Toxicological, enzymatic, and immunochemical characterization of Bothrops asper (Serpentes: Viperidae) reference venom from Panama / Caracterización toxicológica, enzimática e inmunoquímica del veneno de referencia de Bothrops asper (Serpentes: Viperidae) de Panamá

Introduction: It is estimated that 2 000 snakebites occur in Panama every year, 70 % of which are inflicted by Bothrops asper. Objective: To determine the biochemical and toxicologic effects and to assess the immunochemical characteristics of a reference pool of B. asper venom representative of Panama. Methods: The reference venom was prepared as a homogeneous mixture of the venoms obtained from 78 adult snakes collected in four geographic areas of Panama. Enzymatic and toxicological activities were assessed. The electrophoretic pattern was studied by SDS-PAGE. Immunoreactivity of various antivenoms was analyzed by Western blot. Results: B. asper reference venom has lethal, hemorrhagic, myotoxic, edema-forming, coagulant, defibrinating, proteinase and phospholipase A2 activities. SDS-PAGE showed the presence of protein bands with molecular weights ranging from 8 to 70 kDa, with the presence of predominant bands at ≈ 15 kDa and ≈ 30 to 66 kDa, which likely correspond to phospholipases A2 and metalloproteinases, respectively. Immunoblotting showed a high degree of recognition by various antivenoms, especially by antivenoms from Colombia and Costa Rica. Conclusions: Following recommendations by the World Health Organization, this reference venom of B. asper of Panama will become a useful tool for the preclinical evaluation of antivenoms distributed in this country.

Introducción: Se estima que 2 000 mordeduras de serpiente ocurren en Panamá cada año, el 70 % de las cuales son infligidas por Bothrops asper. Objetivo: Determinar los efectos bioquímicos y toxicológicos y evaluar las características inmunoquímicas del veneno de referencia de B. asper representativo de Panamá. Métodos: El veneno de referencia se preparó como una mezcla homogénea de los venenos obtenidos de 78 serpientes adultas recolectadas en cuatro áreas geográficas de Panamá. Se evaluaron las actividades enzimáticas y toxicológicas. El patrón electroforético se estudió mediante SDS-PAGE. La inmunoreactividad de varios antivenenos se analizó mediante transferencia de Western. Resultados: El veneno de referencia de B. asper tiene actividades letales, hemorrágicas, miotóxicas, formadoras de edema, coagulantes, desfibrinante, proteolítica y de fosfolipasa A2. El análisis de SDS-PAGE mostró la presencia de bandas de proteínas con pesos moleculares que varían de 8 a 70 kDa, con la presencia de bandas predominantes a ≈ 15 kDa y ≈ 30 a 66 kDa, que probablemente corresponden a fosfolipasas A2 y metaloproteinasas, respectivamente. La inmunotransferencia mostró un alto grado de reconocimiento por varios antivenenos, especialmente por antivenenos de Colombia y de Costa Rica. Conclusiones: Siguiendo las recomendaciones de la Organización Mundial de la Salud, este veneno de referencia de B. asper de Panamá se convertirá en una herramienta útil para la evaluación preclínica de antivenenos distribuidos en este país.

Doença de Creutzfeldt-Jakob: relato de caso / Creutzfeldt-Jakob disease: case report

As doenças priônicas fazem parte do grupo das síndromes de demência rapidamente progressiva com neurodegeneração. Em humanos, a doença de Creutzfeldt-Jakob é a mais prevalente. Atualmente, seu diagnóstico pode ser baseado em uma combinação do quadro clínico, ressonância magnética e eletroencefalograma com alterações típicas, juntamente da detecção de proteína 14- 3-3 no líquido cefalorraquidiano. Este relato descreve o caso de uma paciente de 74 anos, natural de Ubá (MG), admitida em um hospital da mesma cidade com quadro de demência de rápida progressão, com declínio cognitivo, ataxia cerebelar e mioclonias. No contexto clínico, aventou-se a possibilidade de doença de Creutzfeldt-Jakob e, então, foi iniciada investigação para tal, com base nos critérios diagnósticos. Também foram realizados exames para descartar a possibilidade de doenças com sintomas semelhantes. O caso foi diagnosticado como forma esporádica de doença de Creutzfeldt-Jakob. (AU)

