Kappa and lambda determination by single colored immunohistochemistry and chromogenic in situ hybridization techniques in B-cell malignancies

Background: kappa and lambda light chains detection in bone marrow trephine sections help in the determination of B-cell clonality through evaluation of light chain restriction

Aim of the Work: was to compare the efficacy of single color detection-based immunohistochemistry [IHC] and chromogenic in situ hybridization [CISH] in evaluating kappa/lambda expression in tissues harboring B-lymphoid lesions

Patients and Methods: Forty patients were enrolled in this study. They were divided into three groups chronic lymphocytic leukemia [CLL/SLL] group I [n=13], non-Hodgkin lymphoma [NHL] group II [n=24] and hairy cell leukemia [HCL] group III [n=3]. The 24 NHL cases comprised of [11 diffuse large B-cell lymphoma, 6 mantle cell lymphomas, 3 marginal zone lymphoma, 2 lymphoplasmacytic lymphoma, 1 follicular lymphomas and 1 Burkitt's lymphoma]. Kappa and lambda light chains were detected in their bone marrow trephine sections using single colored immunohistochemistry, chromogenic in situ hybridization and the results were compared to the flowcytometry as reference method

Results: Light chain restriction [LCR] was detected by FCM in 100% of the cases followed by CISH [52.1%; 12/23] of the cases and finally IHC [43%; 18/40]

Conclusion: Both conventional CISH and IHC are effective in determining monoclonality in cases of mature B- cell neoplasm that has plasmacytic differentiation and with high amount of cytoplasmic Ig light chains such as MZL and LP. However, they are not effective in determining monoclonality in cases with low amount of Ig light chain such as cases of pregerminal and germinal center lymphoma. Yet, CISH is more informative than IHC due to the lack of background staining which allowed for greater discrimination between absence and presence of monoclonality

La rizobacteria promotora del crecimiento vegetal Arthrobacter agilis UMCV2 coloniza endofíticamente a Medicago truncatula / The plant growth-promoting rhizobacterium Arthrobacter agilis UMCV2 endophytically colonizes Medicago truncatula

Arthrobacter agilis UMCV2 es una bacteria rizosférica que promueve el crecimiento vegetal de plantas leguminosas proveyéndoles hierro soluble. Un segundo mecanismo de promoción se da a través de la producción de compuestos volátiles que estimulan los mecanismos de absorción de hierro. Adicionalmente, A. agilis UMCV2 tiene la capacidad de inhibir el crecimiento de organismos fitopatógenos. En el presente trabajo se emplea una combinación de las técnicas de reacción en cadena de la polimerasa cuantitativa e hibridación in situ con fluorescencia para detectar y cuantificar la presencia de la bacteria en los tejidos internos de la planta leguminosa Medicago truncatula. Nuestros resultados demuestran que A. agilis UMCV2 se comporta como una bacteria endófita de M. truncatula especialmente en medios donde el hierro está disponible.

Arthrobacter agilis UMCV2 is a rhizosphere bacterium that promotes legume growth by solubilization of iron, which is supplied to the plant. A second growth promotion mechanism produces volatile compounds that stimulate iron uptake activities. Additionally, A. agilis UMCV2 is capable of inhibiting the growth of phytopathogens. A combination of quantitative polymerase chain reaction and fluorescence in situ hybridization techniques were used here to detect and quantify the presence of the bacterium in the internal tissues of the legume Medicago truncatula. Our results demonstrate that A. agilis UMCV2 behaves as an endophytic bacterium of M. truncatula, particularly in environments where iron is available.

Expression of TIMP-2 in HPV-16 infected oral squamous cell carcinoma in patients in Iraq

Aim: To determine the expression of tissue inhibitors of metalloproteinases (TIMP-2) in oralsquamous cell carcinoma (OSCC) and the difference in its expression level between positiveand negative HPV-16 (human papilloma virus- 16) OSCC patients. Methods: This study wasconducted on 33 biopsies obtained from patients with OSCC and 10 normal oral mucosa ascontrols. In situ hybridization (ISH) was used to investigate the presence of HPV-16, whileimmunohistochemistry (IHC) was used to estimate the expression level of TIMP-2. Results: TheTIMP-2 was expressed in 27 (81.8%) of OSCC sections with no significant difference betweenits expression level in HPV-16 positive and HPV-16 negative OSCC cases (p=0.058). TIMP-2was found to be highly expressed in OSCC sections, and the presence of HPV was not relatedto its overexpression. Conclusions: The percentage of samples that appeared to accommodatedetectable HPV-16 was high, but no significant difference was observed in relation to TIMP-2expression level. Future studies with a larger number of patients are highly recommended toaddress the possible association between TIMp-2 and OSCC positive HPV-16.

