group of animals was untreated (N = 8). Before each administration and at different times after treatment, bone marrow was obtained and analyzed for antimony level (LMA group) by electrothermal atomic absorption spectrometry, and for the presence of Leishmania parasites (all groups). There was a significant increase of antimony concentration from 0.76 µg/kg wet organ (4 days after the first dose) to 2.07 µg/kg (4 days after the fourth dose) and a half-life of 4 days for antimony elimination from the bone marrow. Treatment with LMA significantly reduced the number of dogs positive for parasites (with at least one amastigote per 1000 host cells) compared to controls (positive dogs 30 days after treatment: 0 of 9 in the LMA group, 3 of 9 in the group treated with empty liposomes and 3 of 8 in the untreated group). However, complete elimination of parasites was not achieved. In conclusion, the present study showed that multiple dose treatment with LMA was effective in improving antimony levels in the bone marrow of dogs with visceral leishmaniasis and in reducing the number of positive animals, even though it was not sufficient to achieve complete elimination of parasites.

Rapid test for the evaluation of the activity of the prodrug hydroxymethylnitrofurazone in the processing of Trypanosoma cruzi messenger RNAs

No fully effective treatment has been developed since the discovery of Chagas' disease by Carlos Chagas in 1909. Since drug-resistant Trypanosoma cruzi strains are occurring and the current therapy is effectiveness in the acute phase but with various adverse side effects, more studies are needed to characterize the susceptibility of T. cruzi to new drugs. Many natural and/or synthetic substances showing trypanocidal activity have been used, even though they are not likely to be turned into clinically approved drugs. Originally, drug screening was performed using natural products, with only limited knowledge of the molecular mechanism involved in the development of diseases. Trans-splicing, which is unusual RNA processing reaction and occurs in nematodes and trypanosomes, implies the processing of polycistronic transcription units into individual mRNAs; a short transcript spliced leader (SL RNA) is trans-spliced to the acceptor pre-mRNA, giving origin to the mature mRNA. In the present study, permeable cells of T. cruzi epimastigote forms (Y, BOL and NCS strains) were treated to evaluate the interference of two drugs (hydroxymethylnitrofurazone - NFOH-121 and nitrofurazone) in the trans-splicing reaction using silver-stained PAGE analysis. Both drugs induced a significant reduction in RNA processing at concentrations from 5 to 12.5 µM. These data agreed with the biological findings, since the number of parasites decreased, especially with NFOH-121. This proposed methodology allows a rapid and cost-effective screening strategy for detecting drug interference in the trans-splicing mechanism of T. cruzi.

In vitro evaluation of the cytotoxic and trypanocidal activities of Ampelozizyphus amazonicus (Rhamnaceae)

Ampelozizyphus amazonicus Ducke is a tree commonly found in the Amazon region and an extract of its stem bark is popularly used as an antimalarial and anti-inflammatory agent and as an antidote to snake venom. Ursolic acid; five lupane type triterpenes: betulin, betulinic acid, lupenone, 3ß-hydroxylup-20(29)-ene-27,28-dioic acid, and 2a,3ß-dihydroxylup-20(29)-ene-27,28-dioic acid, and three phytosteroids: stigmasterol, sitosterol and campesterol, have been isolated from stem extracts of A. amazonicus Ducke. Their structures were characterized by spectral data including COSY and HMQC. In an in vitro biological screening of the isolated compounds, 3ß-hydroxylup-20(29)-ene-27,28-dioic acid was cytotoxic against the SKBR-3 human adenocarcinoma cell line (1 to 10 mg/mL), while 2a,3ß-dihydroxylup-20(29)-ene-27,28-dioic acid exhibited cytotoxicity against both SKBR-3 human adenocarcinoma and C-8161 human melanoma tumor cell lines (>0.1 mg/mL). In the present study, different extracts and some fractions of this plant were also investigated for trypanocidal activity due to the presence of pentacyclic triterpenes. The triterpene classes are potent against Trypanosoma cruzi. The bioassays were carried out using blood collected from Swiss albino mice by cardiac puncture during the parasitemic peak (7th day) after infection with the Y strain of T. cruzi. The results obtained showed that A. amazonicus is a potential source of bioactive compounds since its extracts and fractions isolated from it exhibited in vitro parasite lysis against trypomastigote forms of T. cruzi at concentrations >100 µg/mL. Fractions containing mainly betulin, lupenone, 3ß-hydroxylup-20(29)-ene-27,28-dioic acid, and 2a,3ß-dihydroxylup-20(29)-ene-27,28-dioic acid showed more activity than crude extracts.