Prion diseases are part of the rapidly progressive dementia syndromes with neurodegeneration. In humans, Creutzfeldt-Jakob disease is the most prevalent. Currently, its diagnosis may be based on a combination of clinical picture, magnetic resonance imaging, and electroencephalogram with typical changes, along with the detection of 14-3-3 protein in cerebrospinal fluid. This report describes the case of a 74-year-old woman from the city of Ubá, in the state of Minas Gerais, who was admitted to a hospital in the same city with a rapidly progressive dementia, cognitive decline, cerebellar ataxia and myoclonus. In the clinical context, the possibility of Creutzfeldt-Jakob disease was raised, and then investigation was started for this disease, based on the its diagnostic criteria. Tests have also been conducted to rule out the possibility of diseases with similar symptoms. The case was diagnosed as a sporadic form of Creutzfeldt-Jakob disease. (AU)

The correlation between serum vascular endothelial growth factor (VEGF) and tumor VEGF receptor 3 in colorectal cancer

PURPOSE: Despite plasma biomarkers offering a number of advantages over tissue-based markers, the relationship between serum vascular endothelial growth factor (VEGF) and VEGF receptor (VEGF-R) tumor expression in colorectal cancer (CRC) is still unclear. This study was designed to establish the relationship between the concentration of serum VEGF and tumor VEGF-R expression in patients with CRC. METHODS: A prospective study of consecutive patients undergoing elective colorectal surgery during 1 year. Preoperative VEGF was determined by enzyme-linked immunosorbent assay and VEGF-R3 by immunochemistry. RESULTS: The initial sample included 134 patients with CRC diagnosis. Results showed significant association of serum values of VEGF with VEGF-R3 expression (P < 0.001), even in the presence of confounders (sex, age, body mass index, tumor location, and surgical approach). The estimated effect size was high (η² = 0.35). CONCLUSION: Serum VEGF has a significant correlation with tumoral VEGF-R3 expression in CRC.

Imaging Cancer Metabolism

It is widely accepted that altered metabolism contributes to cancer growth and has been described as a hallmark of cancer. Our view and understanding of cancer metabolism has expanded at a rapid pace, however, there remains a need to study metabolic dependencies of human cancer in vivo. Recent studies have sought to utilize multi-modality imaging (MMI) techniques in order to build a more detailed and comprehensive understanding of cancer metabolism. MMI combines several in vivo techniques that can provide complementary information related to cancer metabolism. We describe several non-invasive imaging techniques that provide both anatomical and functional information related to tumor metabolism. These imaging modalities include: positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS) that uses hyperpolarized probes and optical imaging utilizing bioluminescence and quantification of light emitted. We describe how these imaging modalities can be combined with mass spectrometry and quantitative immunochemistry to obtain more complete picture of cancer metabolism. In vivo studies of tumor metabolism are emerging in the field and represent an important component to our understanding of how metabolism shapes and defines cancer initiation, progression and response to treatment. In this review we describe in vivo based studies of cancer metabolism that have taken advantage of MMI in both pre-clinical and clinical studies. MMI promises to advance our understanding of cancer metabolism in both basic research and clinical settings with the ultimate goal of improving detection, diagnosis and treatment of cancer patients.

Functional Analysis and Immunochemical Analyses of Ca²⁺ Homeostasis-Related Proteins Expression of Glaucoma-Induced Retinal Degeneration in Rats

The retinal degeneration resulting from elevated intraocular pressure was evaluated through functional and morphological analyses, for better understanding of the pathophysiology of glaucoma. Ocular hypertension was induced via unilateral episcleral venous cauterization in rats. Experimental time was set at 1 and 3 days, and 1, 2, 4, and 8 weeks post-operation. Retinal function was analyzed using electroretinography. For morphological analysis, retinal tissues were processed for immunochemistry by using antibodies against the calcium-sensing receptor and calcium-binding proteins. Apoptosis was analyzed using the TUNEL method and electron microscopy. Amplitudes of a- and b-wave in scotopic and photopic responses were found to be reduced in all glaucomatous retinas. Photopic negative response for ganglion cell function significantly reduced from 1-day and more significantly reduced in 2-week glaucoma. Calcium-sensing receptor immunoreactivity in ganglion cells remarkably reduced at 8 weeks; conversely, protein amounts increased significantly. Calcium-binding proteins immunoreactivity in amacrine cells clearly reduced at 8 weeks, despite of uneven changes in protein amounts. Apoptosis appeared in both photoreceptors and ganglion cells in 8-week glaucomatous retina. Apoptotic feature of photoreceptors was typical, whereas that of ganglion cells was necrotic in nature. These findings suggest that elevated intraocular pressure affects the sensitivity of photoreceptors and retinal ganglion cells, and leads to apoptotic death. The calcium-sensing receptor may be a useful detector for alteration of extracellular calcium levels surrounding the ganglion cells.