Hibridación in situ fluorescente: herramienta en el diagnóstico de las hemopatías malignas / Fluorescence in situ hybridization: diagnostic tool for hematological malignancies

Introducción: las neoplasias hematológicas tienen origen clonal y se caracterizan por presentar gran heterogeneidad genética. El desarrollo de la citogenética molecular a través de la hibridación in situ por fluorescencia (FISH, por su sigla en inglés) se convirtió en un avance importante en el diagnóstico citogenético de estas neoplasias. Objetivo: describir las alteraciones cromosómicas detectadas en pacientes con neoplasias hematológicas a partir de la introducción de esta técnica. Métodos: se realizó un estudio descriptivo de tipo transversal de pacientes con neoplasias hematológicas en el Laboratorio de Citogenética del Instituto de Hematología e Inmunología (IHI), en el período comprendido entre julio de 2014 y abril de 2015. Se utilizó la técnica de FISH con las sondas fluorescentes específicas. Resultados: se estudiaron 87 muestras correspondientes a diferentes tipos de neoplasias hematológicas. Con la sonda LSI BCR/ABL se observaron 18 casos positivos de leucemia mieloide crónica y los ocho pacientes con leucemia linfoide aguda fueron negativos. Se marcaron con sonda PML/RARα 17 muestras con diagnóstico de leucemia promielocítica: 10 fueron positivas. Se procesaron 8 muestras con la sonda LSI RUNX1/RUNX1T1, una resultó positiva. Dos muestras marcadas con sonda LSI RB1 (13q14) y una con LSI TP53 (17p13.1), resultaron negativas. se observó un caso positivo de deleción 7q31. Conclusiones: a pesar de que la muestra estudiada es pequeña, resulta importante reportar los primeros resultados como evidencia de la incorporación de la técnica de FISH en el IHI, lo que constituye una nueva herramienta para el diagnóstico, pronóstico y seguimiento de las neoplasias hematológicas(AU)

Introduction: hematological neoplasias have clonal origin and are characterized by great genetic heterogeneity. The development of molecular cytogenetic through fluorescence in situ hybridization (FISH) became a major advance in the cytogenetic diagnosis of these neoplasias. Aim: to describe chromosomal abnormalities detected in patients with hematological malignancies after the introduction of this technique. Methods: a descriptive cross-sectional study of patients with hematological malignancies was performed. Their bone marrow samples were processed at the Laboratory of Cytogenetics of the Institute of Hematology and Immunology, between July 2014 and April 2015. FISH technique was used along with various fluorescent probes. Results: 87 samples were studied. With LSI BCR / ABL probe, 18 samples were positive of chronic myeloid leukemia and 8 patients with diagnostic of acute lymphoblastic leukemia were negative. With PML/RARα probe 17 samples of patients with promyelocytic leukemia were labeled, 10 were positive. Eight samples were labeled with probe RUNX1 / RUNX1T1, one was positive. Two samples for LSI probes labeled RB1 (13q14) and one with LSI TP53 (17p13.1) were negative. One positive case 7q31 deletion was observed. Conclusions: despite the sample is small, we consider it important to report our first results as evidence of the incorporation of the FISH technique at the IHI, which constitutes a new tool for the diagnosis, prognosis and monitoring of hematological malignances(AU)

The first report of the vanC1 gene in Enterococcus faecium isolated from a human clinical specimen