Evaluation of genotype resistance testing for salvage antiretroviral therapy at AIDS care centers from Ribeirão Preto, São Paulo, Brazil

The availability of HIV-1 genotype resistance testing (GRT) to clinicians has been insufficiently studied outside randomized clinical trials. The present study evaluated the outcome of salvage antiretroviral therapy (ART) recommended by an expert physician based on GRT in a non-clinical trial setting in Ribeirão Preto, Brazil. A prospective, open, nonrandomized study evaluating easy access to GRT at six Brazilian AIDS Clinics was carried out. This cooperative study analyzed the efficacy of treatment recommended to patients whose salvage ART was guided by GRT with that of treatment with ART based only on previous ART history. A total of 112 patients with ART failure were included in the study, and 77 of them were submitted to GRT. The median CD4 cell count and viral load for these 77 patients at baseline were (mean ± SD) 252.1 ± 157.4 cells/µL and 4.60 ± 0.5 log10 HIV RNA copies/mL, respectively. The access time, i.e., the time elapsed between ordering the GRT and receiving the result was, on average, 71.9 ± 37.3 days. The study results demonstrated that access to GRT followed by expert recommendations did not improve the time to persistent treatment failure when compared to conventional salvage ART. Access to GRT in this Brazilian community health care setting did not improve the long-term virologic outcomes of HIV-infected patients experiencing treatment failure. This result is probably related to the long time required to implement ART guided by GRT.

Evaluation of a 1-h 75-g oral glucose tolerance test in the diagnosis of gestational diabetes

In order to evaluate the performance of a 1-h 75-g oral glucose tolerance test (OGTT) for the diagnosis of gestational diabetes mellitus (GDM), a cohort of 4998 women, 20 years or older, without previous diabetes being treated in prenatal care clinics in Brazil answered a questionnaire and performed a 75-g OGTT including fasting, 1-h and 2-h glucose measurements between their 24th and 28th gestational weeks. Pregnancy outcomes were transcribed from medical registries. GDM was defined according to WHO criteria (fasting: ≥126 mg/dL; 2-h value: ≥140 mg/dL) and macrosomia as a birth weight equal to or higher than 4000 g. Areas under the receiver operator characteristic curve (AUC) were compared and diagnostic properties of various cut-off points were evaluated. The AUCs for the prediction of macrosomia were 0.606 (0.572-0.637) for the 1-h and 0.589 (0.557-0.622) for the 2-h plasma glucose test. Similar predictability was demonstrable regarding combined adverse outcomes: 0.582 (0.559-0.604) for the 1-h test and 0.572 (0.549-0.595) for the 2-h test. When the 1-h glucose test was evaluated against a diagnosis of GDM defined by the 2-h glucose test, the AUC was 0.903 (0.886-0.919). The cut-off point that maximized sensitivity (83%) and specificity (83%) was 141 mg/dL, identifying 21% of the women as positive. A cut-off point of 160 mg/dL, with lower sensitivity (62%), had higher specificity (94%), labeling 8.6% as positive. Detection of GDM can be done with a 1-h 75-g OGTT: the value of 160 mg/dL has the same diagnostic performance as the conventional 2-h value (140 mg/dL). The simplification of the test may improve coverage and timing of the diagnosis of GDM.

Quantitative evaluation of experimental choroidal neovascularization by confocal scanning laser ophthalmoscopy: fluorescein angiogram parallels heparan sulfate proteoglycan expression

The objective of the present study was to develop a quantitative method to evaluate laser-induced choroidal neovascularization (CNV) in a rat model using Heidelberg Retina Angiograph 2 (HRA2) imaging. The expression of two heparan sulfate proteoglycans (HSPG) related to inflammation and angiogenesis was also investigated. CNV lesions were induced with argon laser in 21 heterozygous Zucker rats and after three weeks a fluorescein angiogram and autofluorescence exams were performed using HRA2. The area and greatest linear dimension were measured by two observers not aware of the protocol. Bland-Altman plots showed agreement between the observers, suggesting that the technique was reproducible. After fluorescein angiogram, HSPG (perlecan and syndecan-4) were analyzed by real-time RT-PCR and immunohistochemistry. There was a significant increase in the expression of perlecan and syndecan-4 (P < 0.0001) in retinas bearing CNV lesions compared to control retinas. The expression of these two HSPG increased with increasing CNV area. Immunohistochemistry demonstrated that the rat retina damaged with laser shots presented increased expression of perlecan and syndecan-4. Moreover, we observed that the overexpression occurred in the outer layer of the retina, which is related to choroidal damage. It was possible to develop a standardized quantitative method to evaluate CNV in a rat model using HRA2. In addition, we presented data indicating that the expression of HSPG parallels the area of CNV lesion. The understanding of these events offers opportunities for studies of new therapeutic interventions targeting these HSPG.