Value of combined detection of claudin 4 and high-risk human papilloma virus in high-grade squamous intraepithelial lesion and cervix squamous cell carcinoma / 浙江大学学报·医学版

OBJECTIVE@#To investigate the expression of claudin 4 (CLDN4) in cervical tissues from patients with different cervical lesions, and to explore the value of combined detection of CLDN4 and high risk human papilloma virus (HR-HPV).@*METHODS@#The cervical tissue specimens of low-grade squamous intraepithelial lesion (LSIL, =30), high-grade squamous intraepithelial lesion (HSIL, =30), squamous cell carcinoma (SCC, =30) as well as chronic cervicitis (control, =30) were collected from the Sir Run Run Shaw Hospital of Zhejiang University during June 2015 and December 2016. The expression of CLDN4 protein in tissue specimens was detected by immunohistochemistry, HR-HPV was detected by real-time quantitative PCR, and the cervical exfoliated cells were examined by thinprep cytologic test (TCT). The ROC curve was applied to analyze the diagnostic value of TCT combined with HR-HPV and CLDN4 combined with HR-HPV tests for HSIL and SCC of the cervix.@*RESULTS@#With the increase of the severity of cervical lesions, the positive rate of CLDN4 expression rose (=0.832, <0.05). Positivity of both HR-HPV infection and CLDN4 expression was found mainly in the HSIL and SCC groups. The areas under curve (AUC) of TCT combined with HR-HPV and CLDN4 combined with HR-HPV tests for diagnosis of HSIL and SCC were 0.683 and 0.633, respectively; the sensitivity, specificity, positive predictive value, negative predictive value and accuracy of TCT combined with HR-HPV test for diagnosis of HSIL and SCC were 100.0%, 36.7%, 61.2%, 100.0% and 46.7% respectively; those of CLDN4 combined with HR-HPV test were 96.7%, 30.0%, 58.0%, 90.0% and 55.0%, respectively.@*CONCLUSIONS@#CLDN4 expression may be related to the occurrence and development of cervical carcinoma and precancerous lesions. CLDN4 combined with HR-HPV test may be used for diagnosis of HSIL and SCC of the cervix clinically.

E2/E6 ratio and L1 immunoreactivity as biomarkers to determine HPV16-positive high-grade squamous intraepithelial lesions (CIN2 and 3) and cervical squamous cell carcinoma / 부인종양

OBJECTIVE: Human papillomavirus (HPV) 16 is the most carcinogenic HPV genotype. We investigated if HPV16 L1 capsid protein and E2/E6 ratio, evaluated by cervical cytology, may be used as biomarkers of ≥cervical intraepithelial neoplasia (CIN) 2 lesions. METHODS: Cervical specimens were obtained from 226 patients with HPV16 single infection. Using cytology specimen, L1 capsid protein and E2/E6 ratio were detected and the results were compared with those of the conventional histologic analysis of cervical tissues (CIN1–3 and squamous cell carcinoma [SCC]) to evaluate the association. RESULTS: The L1 positivity of CIN2/3 was significantly lower than that of normal cervical tissue (p < 0.001) and SCC demonstrated significantly lower L1 positivity than CIN1 (p < 0.001). The mean E2/E6 ratios of specimens graded as SCC (0.356) and CIN2/3 (0.483) were significantly lower than those of specimens graded as CIN1 (0.786) and normal (0.793) (p < 0.05). We observed that area under the receiver operating characteristic curve (AUC) for E2/E6 ratio (0.844; 95% confidence interval [CI]=0.793–0.895) was higher than that for L1 immunochemistry (0.636; 95% CI=0.562–0.711). A combination of E2/E6 ratio and L1 immunocytochemistry analyses showed the highest AUC (0.871; 95% CI=0.826–0.917) for the prediction of ≥CIN2 lesions. CONCLUSION: To our knowledge, this is the first study to validate HPV L1 capsid protein expression and decreased HPV E2/E6 ratio as valuable predictive markers of ≥CIN2 cervical lesions. Cervical cytology may be analyzed longitudinally on an outpatient basis with noninvasive procedures as against invasive conventional histologic analysis.

Morphine-alcohol treatment impairs cognitive functions and increases neuro-inflammatory responses in the medial prefrontal cortex of juvenile male rats / 대한해부학회지

In the developed and developing world, opioid consumption in combination with alcohol has become one of the substances abused. In this experiment, we examined the effects of alcohol, morphine, and morphine+alcohol combination on cognitive functions and neuroinflammatory responses in the medial prefrontal cortex (mPFC) of juvenile male rats. Alcohol (1.0 ml of 15% v/v ethanol twice daily, subcutaneously, 7 hours apart), morphine (0.5 ml/kg of 0.4 mg/kg morphine chlorate twice daily, subcutaneously, 7 hours apart), morphine+alcohol co-treatment (0.5 ml/kg of 0.4 mg/kg morphine chlorate+1.0 ml of 15% v/v ethanol twice daily, subcutaneously, 7 hours apart) were administered for 21 days. Treatment with morphine+alcohol significantly impairs cognition functions in the Morris water maze, passive avoidance, and novel object recognition tests, furthermore, the treatment significantly increased the quantitative count of astrocytic cells and also conferred marked neuronal cell death in the mPFC, which were studied by glial fibrillary acidic protein immunochemistry for astrocytes and Cresyl violet for Nissl's substance distribution in neurons respectively. These results suggest that alcohol, morphine, and morphine+alcohol co-treatment may trigger cognitive deficits and neuroinflammatory responses in the brain.