The vanC1 gene, which is chromosomally located, confers resistance to vancomycin and serves as a species marker for Enterococcus gallinarum. Enterococcus faecium TJ4031 was isolated from a blood culture and harbours the vanC1gene. Polymerase chain reaction (PCR) assays were performed to detect vanXYc and vanTc genes. Only the vanXYc gene was found in the E. faecium TJ4031 isolate. The minimum inhibitory concentrations of vancomycin and teicoplanin were 2 µg/mL and 1 µg/mL, respectively. Real-time reverse transcription-PCR results revealed that the vanC1and vanXYc genes were not expressed. Pulsed-field gel electrophoresis and southern hybridisation results showed that the vanC1 gene was encoded in the chromosome. E. faecalis isolated from animals has been reported to harbour vanC1gene. However, this study is the first to report the presence of the vanC1gene in E. faecium of human origin. Additionally, our research showed the vanC1gene cannot serve as a species-specific gene of E. gallinarum and that it is able to be transferred between bacteria. Although the resistance marker is not expressed in the strain, our results showed that E. faecium could acquire the vanC1gene from different species.

Clasificación molecular del cáncer de mama, obtenida a través de la técnica de hibridación in situ cromogénica (CISH) / Molecular classification of breast cancer patients obtained through the technique of chromogenic in situ hybridization (CISH)

El cáncer de mama es una enfermedad heterogénea compuesta de un número creciente de subtipos biológicos, con una sustancial variabilidad en la evolución de la enfermedad dentro de cada categoría. El objetivo del presente trabajo fue clasificar las muestras objeto a estudio de acuerdo a las clases moleculares de carcinoma de mama: luminal A, luminal B, HER2 y triple negativo, considerando el estado de amplificación de HER2 obtenido a través de la técnica de hibridación in situ cromogénica (CISH). La muestra estuvo constituida por 200 biopsias fijadas en formol al 10%, procesadas por las técnicas habituales hasta la inclusión en parafina, correspondientes a pacientes diagnosticadas con carcinoma ductal infiltrante de la mama, procedentes de consulta privada y del Instituto de Oncología “Dr. Miguel Pérez Carreño”, con estudio inmunohistoquímico (IHQ) para receptores hormonales y HER2 realizado en el Hospital Metropolitano del Norte de Valencia, Venezuela. La clasificación molecular de los tumores de las pacientes, considerando la expresión de los Receptores de Estrógeno (RE) y Receptores de Progesterona (RP) a través de IHQ y la amplificación de HER2 por CISH, permitió agrupar en las diferentes clases moleculares los casos calificados inicialmente como desconocidos, debido a que tenían un resultado indeterminado (2+) para la expresión de HER2 por IHQ; asimismo, esta clasificación ocasionó que algunos casos considerados inicialmente en una clase molecular pasaron a otra clase, posterior a la revaloración del estado de HER2 a través de CISH.

Breast cancer is a heterogeneous disease composed of a growing number of biological subtypes, with substantial variability of the disease progression within each category. The aim of this research was to classify the samples object of study according to the molecular classes of breast cancer: luminal A, luminal B, HER2 and triple negative, as a result of the state of HER2 amplification obtained by the technique of chromogenic in situ hybridization (CISH). The sample consisted of 200 biopsies fixed in 10% formalin, processed by standard techniques up to paraffin embedding, corresponding to patients diagnosed with invasive ductal carcinoma of the breast. These biopsies were obtained from patients from private practice and the Institute of Oncology “Dr. Miguel Pérez Carreño", for immunohistochemistry (IHC) of hormone receptors and HER2 made in the Hospital Metropolitano del Norte, Valencia, Venezuela. The molecular classification of the patient’s tumors considering the expression of estrogen and progesterone receptors by IHC and HER2 amplification by CISH, allowed those cases originally classified as unknown, since they had an indeterminate (2+) outcome for HER2 expression by IHC, to be grouped into the different molecular classes. Also, this classification permitted that some cases, initially considered as belonging to a molecular class, were assigned to another class, after the revaluation of the HER2 status by CISH.

