Evaluación de reactividad inmunohistoquímica con método del Complejo Avidina­Biotina (ABC) / Evaluation of immunohistochemical reactivity with Avidin-Biotin Complex (ABC) method

Inmunohistoquímica es toda técnica que permite detectar in situ componentes celulares y extracelulares por medio de anticuerpos específicos, empleando sistemas de detección enzimáticos. Dentro de los métodos inmunohistoquímicos, la técnica del complejo avidina­biotina(ABC) es ampliamente utilizada debido a su alta sensibilidad. El objetivo del presente estudio fueevaluar la reactividad inmunohistoquímica del anticuerpo 4C4.9 para la detección de la proteínaS-100, utilizando el método ABC. Para la evaluación de la reactividad inmunohistoquímica se utilizaron 2 biopsias de piel humana con diagnóstico histopatológico de melanoma maligno nodular ulcerado y nevus melanocítico intradérmico, provenientes del Laboratorio de Investigación en Biotecnología Animal de la Universidad de La Frontera, Temuco, Chile. Se utilizó el Kit VECTASTAIN®como método de detección, la dilución del anticuerpo 4C4.9 fue 1/250 y la temperatura de incubación fue a 4 ºC ó 37 ºC por 18 horas. Para validar la técnica, se realizó un control positivo y otro negativo para 4C4.9. Los resultados de la tinción inmunohistoquímica por el método del complejo ABC mostraron tinción positiva para la proteína S-100, tanto en melanoma maligno nodular ulcerado, como en nevus melanocítico intradérmico, incubados durante 18 horas a 4 ºC ó 37 ºC. Sin embargo, la inmunotinción fue más intensa cuando el anticuerpo primario se incubó a 37 ºC. Para una correcta interpretación de los resultados, es necesario tener en consideración que la reacción antígeno-anticuerpo se ve influenciada por diversos factores, como la concentración del anticuerpo, el tiempo y la temperatura de incubación. En conclusión, nuestros resultados sugieren incubarlas muestras con el primer anticuerpo (4C4.9) en una dilución de 1/250 en agua destilada, incu-bando durante 18 h a 37 ºC. Se recomienda la utilización del anticuerpo 4C4.9 como apoyo al diagnóstico y diagnóstico diferencial.

Immunohistochemistry is anytechnique that can detect cellular and extracellular components in situ by means of specific antibodies,using enzymatic detection systems. Among immunohistochemical methods, the technique ofavidin - biotin complex (ABC) is widely used because of its high sensitivity. The aim of this study was to evaluate the immunohistochemical reactivity of the4C4.9 antibody for detection of S-100 protein using the ABC method. For the evaluation ofimmunohistochemical reactivity 2 biopsies of humanskin were used with histopathological diagnosis ofulcerated malignant melanoma and melanocyticintradermal nevi from the Research Laboratory onAnimal Biotechnology of the Universidad de La Fron-tera, Chile. The Kit VECTASTAIN® was used asdetection method, the dilution the 4C4.9 antibodywas 1/250 and incubation temperature was at 4 °Cor 37 °C for 18 hours. To validate the technique, apositive control and a negative for 4C4.9 was performed. The results of immunohistochemicalstaining by the method of ABC complex showed positive staining for protein S-100 both in ulcerated malignant melanoma and melanocytic intradermalnevi, incubated for 18 hours at 4 °C or 37 °C.However, immunostaining was more intense when the primary antibody was incubated at 37° C. For acorrect interpretation of the results, it is necessary to take into consideration that the antigen-antibody reaction is influenced by various factors such as the concentration of antibody, time and temperature ofincubation. In conclusion, our results suggest incubating the samples with the first antibody (4C4.9)at 1/250 dilution in distilled water, incubating for 18h at 37 ºC. However, immunostaining was moreintense when the primary antibody was incubated at37° C. For a correct interpretation of the results, it isnecessary to take into consideration that antigen-antibody reaction is influenced by various factors suchas the concentration of antibody, time and temperature of incubation. In conclusion, our results suggest incubating the samples with the first antibody(4C4.9) at 1/250 dilution in distilled water, incubating for 18 h at 37 ºC. The use of the antibody 4C4.9 is recommended to support the diagnosis and differential diagnosis.