Estudio comparativo de la determinación de vitamina D por dos inmunoensayos / Comparative study of the determination of vitamin D by two immunoassays / Estudo comparativo da determinação de vitamina D por dois imunoensaios

La vitamina D es un metabolito que tiene gran importancia en la actualidad, debido al descubrimiento de nuevas funciones. La mejor manera de medir los niveles de vitamina D en el organismo es medir los niveles de 25(OH)vitamina D, pero hoy en día no existe estandarización entre métodos y, por lo tanto, existe gran variabilidad entre ellos. En los laboratorios clínicos los métodos más utilizados son los inmunoensayos y sólo ADVIA Centaur Vitamina D Total assay e IDS-iSYS 25-Hydroxy Vitamin D pasaron la primera fase del Vitamin D Standardization Certification Program (VDSCP) diseñado por el Centre for Diseases Control (CDC). En este estudio se compararon estos 2 inmunoensayos, pero a pesar de haber pasado la primera fase del VDSCP los resultados reflejaron que sigue existiendo variabilidad entre métodos y, por lo tanto, no son intercambiables, siendo necesario definir valores de referencia para cada método a la espera que el proceso de estandarización termine y exista buena correlación entre los diferentes métodos.

Currently, Vitamin D is a highly important metabolite as a result of the discovery of its new functions. The best way to measure the levels of vitamin D in the body is by measuring the levels of 25(OH)vitamin D, but currently there is no standardization between methods and therefore there is a great variability between them. In clinical laboratories, immunoassays are the most used methods, and only ADVIA Centaur Vitamin D Total assay© and IDS-iSYS 25-Hydroxy Vitamin D©passed the first phase of the VDSCP designed by the CDC. In this research study, these 2 immunoassays were compared, and although both passed the first phase of the VDSCP, the results showed that there still exists variability between methods. Therefore, they are not interchangeable, being it necessary to define some reference values for each method until the standardization method is finishe, and there is a good correlation between methods.

A vitamina D é um metabólito que tem muita importância na atualidade, devido ao achado de novas funções. A melhor maneira de medir os níveis de vitamina D no organismo é medir os níveis de 25(OH)vitamina D, mas hoje em dia não existe padronização entre métodos e, por isso, existe uma grande variabilidade entre eles. Nos laboratórios clínicos, os métodos mais utilizados são os imunoensaios e só a ADVIA Centaur Vitamina D Total assay e IDS-iSYS 25-Hydroxy Vitamin D, passaram a primeira fase do Vitamin D Standardization Certification Program (VDSCP) desenhado pelo Centre for diseases control (CDC). No estudo foram comparados esses dois imunoensaios, porém, apesar de ter passado a primeira fase do VDSCP os resultados mostraram que continua existindo variabilidade entre métodos e, por isso, não são intercambiáveis, sendo necessário definir valores de referência para cada método à espera de que o processo de padronização finalize, e exista boa correlação entre os diferentes métodos.

Malignant Solitary Fibrous Tumor of the Parotid Gland / 대한이비인후과학회지

Solitary Fibrous tumor is usually arising in the pleura of the thoracic cavity and it has been described in a variety of extrapleural sites including the abdominal cavity, soft tissue, upper respiratory tract and rarely in head and neck region. We experienced a rare case of solitary fibrous tumor in the parotid gland. A 49-year-old woman presented with a painless mass in the left parotid. The mass was well-demarcated and easily dissected, and it showed a patternless arrangement of potential malignant spindle cells in a fibrotic background and prominent vascular structures. In immunochemistry, the mass cells were positive for CD34, vimentin, and negative for actin, S-100 protein, and cytokeratins. Therefore, the parotid mass diagnosed as SFT. During the 24 months of follow-up, there was no recurrence of tumor growth.