Quick and inexpensive method to elaborate tissue punches useful in paraffin tissue microarrays / Método rápido y económico para la elaboración de dispositivos de punch útiles en microarreglos de tejidos en parafina

The tissue microarrays (TMAs) were first called multitumor block. In 1998 was described the current technique, that uses an innovated sampling method for more than 1,000 cylindrical paraffin tissue core biopsies in a single paraffin block. TMAs are now considered as a useful powerful research tool in Histology and Pathology laboratories, for the standardization of immunohistochemical techniques along with in situ hybridization. However, one disadvantage to its widespread use is the high cost of professional paraffin tissue punches, and the complexity in the development of homemade devices previously described in other studies. This study describes a step by step process to develop four different home-made devices made with materials that are common in hospitals and offices. These devices are useful in Histopathology laboratories to obtain paraffin blocks with until 360 samples of tissue, investing from two to fifteen dollars in the development of each device described.

Los microarreglos de tejido (TMAs) fueron llamados por primera vez como bloque multitumor. En 1998 se describió la técnica actual, que utiliza un novedoso método de muestreo para obtener más de 1,000 cilindros de biopsias de tejidos incluidos en un solo bloque de parafina. Actualmente, los TMAs se consideran una poderosa herramienta de investigación en laboratorios de Histología y Patología, para la estandarización de técnicas inmunohistoquímica e hibridación in situ entre otras. Sin embargo, uno de los inconvenientes para su uso generalizado es el alto costo de los dispositivos profesionales para tejidos en parafina, y la complejidad en la elaboración de los dispositivos caseros descritos previamente en otros estudios. Este estudio describe paso a paso el proceso de elaboración de cuatro dispositivos caseros útiles para la obtención de matrices de tejido elaborados con materiales que son comunes en hospitales y oficinas. Estos dispositivos son útiles en laboratorios de Histopatología con el fin de obtener bloques de parafina de hasta 360 muestras de tejido, con una inversión de 2 a 15 dólares en la elaboración de cada uno de los dispositivos descritos.

Identificação molecular do HPV em infecções do colo uterino no Brasil: revisão / Molecular identification of HPV infections in cervical cancer in Brazil: review

A infecção pelo Papilomavírus humano (HPV) é o principal responsável pela ocorrência do câncer cervical, sendo que o seu estudo por meio de técnicas moleculares sofreu um aumento significativo na última década. Objetivo: Analisar as publicações sobre a identificação molecular do HPV no colo uterino no Brasil. Metodologia:Trata-se de revisão sistemática nos portais PubMed e Biblioteca Virtual em Saúde entre os anos de 2007 a 2012, utilizando os termos: “human papillomavirus”, “cervical cancer”, “polymerase chain reaction” e “Brazil”. Dos 37 artigos identificados, 16 permaneceram após leitura dos mesmos na integra, sendo excluídos: os disponíveis apenas o resumo; os estudos que focaram a população masculina; os retrospectivos; com dados dos tipos de HPV indisponíveis; estudos experimentais; comparativo de técnicas; e de variantes intratípicas. De posse desses artigos, realizou-se a distribuição entre a frequência dos tipos de HPV em relação às diferentes técnicas de genotipagem e regiões do Brasil onde ocorreram as pesquisas que originaram os artigos. Resultados: Houve um predomínio das publicações na região Sudeste (43,7%), seguido pelo Nordeste (25,0%) e Sul (18,7%). Dos 16 artigos incluídos, observou-se uma maior frequência pelo HPV tipo 16 seguido do 31. Conclusão:As técnicas de diagnóstico moleculares são importantes ferramentas para a identificação dos tipos de HPV presentes em infecções do colo uterino, observando a necessidade de identificação dos genótipos que predominam na população brasileira, com a finalidade de melhoria na elaboração de políticas públicas em saúde.

he infection by human papillomavirus (HPV) is the main responsible for the occurrence of cervical cancer, and their study using molecular techniques has increased significantly in the last decade. Objective: To analyze the publications on the molecular identification of HPV in cervical cancer in Brazil. Methodology: This is a systematic review in PubMed and Virtual Health Library postals between the years 2007 and 2012 using the terms: “humanpapillomavirus”, “cervical cancer”, “polymerasechainreaction” and “Brazil.” Out of the 37 articles identified, 16 remained after their fully reading, being excluded: available only the summary, the studies that focused on the male population; retrospective; with data of HPV types unavailable; experimental studies, comparative techniques; and intratypical variants. Based on these articles the frequency distribution of HPV types was held relative to different genotyping techniques and regions of Brazil where the articles research took place. Results: There was a predominance of publications in the Southeast (43.7%), followed by the Northeast (25.0%) and South (18.7%) out of the 16 articles included, we observed a higher frequency of HPtion of HPV types in cervical infections, observing the need for identification of genotypes that predominate in the Brazilian population, with the aim of improving the development of public health policies.