Radioimmunoimaging of lymphoma in mice with a two-step pretargeting strategy using biotinyled CD45 monoclonal antibody and (188)Re-avidin / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a two-step pretargeting approach to lymphoma radioimmunoimaging in mice using biotinynaled CD45 monoclonal antibody (McAb) and (188)Re-avidin in a tumor-bearing mouse model.</p><p><b>METHODS</b>Six Nod-Scid mice bearing lymphoma cell xenograft were randomized to receive either an intravenous injection of 50 µg/200 µL biotinyled CD45 McAb followed 24 h later by an intraperitoneal injection of 3.7 MBq (50 µg/100 µL) (188)Re-avidin (two-step pretargeting group), or a single intravenous injection of 3.7 MBq (100 µg/100 µL) (188)Re-CD45 McAb (control group). SPECT was performed at 0.5, 1, 6 and 23 h post-injection to characterize (188)Re isotope biodistribution. At 24 h pos-injection, the mice were sacrificed for measurement of radioactivity uptake in the tumor and normal tissues and calculation of the tumor-to-non-tumor (T/NT) ratios.</p><p><b>RESULTS</b>SPECT showed that the two-step pretargeting method resulted in a low radioactivity in the blood pool during the imaging and a concentrated radioactivity in the liver and spleen. The transplanted tumor began to be displayed at 1 h post-injection and was clearly displayed at 1-6 h; the images were clear even at 23 h. With the two-step pretargeting method, the radioactive uptake at 24 h post-injection were (1.34∓0.52)%, (6.77∓2.32)%, and (2.81∓1.25)% in the tumor, kidney and liver, respectively, with low radioactivity levels in other organs and high tumor/blood and tumor/muscle ratios (4.28∓0.82 and 8.00∓0.88, respectively). In the control group, SPECT revealed intense radioactivity in the liver, spleen, and kidneys with obscure display of the tumor; at 20 h, the radioactivity in the blood pool remained high but that in the tumor was low, and the tumor/blood and tumor/muscle ratios at 24 h were only 0.58∓0.06 and 3.21∓0.24, respectively.</p><p><b>CONCLUSION</b>Compared with (188)Re-CD45 McAb, the two-step pretargeting approach exhibits a good specificity in targeting lymphoma with an increased T/NT ratio in mice and allows early tumor display at 1 h post-injection.</p>

Detection and identification of huwentoxin-IV interacting proteins by biotin-avidin chemistry combined with mass spectrometry

Numerous spider toxins are of interest as tools for neurophysiological research or as lead molecules for the development of pharmaceuticals and insecticides. Direct detection and identification of the interacting proteins of a spider toxin are helpful for its action-mechanism analysis and practical application. The present study employed a combinative strategy for the analysis of interacting proteins of huwentoxin-IV (HWTX-IV), a peptidic neurotoxin from the venom of the spider Selenocosmia huwena.

Detection and identification of huwentoxin-IV interacting proteins by biotin-avidin chemistry combined with mass spectrometry

Numerous spider toxins are of interest as tools for neurophysiological research or as lead molecules for the development of pharmaceuticals and insecticides. Direct detection and identification of the interacting proteins of a spider toxin are helpful for its action-mechanism analysis and practical application. The present study employed a combinative strategy for the analysis of interacting proteins of huwentoxin-IV (HWTX-IV), a peptidic neurotoxin from the venom of the spider Selenocosmia huwena.

Imunocitoquímica e imunohistoquímica para o diagnóstico laboratorial da leishmaniose tegumentar / Immunocytochemistry and immunohistochemistry for laboratorial diagnosis of cutaneous leishmaniasis

0 aumento significativo do número de casos notificados da Leishmaniose Tegumentar Americana (LTA) e a expansão geográfica da endemia tem motivado o desenvolvimento de novas tecnologias para auxiliar no diagnóstico das leishmanioses, visando minimizar as restrições apresentadas pelos testes diagnósticos disponíveis nos serviços de saúde. 0 presente trabalho empregou imunocitoquimica e imunohistoquímica (ICQ/IHQ) como métodos diagnósticos laboratoriais para LTA. Amostras de culturas de Leishmania in vitro e cortes histológicos de lesões em animais infectados experimentalmente foram submetidos a ICQ/IHQ, utilizando anticorpos policlonais desenvolvidos para este estudo e o complexo avidina-biotina modificado (Ultra Streptavidin®). Em comparação com outras técnicas empregadas para o diagnóstico da LTA, nos casos avaliados, a IHQ apresentou resultados semelhantes aos da histopatologia com coloração HE, com sensibilidade de 33,3% para formas amastigotas. Quando considerada a presenva de antígenos de Leishmania no padrão celular, a IHQ apresentou uma sensibilidade de 83,3%, significativamente maior que na histopatologia e compatível com metodos padrão ouro de cultura e PCR. As metodologias de ICQ/IHQ desenvolvidas neste trabalho foram capazes de demonstrar em biópsias de lesões, a presença de formas amastigotas e antígenos de Leishmania, oferecendo contribuição adicional ao diagnóstico da LTA, sendo de facil aplicação e podendo ser utilizada no sistema público de saúde.