Notalgia Paresthetica of the Back Successfully Controlled by Pregabalin / 대한피부과학회지

Notalgia paresthetica (NP) is a chronic localized itch, affecting mainly the inter-scapular area particularly between the T2-T6 dermatomes. Occasionally it has a more widespread distribution and involves the shoulders, back, and upper chest. There are no specific cutaneous signs, apart from those attributed to scratching and rubbing. Various etiologies have been reported, but the cause of NP is not established. The current hypothesis regarding its etiology postulates that a neuropathic itch develops due to nerve entrapment of the posterior rami of spinal nerve arising at T2-T6. Another recent documented case showed an increase in the number of intradermal nerves by neural immunochistochemistry staining of S-100 protein, protein gene product 9.5 (PGP 9.5). Herein, we experienced an uncommon case of NP of the back and tried to clarify pathogenesis by using quantitative sensory testing, such as neurometer and Von-Frey filaments. Also, we performed neural immunochemistry to confirm an increase in nerve fibers at the site of the lesion.

Evaluation of expression of the Wnt signaling components in canine mammary tumors via RT² Profiler PCR Array and immunochemistry assays

The Wnt signaling pathway and its key component β-catenin have critical roles in the development of diseases such as tumors in mammals. However, little has been reported about involvement of the Wnt/β-catenin signaling pathway in canine mammary tumors (CMTs). The present study detected expression of 30 Wnt signaling pathway-related genes in CMTs; the results are potentially useful for molecular-based diagnosis of CMTs and the development of new targeted therapies. Significant upregulations of dickkopf-1 protein, secreted frizzled-related sequence protein 1 (SFRP1), frizzled 3, β-catenin, and lymphoid enhancer-binding factor 1 (LEF1) were detected in highly malignant CMTs compared to levels in normal mammary gland tissues; moreover, highly significant upregulation of WNT5A was observed in low malignancy CMTs. Downregulation was only detected for SFRP4 in malignant CMT samples. The subcellular location of β-catenin and cyclin D1 in 100 CMT samples was investigated via immunohistochemical analysis, and significantly increased expressions of β-catenin in cytoplasm and cyclin D1 in nuclei were revealed. Western blotting analysis revealed that the expression of β-catenin and LEF1 increased in in the majority of CMT samples. Taken together, the results provide important evidence of the activation status of the Wnt pathway in CMTs and valuable clues to identifying biomarkers for molecular-based diagnosis of CMT.

La problemática del endosulfán: aspectos químicos, analíticos y ambientales / The roblem of enndosulfan: chemical, analytical and environmental issues

Introducción. Dado el carácter residual y el alto nivel de toxicidad del endosulfán, se ha incrementado la preocupación a nivel mundial por su impacto ambiental. Por eso la búsqueda de metodologías analíticas que permitan su detección y cuantificación es un tema de investigación vigente. Las propiedades del endosulfán y su patrón de degradación le confieren características que hacen muy compleja la problemática, al tiempo que el pesticida y sus metabolitos se expanden extensivamente por la atmósfera y contaminan tanto matrices lipofílicas como hidrofílicas. Objetivo. Llevar a cabo una revisión sobre las investigaciones más recientes acerca de la problemática del endosulfán, que incluye aspectos químicos, su problemática ambiental y técnicas inmunoquímicas para su análisis, tema en el que ha estado interesado nuestro grupo de investigación en la Universidad de Caldas. Método. El presente artículo de revisión analiza, cualitativamente, la literatura científica disponible en las bases de datos Science Direct, PubMed, Scielo y páginas Web oficiales, empleando como palabras de búsqueda: endosulfán, haptenos, análisis inmunoquímico del endosulfán, problemática ambiental del endosulfán, regulaciones internacionales sobre el endosulfán, normatividad vigente sobre el endosulfán, y métodos instrumentales de análisis. Resultados. Se obtuvo información pertinente relacionada con el objetivo propuesto, la cual se presenta en 3 secciones: aspectos químicos del endosulfán, problemática ambiental y técnicas inmoquímicas para su análisis. Conclusión. El endosulfán ha sido prohibido por los organismos internacionales. Sin embargo, el pesticida se continúa usando en algunos países y éste se difunde fácilmente hacia otras regiones, de tal manera que se ha convertido en un problema global. Hay investigaciones recientes sobre métodos de remediación química o biológica, pero los esfuerzos son modestos frente a la enorme problemática de este pesticida. En lo pertinente a análisis químicos de endosulfán, en los últimos 15 años se han investigado técnicas inmunoquímicas de análisis (basadas en haptenos), como alternativa a los métodos cromatográficos instrumentales.