Análise crítica dos métodos moleculares para detecção do papilomavírus humano: revisão da literatura / Molecular methods for human papillomavirus detection: literature review

O papilomavírus humano (HPV) é um vírus que infecta o epitélio cutâneo e das mucosas. Células infectadas por este vírus perdem a capacidade de controlar o ciclo celular e passam a proliferar descontroladamente gerando alterações displásicas que podem progredir para lesões malignas. Existem mais de 200 tipos de HPV e entre eles aqueles que apresentam maior ou menor risco de causar câncer. Dessa forma, o HPV pode ser classificado como sendo de baixo risco ou alto risco. Os métodos mais utilizados em pesquisa para análise molecular do HPV é a hibridização in situ (ISH), reação em cadeia da polimerase (PCR) que varia desde a PCR alelo específica, passando pelo tipo Nested e a PCR multiplex, e a mais nova técnica baseada na tecnologia de microarray. A maioria destes testes é realizada apenas em centros de pesquisa, e não rotineiramente na clínica. Por outro lado, o teste de captura híbrida II para HPV, baseado na hibridização do DNA, é comercialmente disponível. As técnicas para detecção do DNA do HPV e sua genotipagem variam quanto a sua sensibilidade e especificidade. Técnicas que utilizam sondas como a hibridização in situ e o Southern blotting são as menos sensíveis para detecção da sequência do DNA, enquanto que as mais sensíveis são as técnicas que utilizam a amplificação do DNA alvo, como a PCR e a qPCR. À medida que a tecnologia avança, as técnicas moleculares vão se aprimorando para a detecção do HPV. O objetivo final é desenvolver uma metodologia de baixo custo que apresente resultados rápidos e eficientes.

The human papillomavirus (HPV) is a virus that infects the skin and mucosal epithelium. Infected cells lost the ability to control cell cycle and begin to proliferate uncontrollably causing dysplastic alterations that can progress to malignant lesions. There are over 200 types of HPV with higher or lower risk of causing cancer. Thereby, HPV can be classified as high risk or low risk. The methods used in research for molecular analysis of HPV is the in situ hybridization (ISH), polymerase chain reaction (PCR) that varies from the allele specific PCR, Nested, PCR multiplex, and the newest technique based on microarray technology. Most of these tests are performed only in research centers, and not routinely in the clinic. An exception is the Hybrid Capture II test for HPV. The detection techniques of HPV and its genotyping vary in their sensitivity and specificity. Techniques that use probes, as in situ hybridization and Southern blotting are less sensitive for detection of DNA sequence, while the most accurate are the techniques based on DNA amplification, such as PCR and qPCR. As technology advances, molecular techniques become more accurate for the detection of HPV. The ultimate goal is to develop an inexpensive method to provide rapid and efficient results.

HER 2 status in invasive breast cancer: Immunohistochemistry, fluorescence in-situ hybridization and chromogenic in-situ hybridization.

Introduction : HER2/neu gene status in breast cancers can be evaluated by targeting protein and gene - immunohistochemistry (IHC) and fluorescence in-situ hybridization (FISH). Recent studies have shown chromogenic in-situ hybridization (CISH) as a relatively cheaper alternative. Materials and Methods : Forty-three nonconsecutive, randomly selected primary invasive breast cancer cases were evaluated for c-erbB-2 (HER2 protein) by IHC and gene amplification by FISH and CISH. Results of each of the same were compared. Results : CISH showed approximately 90% and 100% concordance for IHC negative and positive cases, respectively; while approximately 94.4% and 91% concordance with FISH amplified and non-amplified cases, respectively. Conclusion : This study showed feasibility of incorporation of CISH as a low cost option in routine management of breast carcinoma in the Indian setting. Secondly, reconfirmation of IHC negative and positive cases can be done by CISH.