The significant increase in cutaneous leishmaniasis (CL) notified cases and the geographic expansion of this endemy has motivated the development of new techniques to help in leishmaniasis diagnosis, seeking the minimization of the restrictions imposed by the diagnostic tests available at the health services. The current study applied immunocytochemistry and immunohystochemistry methods (ICC/IHC) for laboratory diagnosis of CL. Imprints and histological sections from tissue infected with Leishmania were submitted to ICC/IHC methods using polyclonal antibodies developed for this study and a modified avidin-biotin complex (Ultra Streptavidin®). The samples also were submitted for routinely stained hematoxylin and eosin (H&E) specimens and gold standard methods (culture and PCR). Compared with other useful techniques for the CL diagnosis, ICC/IHC showed the same sensitivity results (33%) as H&E stain for amastigotes recognition. When the presence of Leishmania antigens was evaluated, ICC/IHC presented 83,3% sensitivity, i.e., higher than that detected by histopathology and equivalent with gold standard methods (culture and PCR). The ICC/IHC techniques developed in the current study were able to recognize amastigote forms and also Leishmania antigens in lesion biopsies, offering an additional help to CL diagnosis and it can be easily applied in the public health system.

Effect of seeding using an avidin-biotin binding system on the attachment of periodontal ligament fibroblasts to nanohydroxyapatite scaffolds: three-dimensional culture

PURPOSE: For periodontal tissue engineering, it is a primary requisite and a challenge to select the optimum types of cells, properties of scaffold, and growth factor combination to reconstruct a specific tissue in its natural form and with the appropriate function. Owing to fundamental disadvantages associated with using a two-dimensional substrate, several methods of seeding cells into three-dimensional scaffolds have been reported and the authors have asserted its usefulness and effectiveness. In this study, we explore the cell attachment of periodontal ligament fibroblasts on nanohydroxyapatite (n-HA) scaffold using avidin biotin binding system (ABBS). METHODS: Human periodontal ligament fibroblasts were isolated from the health tooth extracted for the purpose of orthodontic procedure. HA nanoparticles were prepared and Ca(NO3)2-4H2O and (OC2H5)3P were selected as precursors of HA sol. The final scaffold was 8 mm in diameter and 3 mm in height disk with porosity value of 81.55%. 1x10(5) periodontal ligament fibroblasts were applied to each scaffold. The cells were seeded into scaffolds by static, agitating and ABBS seeding method. RESULTS: The number of periodontal ligament fibroblasts attached was greater for ABBS seeding method than for static or agitating method (P<0.05). No meaningful difference has been observed among seeding methods with scanning electron microscopy images. However, increased strength of cell attachment of ABBS could be deduced from the high affinity between avidin and biotin (Kd=10(-15) M). CONCLUSIONS: The high-affinity ABBS enhances the ability of periodontal ligament fibroblasts to attach to three-dimensionally constructed n-HA scaffolds.

Targeted Ultrasound Imaging of Apoptosis with Annexin A5 Microbubbles in Acute Doxorubicin-Induced Cardiotoxicity

BACKGROUND: The aim of this study was to assess the feasibility of targeted ultrasound imaging on apoptosis with annexin A5 microbubbles (A5MB) in acute doxorubicin-induced cardiotoxicity. METHODS: Avidinated and octafluoropropan-filled phospholipid microbubbles were conjugated with biotinylated annexin A5. To confirm the specific binding of A5MB, flow cytometry was performed with hydrogen peroxide induced apoptosis in rat aorta smooth muscle cells incubated with fluorescein-5-isothiocyanate (FITC) labeled annexin A5 and A5MB. Adult male rats were injected intraperitoneally with 5 mg/kg doxorubicin weekly for 3 weeks (n = 5). Control rats were injected with normal saline (n = 5). At 24 hours after the final treatment, triggering imaging was performed 15 min after an intravenous bolus injection of A5MB for washout of freely circulating microbubbles. After echocardiography, the heart was isolated for histological detection of apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. RESULTS: In the in vitro tests, fluorescence intensity was low for healthy cells and high for apoptotic cells when incubated with FITC-labeled annexin A5 and A5MB. Rats treated with doxorubicin showed significant contrast opacification of the myocardium on contrast echocardiography using A5MB. However, no opacification was observed in control rats. Apoptosis was confirmed by TUNEL assay in doxorubicin treated rats. CONCLUSION: Acute doxorubicin-induced cardiomyopathy based on early apoptosis can be assessed and imaged with targeted ultrasound imaging using A5MB in rats.