Introduction. Due to the residual character and high level of toxicity of endosulfan, worldwide concern for its environmental impact has increased. Therefore, the search for analytical methodologies that allow its detection and quantification continues to be a topic of current research. Endosulfan properties and its degradation pattern confer it characteristics that make very complex the problematic while the pesticide and its metabolites are extensively spread throughout the atmosphere and contaminate both lipophilic and hydrophilic matrixes. Objective. To conduct a review on some recent research about the endosulfan problematic including chemical aspects, environmental issues and immunochemical techniques for its analysis, which is an area of current interest in this research group at Universidad de Caldas. Method. This review article analyzes qualitatively scientific literature from Science Direct, PubMed, Scielo databases and official Web pages using key words such as endosulfan, endosulfan immunochemical analysis, endosulfan environmental problematic, endosulfan international regulations, endosulfan current normativity and instrumental methods for the analysis. Results. Relevant information related to the proposed objective was found which is presented in 3 sections: chemical aspects of endosulfan, environmental issues, and immunochemical techniques for its analysis. Conclusions. Endosulfan has been banned by international organizations. However, the pesticide is still being used in some countries and is easily spread to other regions, so that the problem became a global concern. There is some recent research on biological or chemical remediation techniques, but efforts seem insufficient in comparison to the enormous impact of the pesticide. In regard to Endosulfan chemical analyses, during the past 15 years immunochemical hapten-based analysis techniques have been investigated as alternatives to the instrumental chromatographic methods.

Immunohistochemical evaluation of CD20 expression in patients with multiple myeloma

Objective: CD20 expression was reported at different rates in patients with multiple myeloma. The importance of this B-cell antigen for plasma cells is still unknown. This study aimed to investigate CD20 expression of myeloma cells in bone marrow, and any relationship between the stage of disease, isotype and clinical features. Methods: Sixty-one patients who were admitted to the hematology clinic of the Adnan Menderes Medical School with the diagnosis of multiple myeloma according to the criteria of the "International Myeloma Working Group" were enrolled in this study. Age, gender, Durie-Salmon stage, history of autologous hematopoietic stem cell transplantation, and the distribution pattern and positivity of CD20 expression on multiple myeloma cells in bone marrow were evaluated. The Mann-Whitney U and chi-square tests were used for statistical analysis with a p-value < 0.05 being accepted as statistically significant. Results: Thirty patients (48.9%) had positive scores for CD20 with the distribution pattern being most likely interstitial in 55.6% of the cases. There was no statistically significant difference between immunohistochemical positivity for CD20 expression on multiple myeloma cells, immunoglobulin type, and the stage of disease. Conclusion: The combination of immunohistochemical studies with flow cytometry may reveal the importance of CD20 positivity in patients with multiple myeloma more clearly. .

Variations in Matrix Metalloproteinase Expression by Disc Location in Patients with Sequestrated Lumbar Disc Herniation

STUDY DESIGN: In vivo study OBJECTIVES: To evaluate variations in matrix metalloproteinase (MMP) expression levels according to the disc location in patients with sequestrated lumbar disc herniation. SUMMARY OF LITERATURE REVIEW: MMPs are considered to be the major catabolic enzymes in the intervertebral disc. MMPs have been known to be the primary mediators of extracellular matrix (ECM) degradation, to play major roles in disc degeneration by changing the collagens and the extracellular matrix, and to be involved in the processes of apoptosis and autoresorption of herniated disc materials by inducing inflammatory cytokines. MATERIALS AND METHODS: The sequestered and contained disc materials were removed from seven patients with sequestered lumbar disc herniations. The materials from the contained discs were classified into group 1 and those of the sequestered discs into group 2. Immunochemistry tests were conducted for the tissues of both groups. The expression levels of MMP-1, 3, and 13 were checked using a fluorescence microscope. The amount of expression of each MMP was calculated using the percentage of expressed cells and analyzed statistically. RESULTS: In the histological study, increased expression of MMP-1, 3, and 13 was found in group 2. In the statistical analysis after the quantification of MMP expression, the expression of all MMPs was found to have increased significantly in group 2 (p<0.05). CONCLUSIONS: The increased expression of MMP-1, 3, and 13 indicated that the inflammation and degeneration processes, and the spontaneous resorption by the surrounding tissues were more active in the sequestered disc group than in the contained disc group.

Métodos electroforético e inmunoquímicos para detectar proteínas de leche, soja y huevo en premezclas comerciales / Electrophoretic and immunochemical methods for the detection of milk, soy and egg proteins in commercial mix products

The presence of milk, egg and soy proteins was evaluated in thirteen commercial products, which were premixes to make cream caramel, cake, soy "milanesas", "tortas fritas", pizza, gnocchi and complementary foods. Samples were analysed using SDS-PAGE, immunoblotting and ELISA methods. The electrophoretic method and immunoblotting were useful to confirm the presence of milk, egg and soy proteins in four samples that declared them as ingredients. In other samples, both methods showed negative results for some of these proteins, although they were also declared as ingredients. This suggests that those proteins were not added as ingredients in these products. The ELISA kits detected very low concentration of the allergenic proteins in four products with precautionary phrases and also in five samples that made no reference to them in their labels. ELISA methods are useful to detect cross contamination due to their high sensitivity. The food industry should be responsible for the declaration of milk, egg and soy proteins in their food labels.