Aplicabilidad de un nuevo dispositivo mecánico para hibrid disección submucosal endoscópica en modelos experimentales exvivo / Applicability of a new mechanical device for ebdiscopic sub mucosal hybrid dissection in live experimental models

La disección submucosal endoscópica es una técnica terapéutica prometedora para la resección en bloque de tumores gastrointestinales. Esta técnica tiene sus desventajas, tiempo de intervención largo, complejidad del procedimiento, y tasa de complicaciones. Mostrar la aplicabilidad y seguridad de la Hibrid disección submucosal endoscópica en estómago de cerdos exvivos utilizando dispositivo mecánico para elevar la pieza a disecar. 4 endoscopistas expertos en Hibrid disección submucosal endoscópica en modelos experimentales ex vivos realizaron disección submucosal endoscópica utilizando la técnica Hibrid Knife y dispositivo mecánico por fuera del canal de trabajo para elevar la pieza a disecar. Tiempo de intervención y tasa de complicaciones durante la introducción del dispositivo y la disección submucosal endoscópica (DSE) fue documentada. 11 procedimientos fueron realizados. En los primeros 4 procedimientos, el tiempo promedio de DSE fue 6.5 min (lesiones < 2cms), en los siguientes 4 procedimientos 9.5 min (lesiones entre 2- 3 cms) y en los 3 últimos procedimientos 10.33 min (lesiones > 3 cms). No hubo complicaciones. En nuestro trabajo el uso del dispositivo mecánico pareciera facilitar la técnica Hibrid-DSE haciéndola menos laboriosa, disminuyendo el tiempo y la tasa de complicaciones, se necesitaran estudios in vivo

The endoscopic submucosal dissection is a promising therapeutic technique for bloc resection of gastrointestinal tumors. However, this technique has some disadvantages like long intervention time, complexity of procedure and complications rate. To show the applicability and security of the endoscopic submucosal hybrid dissection in pigs stomach alive, using a mechanical device to lift up the piece to be dissected. 4 endoscopic submucosal hybrid dissection expert physicians, in live experimental models performed the submucosal endoscopic dissection using the Hybrid Knife technique and the mechanical device out of the working channel, in order to lift up the piece to be dissected.The intervention time and difficulty rate during the device introduction and the ESD (Endoscopic Sub mucosal Dissection) were documented. A total of 11 procedures were performed. In the first 4 procedures, the average time of DSE was 6.5 min (lesions < 2cms), in the following 4 procedures 9.5 min (lesions between 2 to 3 cm) and in the last 3 procedures was 10.33 min (lesions > 3 cm). There were no complications. In our work, to use the mechanical device seems to ease the Hybrid ESD, making it less difficult, diminishing time and complication rates. Live study will be necessary

Epstein-Barr virus-associated gastric carcinoma in Brazil: comparison between in situ hybridization and polymerase chain reaction detection

Epstein-Barr virus (EBV) has been associated with 10% of gastric carcinomas. The aim of this study was to determine the frequency of EBV in gastric carcinomas in Brazil assessed by in situ hybridization (ISH) and PCR, which would contribute to the characterization of the clinical and pathological aspects of EBV-associated gastric carcinomas. One hundred and ninety-two gastric carcinoma cases were collected at hospitals in two Brazilian states. Seventy-three out of 151 cases were PCR(+), while 11/160 cases were ISH(+). Nine out of eleven ISH(+) cases displayed a diffuse staining pattern and 2 out of 11 a focal pattern. Both techniques showed that the EBV(+) cases were characterized by their association with males, older patients, lower gastric region, intestinal type, advanced stage and poorly to moderately differentiated tumors. The concordance between the two techniques was 55.8% (Cohen's kappa index = 0.034). Four cases were ISH(+)/PCR(-), while 49 cases were PCR(+)/ISH(-). Only two cases showed stained lymphocytes by ISH and one of them was PCR(-). The observed discrepancy between the two techniques could not be explained just by the elevated accuracy of PCR. ISH(+)/PCR(-) carcinomas may be encountered if EBV is not present in the whole tumor tissue or if there are polymorphisms in the sequences of the viral genome amplified. On the other hand, the high frequency of PCR(+) results associated with the absence of ISH staining in lymphocytes and/or tumors cells suggests that the virus may be present in tumor cells or other cell types without expressing EBER1, the target of the ISH technique.

