Eosinophilic esophagitis auxiliary diagnosis based on a peptide ligand to eosinophil cationic protein in esophageal mucus of pediatric patients.

Eosinophilic esophagitis (EoE) is a chronic inflammatory condition of the esophagus characterized by increased number of eosinophils. Currently, EoE diagnosis is based on endoscopic procedures for histopathological examination, eosinophils' counting and, often, in clinical practice, the challenge is the differentiation between EoE and gastroesophageal reflux disease (GERD). Our aim was to develop novel peptide ligand to Eosinophil cationic protein (ECP) present in EoE biopsies of patients with potential to be used for detection. We performed a comparative proteomic analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) of esophageal biopsies from pediatric patients with eosinophilic esophagitis, gastroesophageal reflux disease and control individuals. Then, phage display technology was used to select peptides against specific up-regulated protein from EoE patients. Twelve phage clones were selected after three biopanning rounds, and the best phage clone reactivity was evaluated by phage-ELISA assay using esophageal mucus samples from 94 pediatric patients. Mass spectrometry showed that eosinophil cationic protein (ECP) was one of the most up-regulated proteins in EoE patients, which is an eosinophil granule protein usually deposited on tissues to mediate remodeling, but in excess may cause fibrosis and hypertrophy, especially in allergic responses. A highly reactive ECP-ligand peptide (E5) was able to distinguish reactive mucus of EoE patients from GERD and the control individuals by Phage-ELISA, achieving a sensitivity of 84.62%, and a specificity of 82.72%. This is the first study that successfully demonstrated an antibody-like peptide targeting ECP at the esophagus mucus as a useful auxilliary tool for EoE diagnosis with a significant association with atopic disorders and dysphagia.ClinicalTrials.gov no.: NCT03069573.

Acute toxicity of Schizolobium parahyba aqueous extract in mice.

The herbal extract of Schizolobium parahyba leaves is used commonly in the Brazil central region to treat snakebites. This study evaluates the acute toxicological effects of Schizolobium parahyba aqueous extract in mice 24 h after intraperitoneal administration. Acute toxicity was evaluated using biochemical, hematological and histopathological assays. Alterations in the levels of transaminases, bilirubin, albumin and prothrombrin time were observed, and these are likely to occur due to hepatic injury, which was confirmed by light microscopy. Liver histopathological analysis revealed the presence of lymph plasmocitary inflammatory infiltrate, but no other histopathological alterations were observed in any of the other organs analysed. The data confirm the low toxicity of the extract of Schizolobium parahyba and provide a model for the selection of a dose that does not cause injuries in the organism.

BthMP: a new weakly hemorrhagic metalloproteinase from Bothrops moojeni snake venom.

In this work, a new weakly hemorrhagic metalloproteinase (BthMP) was purified from Bothrops moojeni snake venom. This enzyme was homogeneous by native and SDS-PAGE. It showed a polypeptide chain of 23.5kDa, pI=7.1, and N-terminal blocked. BthMP is comprised of high proteolytic activity on casein, fibrin and bovine fibrinogen, with no coagulating, esterase or phospholipase A(2) activities; it was inhibited by EDTA, EGTA and 1,10-phenanthroline and maintained its activity on pH from 7.0 to 9.0 and temperature from 5-40 degrees C. Assays with metal ions showed that Ca(2+) is an activator, whereas Zn(2+) and Hg(2+) inhibited about 50 and 80% of its activity, respectively. The edema evidenced the important role of the toxin in the inflammatory activity of the venom. BthMP also caused unclotting, and provoked histological alterations in the gastrocnemius muscle of mice inducing hemorrhage, necrosis and leukocytic infiltrate. The molecular mass and the inhibition assays suggest that the metalloproteinase BthMP belongs to class P-I of SVMPs.

Anti-snake venom properties of Schizolobium parahyba (Caesalpinoideae) aqueous leaves extract.

Many medicinal plants have been recommended for the treatment of snakebites. The aqueous extracts prepared from the leaves of Schizolobium parahyba (a plant found in Mata Atlantica in Southeastern Brazil) were assayed for their ability to inhibit some enzymatic and biological activities induced by Bothrops pauloensis and Crotalus durissus terrificus venoms as well as by their isolated toxins neuwiedase (metalloproteinase), BnSP-7 (basic Lys49 PLA(2)) and CB (PLA(2) from crotoxin complex). Phospholipase A(2), coagulant, fibrinogenolytic, hemorrhagic and myotoxic activities induced by B. pauloensis and C. d. terrificus venoms, as well as by their isolated toxins were significantly inhibited when different amounts of S. parahyba were incubated previously with these venoms and toxins before assays. However, when S. parahyba was administered at the same route as the venoms or toxins injections, the tissue local damage, such as hemorrhage and myotoxicity was only partially inhibited. The study also evaluated the inhibitory effect of S. parahyba upon the spreading of venom proteins from the injected area into the systemic circulation. The neutralization of systemic alterations induced by i.m. injection of B. pauloensis venom was evaluated by measuring platelet and plasma fibrinogen levels which were significantly maintained when S. parahyba extract inoculation occurred at the same route after B. pauloensis venom injection. In conclusion, the observations confirmed that the aqueous extract of S. parahyba possesses potent snake venom neutralizing properties. It may be used as an alternative treatment to serum therapy and as a rich source of potential inhibitors of toxins involved in several physiopathological human and animal diseases.