Clinical application of avidin-biotin ELISA to detect serum hepatoma-specific gamma-glutamyltransferase in patients with primary hepatic cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To detect serum hepatoma-specific datura stramonium lectin-tightly binding gamma-glutamyl transferase (DSA-GGT) in patients with primary hepatic cancer (PHC) by avidin-biotin ELISA method which was established in our laboratory, and carry on a study of its clinical application.</p><p><b>METHODS</b>To detect serum DSA-GGT in 45 healthy control subjects, 58 PHC patients and 203 non-PHC patients (including 36 patients with other tumors and 167 patients with benign liver diseases) with the method was established; meanwhile, AFP was detected by ELISA method.</p><p><b>RESULTS</b>38 individuals were DSA-GGT positive in 58 PHC patients, the sensitivity was 65.5%. 18 individuals were DSA-GGT positive in 203 patients without PHC, the specificity was 91.1%. The sensitivity and specificity of AFP in diagnosis of PHC patients was 69.0% and 90.6%, respectively. The sensitivity and specificity of combination of DSA-GGT and AFP was 93.1% and 85.7%, respectively. The average intra-CV and inter-CV of DSA-GGT ELISA was 8.9% and 11.5%, respectively.</p><p><b>CONCLUSION</b>The sensitivity and specificity of DSA-GGT ELISA method established in our lab is similar with that of AFP assay and the accuracy is good. Combination of DSA-GGT and AFP may improve the diagnostic sensitivity. The method should be potentially as a new way to improve diagnosis of PHC.</p>

A novel protein microarray detection technique based on biotin-avidin conjugation probe / 生物工程学报

In this experiment, a novel biotin-avidin conjugation probe was synthesized and employed in the detection of reverse-phase protein microarray. Firstly, the proportion of the biotin-avidin conjugation probe was optimized. Then the rat IgG and goat anti-rat IgG system was served as a model to optimize the fabrication conditions of reverse-phase protein microarray, including the non-specific absorption of streptavidin-Cy3 molecules, spotting buffer as well as protein activities. At last, the biotin-avidin conjugation probe was applied to the detection of the reverse-phase protein microarray. The results show that the protein microarray prepared by using BSA spotting buffer could prevent non-specific absorptions of fluorescent molecules and improve the sensitivity, effectively. In addition, compared with traditional biotin-avidin system, the detection limit could be improved four times using the biotin-avidin conjugation probe. In conclusion, the biotin-avidin conjugation probe has its merits of easy synthesis, low price and could be further conjugated with other signal amplification techniques, which is promising to be used in the detection of protein microarray.

Biotin/avidin sandwich enzyme-linked immunosorbent assay for Culicidae mosquito blood meal identification

The knowledge of mosquitoes Culicidae host feeding patterns is basic to understand the roles of different species and to indicate their importance in the epidemiology of arthropod-borne diseases. A laboratory assay was developed aiming at standardizing the biotin-avidin sandwich enzyme-linked immunosorbent assay, which was unprecedented for mosquito blood meal identification. The enzyme-linked immunosorbent assay (ELISA) activity was evaluated by the detection of titers on each sample of the 28 blood-fed Culex quinquefasciatus. In light of the high sensitivity that the technique permits, by means of small quantities of specific antibodies commercially provided and phosphatase substrate which reinforces additional dilutions, human and rat blood meals were readily identified in all laboratory-raised Culex quinquefasciatus tested. The assay was effective to detect human blood meal dilutions up to 1:4,096, which enables the technique to be applied in field studies. Additionally, the present results indicate a significant difference between the detection patterns recorded from human blood meal which corroborate the results of host feeding patterns.