Uso de la hibridación IN SITU fluorescente (FIH) para la identificación de bacteririemias: evaluación del impacto de una técnica rápida de diagnóstico bacteriologico / Using fluorescence in situ hybridization (FIH) to identify bacteririemias: assessing the impact of rapid diagnostic technique bacteriological

La detección e identificación de microorganismos en la sangre de un paciente es uno de los diagnóstico más importantes del Laboratorio de Microbiología Clínica. La bacteriemia se define como la presencia de bacterias viables en la sangre circulante. Cuando las bacterias se multiplican a una velocidad tal que excede la capacidad del sistema retículo endotelial de removerlas se produce la septicemia, que puede originar infecciones graves y generalizadas como el fallo multiogánico séptico. Las técnicas microbiológicas tradicionales se basan en el cultivo de sangre - hemocultivo - con el objeto de recuperar las bacterias viables, identificarlas y realizar los ensayos de sensibilidad, El primer informe de un hemocultivo positivo está basado en la realización de una coloración de Gram sobre el caldo de cultivo. La variabilidad de las caracteristicas morfológicas y de tinción de los gérmenes, ocasionada por las condiciones de cultivo, y la falta de sensibilidad son los principales condicionantes para que la coloración de Gram sólo tenga un valor orientativo más para el microbiólogo que para el médico.

SUMMARY: The detection and identification fo microorganisms in a patient´s blood in one of the most important diagnostic tasks of the clinical microbiology laboratory. Bacteremia was defined as the presence of viable bacteria in the blood stream. When remove them, septicemia occurs. This can cause widespread and serious infections such as septic multi-organic failure. The traditional microbiological techniques based on blood culture, in order to recover viable bacteria, identify and perform susceptibility testing. The first report of a positive blood culture is based on performing a Gram stai directly from the broth culture.

RIC-8B, uma GEF putativa do sistema olfatório, interage com G´ALFA´olf, G´BETA´1, e G´GAMA´13 / RIC-8B, a putative GEF of the olfactory system, interacts with G´ALFA´olf, G´BETA´1, e G´GAMA´13

O sistema olfatório de mamíferos é capaz de detectar milhares de substâncias químicas diferentes, mesmo em baixas concentrações. Um odorante disperso no ar pode se ligar a um receptor olfatório (OR) iniciando o processo de detecção. Os ORs são membros da super família de receptores acoplados a proteína G (GPCRs). Apesar de a via de transdução de sinal de odorantes estar bem descrita, pouco se sabe sobre os seus moduladores. Em 2005, nosso laboratório identificou RIC-8B como um possível fator de troca de nucleotídeos de guanina (GEF) que poderia amplificar a atividade da proteína G olfatória (Golf). No presente trabalho mostramos que RIC-8B é capaz de interagir com Gγ13. Procurando os outros componentes desse complexo identificamos Gβ1 como sendo a subunidade Gβ mais expressa no epitélio olfatório...

Simultaneous presence of bovine papillomavirus and bovine leukemia virus in different bovine tissues: in situ hybridization and cytogenetic analysis

Bovine papillomavirus (BPV) DNA sequences were detected in different tissues, in addition to epithelium. Cytogenetic abnormalities were observed in blood lymphocytes. The presence of more than one virus in a single tissue is a difficult aspect to evaluate, especially when the DNA sequences are detected in tissues that are not specifically targeted by the virus. BPV and bovine leukemia virus (BLV) are clastogenic, causing chromosome aberrations in peripheral blood lymphocytes. In the present study, we investigated the simultaneous presence of DNA sequences of both viruses and the possibility of vertical transmission and compared the types of chromosome aberrations related to viral action. BPV 1, 2, and 4 DNA sequences were found in three females of the herd and in their offspring. BLV DNA sequences were not detected in their progeny. A newborn calf that was negative for BLV infection showed specific chromosome rearrangements possibly related to the effect of infection with BPV.