Cytogenetic characterization and evaluation of c-MYC gene amplification in PG100, a new Brazilian gastric cancer cell line

Gastric cancer is the fourth most frequent type of cancer and the second cause of cancer mortality worldwide. The genetic alterations described so far for gastric carcinomas include amplifications and mutations of the c-ERBB2, KRAS, MET, TP53, and c-MYC genes. Chromosomal instability described for gastric cancer includes gains and losses of whole chromosomes or parts of them and these events might lead to oncogene overexpression, showing the need for a better understanding of the cytogenetic aspects of this neoplasia. Very few gastric carcinoma cell lines have been isolated. The establishment and characterization of the biological properties of gastric cancer cell lines is a powerful tool to gather information about the evolution of this malignancy, and also to test new therapeutic approaches. The present study characterized cytogenetically PG-100, the first commercially available gastric cancer cell line derived from a Brazilian patient who had a gastric adenocarcinoma, using GTG banding and fluorescent in situ hybridization to determine MYC amplification. Twenty metaphases were karyotyped; 19 (95%) of them presented chromosome 8 trisomy, where the MYC gene is located, and 17 (85%) presented a deletion in the 17p region, where the TP53 is located. These are common findings for gastric carcinomas, validating PG100 as an experimental model for this neoplasia. Eighty-six percent of 200 cells analyzed by fluorescent in situ hybridization presented MYC overexpression. Less frequent findings, such as 5p deletions and trisomy 16, open new perspectives for the study of this tumor.

Evaluation of piroxicam-β-cyclodextrin as a preemptive analgesic in functional endoscopic sinus surgery

The preemptive analgesic efficacy and adverse effects of preoperatively administered piroxicam-β-cyclodextrin for post-endoscopic sinus surgery pain was determined in a prospective, double-blind, randomized, clinical study. Seventy-five American Society of Anesthesiologists status I-II patients, aged 18-65 years, were divided into three groups with similar demographic characteristics: group 1 received 20 mg piroxicam-β-cyclodextrin, group 2 received 40 mg piroxicam-β-cyclodextrin and group 3 received placebo orally before induction of general anesthesia. A blinded observer recorded the incidence and severity of pain at admission to the post-anesthesia care unit (PACU), at 15, 30, and 45 min in the PACU, and 1, 2, 4, 6, and 24 h postoperatively. All patients received patient-controlled morphine analgesia during the postoperative period and consumption was recorded for 24 h. During the PACU period, mean visual analogue scale values were significantly lower in groups 1 and 2 compared to group 3 (P < 0.05). During the postoperative period, morphine consumption was 3.03 ± 2.54, 2.7 ± 2.8, and 5.56 ± 3.12 mg for each group, respectively (P < 0.05). As a side effect, bleeding was observed in groups 1 and 3, nausea and vomiting in all groups, and edema only in group 3. However, no significant differences were detected in any of the parameters analyzed, which also included epigastric pain, constipation/diarrhea and headache. Similar hematological test results were obtained for all groups. Preemptive administration of piroxicam-β-cyclodextrin effectively reduced analgesic consumption, and 40 mg of the drug was more effective than 20 mg piroxicam-β-cyclodextrin without side effects during the postoperative period.

Evaluation of anti-Wnt/β-catenin signaling agents by pGL4-TOP transfected stable cells with a luciferase reporter system

Refractory and relapsed leukemia is a major problem during cancer therapy, which is due to the aberrant activation of Wnt/β-catenin signaling pathway. Activation of this pathway is promoted by wingless (Wnt) proteins and induces co-activator β-catenin binding to lymphoid enhancer factor (LEF)/T-cell factor protein (TCF). To provide a convenient system for the screening of anti-Wnt/β-catenin agents, we designed a bi-functional pGL4-TOP reporter plasmid that contained 3X β-catenin/LEF/TCF binding sites and a selectable marker. After transfection and hygromycin B selection, HEK 293-TOP and Jurkat-TOP stable clones were established. The luciferase activity in the stable clone was enhanced by the recombinant Wnt-3A (rWnt-3A; 100-400 ng/mL) and GSK3β inhibitor (2’Z,3’E)-6-bromoindirubin-3’-oxime (BIO; 5 µM) but was inhibited by aspirin (5 mM). Using this reporter model, we found that norcantharidin (NCTD; 100 µM) reduced 80% of rWnt-3A-induced luciferase activity. Furthermore, 50 µM NCTD inhibited 38% of BIO-induced luciferase activity in Jurkat-TOP stable cells. Employing ³H-thymidine uptake assay and Western blot analysis, we confirmed that NCTD (50 µM) significantly inhibited proliferation of Jurkat cells by 64%, which are the dominant β-catenin signaling cells and decreased β-catenin protein in a concentration-dependent manner. Thus, we established a stable HEK 293-TOP clone and successfully used it to identify the Wnt/β-catenin signaling inhibitor NCTD.