A novel highly reactive Fab antibody for breast cancer tissue diagnostics and staging also discriminates a subset of good prognostic triple-negative breast cancers.

The discovery of novel markers for breast cancer (BC) has been recently relied on antibody combinatorial libraries and selection through phage display. We constructed a recombinant Fab library, and after selections against BC tissues, the FabC4 clone was thoroughly investigated by immunohistochemistry in 232 patients with long-term follow-up. The FabC4 ligand was determined by mass spectrometry. The FabC4 expression was associated with younger age, lack of progesterone receptor, higher histological grades and non-luminal subtypes, and it also identified a subset of good prognostic triple-negative BCs, possibly targeting a conformational epitope of Cytokeratin-10 (CK10). This new CK10-epitope specific antibody may open new possibilities in diagnostic and therapeutic strategies.

Combined analysis of multiple mRNA markers by RT-PCR assay for prostate cancer diagnosis.

OBJECTIVES: To develop a semi-quantitative method for prostate cancer diagnosis and to validate this technique in clinical protocols with the use of multiplex RT-PCR assays for five different biomarkers associated with carcinogenesis, including the PCA3 gene. DESIGN AND METHODS: AR, SRD5A2, KLK2, PSMA, and PCA3 transcripts were analyzed by multiplex RT-PCR assay in 73 prostatic tissue samples from patients with prostate cancer (PCa) and benign hyperplasia (BPH). RESULTS: Significant differences were observed between cancerous and hypertrophic tissues in the relative expression of these genes. AR, KLK2, PSMA, and PCA3 genes displayed increased transcriptional levels in the cancer specimens; on the other hand, SRD5A2 mRNA levels were higher in the BPH samples. CONCLUSIONS: Our results suggest that the most promising marker for PCa diagnosis was positive PCA3 detection associated with serum PSA levels, which showed 28-fold higher chances for cancer occurrence, with 92% specificity and 94% positive predictive value.

Punçäo com agulha fina da mama: resultados e dificuldades / Breast fine needle biopsy: results ans dificulties

A punçäo com agulha fina (PAF) da mama é um importante meio diagnóstico de nódulos mamários. Sendo dependente de treinamento do profissional, seja para obter o material, seja para interpretá-lo, a técnica deve ser testada em cada instituiçäo para que se calcule os riscos de falsos diagnósticos positivos e negativos. Analisando 1.321 pacientes determinamos a acuidade diagnóstica deste exame através da comparaçäo de 496 citologias com as respectivas histologias. Obtivemos sensibilidade de 94 por cento, especificidade de 99,3 por cento, VPP de 98 por cento e VPN de 96, 7 por cento. Partindo do pressuposto de que a PAF da mama tem seu valor já estabelecido na literatura, optamos por discutir, neste trabalho, suas dificuldades principalmente falsos-positivos, falsos-negativos e materiais insatisfatórios

Ocorrência de cisticircose em necropsias realizadas em Uberlândia, Minas Gerais, Brasil / Ocurrence of cysticercosis in autopsies performed in Uberlandia, Minas Gerais, Brazil

Foram realizadas 3937 necropsias no período de 1971 a 1993 no Serviço de Anatomia Patológica do Hospital de Clínicas da Fundaçäo de Assistência, Estudo e Pesquisa de Uberlândia Universidade Federal de Uberlândia, Minas Gerais, local para onde convergem todos os casos de autópsias do município. A análise de 2862 registros cujos laudos estavam completos e com idade de um ano revelou 39 casos (1,4 por cento de cisticercose. A idade variou de 16 a 83 anos, sendo 66,5 por cento do sexo masculino. A análise da procedência demonstrou que 82,1 por cento dos indivíduos eram do Estado de Minas Gerais e 15,4 por cento de Goiás; 1 caso (2,5 por cento) näo tinha procedência anotada. Dos 39 indivíduos parasitados, 35 (89,7 por cento) apresentavam comprometimento do sistema nervoso central, isolado ou associado a outras formas clínicas da doença; em 9 ocorreu a forma cardíaca (isolada ou associada), em 4 a forma muscular (isolada ou associada) e em 4 a forma visceral (isolada ou associada) Em somente 7 casos (17,9 por cento) a cisticercose foi a causa básica do óbito