Localization of HSP single-copy genes by inexpensive, permanent non-fluorescent in situ hybridization on meiotic chromosomes of the grasshopper Schistocerca pallens (Acrididae)

There have been many studies on Schistocerca gregaria and Locusta migratoria, which are important grasshopper pests in many parts of the world. However, the main pest grasshopper species in Brazil, S. pallens, Rhammatocerus schistocercoides and Stiphra robusta, are very poorly characterized genetically. We adapted a permanent in situ hybridization method to extend the genetic characterization of S. pallens by mapping the single-copy genes Hsp70, Hsp83, Hsp27, and Ubi on meiotic chromosomes. Hsp70 was mapped on the L2 chromosome, in which 82% of the signals were observed. Hsp83 was mapped on a medium-sized chromosome, on which 81% of the signals were observed, tentatively identified as M7. The hybridization signals for the Hsp27 gene were detected on the L1 chromosome at a frequency of 58%. The main hybridization site of the Ubi probe was on the L2 chromosome, with 73% of the signals. All mapped genes also presented secondary hybridization signals, always at frequencies below 30%. These are the first single-copy genes mapped for S. pallens and also for the Acrididae family. Since the Acrididae generally present very similar karyotypes, these data are useful as new landmarks for chromosome identification and as a tool for phylogenetic studies on the genus Schistocerca and for comparison with other insects.

Detection of HPV16 and 18 by in situ hybridization in precancerous and cancerous lesions of cervix.

Fifty cervical biopsies from women with preinvasive and invasive malignancies of uterine cervix and ten normal cervical biopsies were examined for the presence of human papilloma virus (HPV) 16 and 18 DNA sequences by in situ hybridization (ISH) method with biotinylated DNA probes. The overall positivity of HPV DNA was 48% (24/50). The positivity of HPV 16 DNA for low grade squamous intraepithelial lesion (LSIL), high grade squamous intraepithelial lesions (HSIL) and squamous cell carcinoma (SCC) were 33.33%, 45.45%, 42.30% respectively. The positivity for HPV 18 DNA for LSIL, HSIL and SCC were 0%, 18.18%, 30.76% respectively. Two cases of cervical adenocarcinomas showed positivity for HPV 18 DNA only.

Hibridização in situ com sonda não-radioativa para mRNA: princípios e aplicações em patologia / In situ hybridization with non-radioactive riboprobes: principles and applications in pathology

A técnica de hibridização in situ (ISH) tem sido usada para identificar mRNA (ou DNA) em amostras de tecido de material humano e animal. Embora uma série de protocolos para essa técnica seja utilizada, as descrições não são bem detalhadas. O objetivo deste trabalho é descrever a reação de hibridização in situ em tecido fresco e sua aplicação em patologia, tornando mais compreensível essa técnica tão importante, que possibilita observar a localização tecidual e a expressão temporal e espacial dos transcritos de um determinado gene (mRNA). Resultados de reações com as ribossondas PITX1, SHH e WNT-5A, realizadas em amostras de tecido congelado, são apresentados.

In situ hybridization (ISH) has been used to identify mRNA (or DNA) in fresh tissue samples of humans and animals. Several protocols describing this technique are available, although its description is not usually detailed enough. The present work describes in situ hybridization reaction on fresh tissue in a way to make understandable this important technique, which allows verifying the cellular localization, and the spatial and temporal expression, of gene transcripts (mRNA). Results with PITX1, TGIF, SHH and WNT-5A riboprobes, in fresh tissue samples, are presented.

Detection of hepatitis C virus RNA by in situ hybridization in paraformaldehyde fixed biopsies

Fourteen hepatitis C virus (HCV) chronically infected patients were submitted to routine liver biopsy for histological evaluation. Liver samples were assayed to HCV-RNA by in situ hybridization, using digoxigenin labeled probe. HCV genotypes were found to be predominantly type 1 (71.4 percent), followed by genotype 3 (21.4 percent), and genotype 2 (7.2 percent). Alanine-aminotransferase levels were raised in 10 patients. The histopathological scores were minimal (21.4 percent), mild (57.2 percent), and moderate (21.4 percent). Viral RNA was detected in liver cells from nine patients (64.3 percent). ISH method provides localization and poor confirmation of HCV RNA in the liver tissue of HCV chronic patients.