Longitudinal evaluation of hepatic osteodystrophy in children and adolescents with chronic cholestatic liver disease

Bone mass loss is a major complication of chronic cholestatic liver disease (CCD). However, the long-term impact of CCD on bone mass acquisition is unknown. We longitudinally assessed bone mineral density (BMD) and factors involved in bone remodeling in 9 children and adolescents with CCD Child-Pugh A (5 boys/4 girls) and in 13 controls (6 boys/7 girls). The groups were evaluated twice, at baseline (T0) and after 3 years (T1), when osteocalcin, deoxypyridinoline, 25-hydroxyvitamin-D, parathyroid hormone, insulin-like growth factor-I (IGF-I), and BMD (L1-L4, proximal femur and total body) were determined. Serum levels of receptor activator for nuclear factor kB ligand (RANKL) and osteoprotegerin were measured only at T1. Lumbar spine BMD was reanalyzed twice: after adjustment for bone age and to compensate for the height factor. Volumetric density was also estimated mathematically in L2-L4. The BMD of L1-L4 was lower in the CCD group (Z-score at T0: control = -1.2 ± 0.8 vs CCD = -2.2 ± 1.4, P < 0.05; T1: control = -0.7 ± 0.8 vs CCD = -2.1 ± 1.1, P < 0.05). Osteocalcin and deoxypyridinoline were similar for the two groups. The CCD group presented lower IGF-I (Z-score at T1: control = 1.4 ± 2.8 vs CCD = -1.5 ± 1.0, P < 0.05) and RANKL (control = 0.465 ± 0.275 vs CCD = 0.195 ± 0.250 pM, P < 0.05) than control. Children with compensated CCD Child-Pugh A showed early impairment of bone acquisition, with the impact being more severe in an initial phase and then tapering in a slowly progressive way. Reduction in endocrine IGF-I has a crucial role in this process.

Evaluation of the immune humoral response of Brazilian patients with Rubinstein-Taybi syndrome

Rubinstein-Taybi syndrome (RTS) is a rare developmental disorder characterized by craniofacial dysmorphisms, broad thumbs and toes, mental and growth deficiency, and recurrent respiratory infections. RTS has been associated with CREBBP gene mutations, but EP300 gene mutations have recently been reported in 6 individuals. In the present study, the humoral immune response in 16 RTS patients with recurrent respiratory infections of possible bacterial etiology was evaluated. No significant differences between patients and 16 healthy controls were detected to explain the high susceptibility to respiratory infections: normal or elevated serum immunoglobulin levels, normal salivary IgA levels, and a good antibody response to both polysaccharide and protein antigens were observed. However, most patients presented high serum IgM levels, a high number of total B cell and B subsets, and also high percentiles of apoptosis, suggesting that they could present B dysregulation. The CREBBP/p300 family gene is extremely important for B-cell regulation, and RTS may represent an interesting human model for studying the molecular mechanisms involved in B-cell development.

Evaluation of the antischistosomal activity of sulfated α-D-glucan from the lichen Ramalina celastri free and encapsulated into liposomes

The antischistosomal activity of the sulfated polysaccharide α-D-glucan (Glu.SO4) extracted from Ramalina celastri was evaluated after encapsulation into liposomes (Glu.SO4-LIPO) in Schistosoma mansoni-infected mice. The effect of treatment with Glu.SO4 and Glu.SO4-LIPO (10 mg/kg) on egg elimination, worm burden and hepatic granuloma formation was assessed using female albino Swiss mice, 35-40 days of age, weighing 25 ± 2 g, infected with 150 cercariae/animal (Biomphalaria glabrata, BH strain). Four groups (N = 10) were studied, two controls (empty liposomes and NaCl) and two treated groups (Glu.SO4-LIPO and Glu.SO4) using a single dose. Parasitological analysis revealed that Glu.SO4-LIPO was as efficient as Glu.SO4 in reducing egg elimination and worm burden. Treatment with free Glu.SO4 and Glu.SO4-LIPO induced a statistically significant reduction in the number of granulomas (62 and 63%, respectively). Lectin histochemistry showed that wheat germ agglutinin intensely stained the egg-granuloma system in all treated groups. On the other hand, peanut agglutinin stained cells in the control groups, but not in the treated groups. The present results suggest a correlation between the decreasing number of hepatic egg-granulomas and the glycosylation profile of the egg-granuloma system in animals treated with free Glu.SO4 or Glu.SO4-LIPO.