Se evaluó la detección de proteínas de leche, huevo y soja en trece productos comerciales, correspondientes a premezclas para preparar flanes, bizcochuelo, milanesas de soja, tortas fritas, pizza, ñoquis y productos en polvo a base de harinas para niños pequeños. Las muestras fueron analizadas por SDS-PAGE, inmunoblotting y métodos de ELISA. El método electroforético e inmunoblotting resultaron útiles para confirmar la presencia de las proteínas alergénicas en algunas muestras que las declaraban como ingredientes. En cuatro muestras que también declaraban como ingredientes alguna de estas proteínas, tanto con SDS-PAGE como con Inmunoblotting, los resultados fueron negativos, sugiriendo que dichas proteínas no fueron agregadas como ingredientes en dichos alimentos. Los kits de ELISA permitieron la detección de concentraciones muy bajas de estos alérgenos en cuatro productos que presentaban frases de advertencia e incluso en otros cinco que no tenían ninguna declaración de alérgenos. Los métodos de ELISA son útiles para detectar contaminaciones cruzadas, dada su elevada sensibilidad. Se concluye que resulta necesario una declaración responsable de estas proteínas alergénicas, en los rótulos de este tipo de alimentos por parte de los fabricantes.

Normalización de un método inmunoquímico para detectar Papilomavirus humano tipo 16 en lesiones cérvico-uterinas / Standardization of immunochemical method for detection of type 16 human papilomavirus in cervix uterine lesions

Introducción: la infección por Papilomavirus Humano (PVH) es la condición necesaria para la aparición y desarrollo del cáncer cérvico-uterino. Los genotipos de alto riesgo oncogénico son los causantes de este tipo de neoplasia y dentro de ellos el más frecuente es el PVH 16, que se encuentra aproximadamente en el 60 % de los casos. Los métodos de diagnóstico comerciales resultan costosos para países con escasos recursos económicos, lo que sugiere la búsqueda de alternativas empleando protocolos sencillos y baratos. Objetivos: normalizar un método inmunoquímico para la detección del antígeno L1 de PVH tipo 16 en muestras cérvico-uterinas de pacientes con lesiones intraepiteliales escamosas y determinar la coincidencia entre el método normalizado y la Reacción en Cadena de la Polimerasa en Tiempo Real (RCP-TR), como técnica de referencia, para estimar la utilidad de dicho método en el diagnóstico de la infección por este genotipo viral. Métodos: se compararon tres procedimientos de inmunotinción (Indirecto de inmunoperoxidasa en dos pasos, Estreptavidina-Biotina y Amplificación por polímero) respecto a sensibilidad analítica, tinción inespecífica de fondo y tiempo de terminación, para la detección de la proteína L1 de PVH 16 en líneas celulares derivadas de carcinomas cervicales humanos y en muestras cérvico-uterinas utilizadas como controles. El protocolo normalizado se aplicó a muestras cérvico-uterinas de mujeres entre 30 y 59 años, 82 con lesiones intraepiteliales cervicales y 10 sin antecedentes de alteraciones citológicas, a las que además se les determinó PVH 16 mediante RCP-TR. Resultados: el procedimiento de Estreptavidina-Biotina resultó el más sensible y específico. La coincidencia entre el método inmunoquímico y la RCP-TR fue de un 98,6 por ciento, la sensibilidad fue de un 98,57 por ciento y la especificidad de un 91,67 por ciento, con valores predictivos negativo y positivo por encima del 90 por ciento. Conclusiones: se demostró la validez del método inmunoquímico como prueba confirmatoria de la infección por PVH 16. Dicho método probó ser sensible, sencillo y no requiere de una compleja infraestructura para detectar PVH 16 en muestras cervicales. Además, esta técnica permite obtener información rápidamente y evita el uso de métodos invasivos(AU)

Introduction: Human Papillomavirus (HPV) infection is the necessary condition for the occurernce and development of cervical cancer. The high oncogenic risk genotypes are the responsible for this type of neoplasia and the most frequent is HPV 16 that affects roughly 60 percent of cases. Commercial kits for HPV detection are expensive for resource-poor countries, which suggests the search for alternative throguh non-expensive simple protocoles. Objectives: to standardize an immunochemical method for the detection of HPV 16 L1 antigen in cervical samples of patients with squamous intraepithelial lesions and to determine the diagnostic coincidence between the immunochemical method and the real-time polymerase chain reaction to estimate the usefulness of this method for the detection of cervical infection with this viral genotype. Methods: three immunostaining methods (Two-Step Indirect Immunoperoxidase, Labelled Streptavidin-Biotin and Enhanced Polymer) were compared in terms of analytical sensitivity, nonspecific background staining and time of completion, for the detection of protein L1 of HPV-16 in a cell line derived from human cervical carcinoma and clinical samples from uterine cervix. The optimized protocol was applied to 82 cervical samples from women aged 30-59 years with squamous intraepithelial lesions and to 10 samples of sexually active women without previous signals of positive cytology. The presence of type 16 HPV was also detected with the aid of RT-PCR. Results: the Streptavidin-Biotin system was the most sensitive and specific. The diagnostic agreement between the immunochemical method and the real-time polymerase chain reaction reached 98.6 percent, sensitivity was 98.57 percent and specificity was 91.67 %, with positive and negative predictive values above 90 percent. Conclusions: the validity of the immunochemical method as a confirmatory test for infection by HPV-16 has been demonstrated. The normalized immunochemical method proved to be a sensitive, simple, relatively fast method to detect HPV from clinical samples of cervical cells. Furthermore, this method provides information quickly, avoiding the use of invasive methods in patients(AU)

Immunohistochemical study of polycystin-1 in dentigerous cysts.

Background: The alterations involved in step-wise transformation of a dental follicle to dentigerous cyst (DC) is not clearly known. Primary cilium and its protein have been hypothesized to be associated with DC. Mutation of a ciliary protein, polycystin‑1 (PC1) is associated with autosomal dominant polycystic kidney disease. This study was performed to assess the immunohistochemical expression of PC1 between DC and postfunctional follicular tissue (PFFT). Materials and Methods: Thirty‑one consecutive PFFT and 15 DC formed the study group. The PFFT and DC tissues were stained with antibody against PC1. Statistical Package for Social Service was used to analyze data. Descriptive statistics and Student’s Chi‑square test were appropriately used. P ≤0.05 was taken as significant. Results: Fifteen DC (100%) and 7 (22.58%) PFFT were positive for PC1. The difference was statistically significant (P = 0.000). PC1 expression was observed in the cytoplasm with varying intensity. Discussion and Conclusion: All PC1 positive epithelial cells’ cytoplasm stained diffusely. Abnormal cytoplasmic expression of PC1 in all positive epithelial lining indicates that the PC1 probably is associated with cystic transformation.

Pulmonary adenocarcinoma in cattle / Adenocarcinoma pulmonar en bovinos

The Macroscopic, histological and immunohistochemical aspects of lung acinar adenocarcinoma and the presence of nodules in the abdominal cavity of an adult female bovine are reported. In the necropsy analysis samples were collected from the: lung, heart, spleen, liver, pancreas, kidney, uterus, intestine, brain, and from nodules found in the lung and abdominal cavity, which were routinely processed to be stained by hematoxylin-eosin and for an immunohistochemistry exam with the antibodies: cytokeratin (dilution 1:200 µL) and vimentin (dilution 1:1000 µL). The histopathological examination revealed neoplastic epithelial cells with acini formation. The immunohistochemical examination of the tumor cells showed positive marking for cytokeratin and the absence of marking for vimentin. According to anatomical, morphological, and histopathological findings, as well as the result of the immunohistochemical examination, the tumor was characterized as lung acinar adenocarcinoma.

Se relatan los aspectos macroscópicos, histológicos e inmunohistoquímicos de un adenocarcinoma acinar pulmonar y la presencia de nódulos en la cavidad abdominal en una hembra bovina adulta. En el análisis necroscópico fueron colectados fragmentos de: pulmón, corazón, bazo, hígado, páncreas, riñón, útero, intestino, encéfalo y de los nódulos hallados en pulmón y cavidad abdominal, los cuales fueron procesados rutinariamente para ser teñidos mediante Hematoxilina-Eosina y para examen inmunohistoquímico con los anticuerpos: citoqueratina (con dilución 1:200 µL) y vimentina (dilución 1:1000 µL). El examen histopatológico reveló células epiteliales neoplásicas con formación de acinos. El examen inmunohistoquímico de las células neoplásicas demostró marcación positiva para citoqueratina y ausencia de marcación para vimentina. De acuerdo con los hallazgos anatómicos, morfológicos, histopatológico, y el resultado del examen inmunohistoquímico, se logró caracterizar el tumor como adenocarcinoma acinar pulmonar.