Effect of Avidin and Biotin in Attachment of Human Adipose Stem Cells to Micronized Acellular Dermal Matrix

PURPOSE: In tissue engineering, it is important that the scaffolds have high affinity with cells for making efficient use of cells. The authors studied the binding affinity of human adipose stem cells(ASCs) to micronized acellular dermal matrix(alloderm) using biotin and avidin linkages. METHODS: Human ASCs were harvested from adipose tissue obtained by abdominoplasty. ASCs(1x10(4), 5x10(4), 1x10(5), 5x10(5), 1x10(6), 5x10(6) cells) were attached to micronized alloderm(1mg) in three groups; 1) control group in which no ASCs and alloderm was treated; 2) serum group in which alloderm was exposed to fetal bovine serum; and 3) biotin group in which biotinylated cells were attached to biotinylated alloderm. The binding affinities were determined 1 day after making ASC-alloderm complexes. The proliferation rates were determined by XTT assays in 4, 7, 14, and 21 days and scanning electron microscopic examination was performed in 7 and 21 days after culture of ASC-alloderm complexes. RESULTS: The binding affinities of the biotin group were significantly increased in all cell concentrations. Maximum binding affinity was observed at 5x10(4)/mg of micronized dermal matrix in biotin group. The viabilities were lowest in biotin group in contrast to binding affinity, but the difference was not significant. SEM showed well attachment of cells to micronized dermal matrix in all groups. CONCLUSION: The use of avidin/biotin facilitated human ASCs attaching to micronized acellular dermal matrix. This attachment would not disturb adipose stem cells viabilities. The present study suggests that avidin/ biotin can be used as making efficient use of cells in adipose tissue engineering.

Identificacão do sangue ingerido por Lutzomyia (Lutzomyia) longipalpis (Lutz & Neiva, 1912) e Lutzomyia (Lutzomyia) almerioi (Galati & Nunes, 1999) pela técnica imunoenzimática do ELISA de captura, no sistema avidina-biotina / Blood meals identification of Lutzomyia (Lutzomyia) longipalpis (Lutz & Neiva, 1912) e Lutzomyia (Lutzomyia) almerioi (Galati & Nunes, 1999) by enzime-linked immunossorbent assay biotin-avidin

Lutzomyia longipalpis e Lutzomyia almerioi, espécies integrantes da fauna flebotomínea da Serra da Bodoquena, no Estado de Mato Grosso do Sul, têm sido objeto de estudo devido às suas elevadas abundâncias no Assentamento Guaicurus, foco de leishmaniose tegumentar humana e visceral canina. Em pesquisas que vem sendo realizadas neste acampamento para a identificacão de vetores destas parasitoses, foram capturados no período de 2002 a 2004, com armadilhas automáticas luminosas, instaladas em ambiente peridoméstico (galinheiro), 83 exemplares ingurgitados de Lutzomyia longipalpis e Lutzomyia almerioi. O presente estudo teve como objetivo a investigacão do hábito alimentar para ave das fêmeas de ambas as espécies de flebotomíneos, mediante o emprego da técnica imunoenzimática de captura,comparando-se a reatividade durante os anos de 2002 a 2004. Dentre 57 amostras de Lutzomyia longipalpis e 26 de Lutzomyia almerioi, foram encontradas 72 por cento reagentes para ave em Lutzomyia longipalpis e 96 por cento em Lutzomyia almerioi, o que justifica o estudo do hábito alimentar na região, como medida de prevencão e instituicão de vigilância epidemiológica.

Identification of Leukemia Surface Proteins Using a Proteomic Technique / 대한혈액학회지

BACKGROUND: Numerous cell surface proteins of leukemia cells such as CD33 and CD52 have been identified as diagnostic and therapeutic targets. Thus the profiling of the cell surface proteome and proteins restricted to specific leukemia(s) can provide a way to identify novel targets for leukemia diagnosis and therapy. However, there is a lack of data pertaining to the comprehensive analysis of surface membrane proteins because there are few effective strategies for profiling surface membrane proteomes. METHODS: We report on the application of quantitative proteomic techniques that incorporate affinity-capture and purification on monomeric avidin columns to identify all biotinylated cell surface proteins from leukemia cell lines. RESULTS: An analysis of a subset of biotinylated proteins among the different human leukemia cell lines using matrix-assisted laser desorption ionization and tandem mass spectrometry identified, among others, some widely expressed proteins in leukemia cells, such as CD11a, CD11c, CD18, CD31, CD44, and CD147, as well as a set of proteins identified as chaperone proteins, including HSP90, GRP78, GRP75, HSP70, HSP60 and protein disulfide isomerases. On the basis of their known functional roles, several of these proteins may participate in the progression of leukemogenesis and should be considered as potential markers of leukemia. CONCLUSION: Comprehensive profiling of the leukemia cell surface proteome provides an effective approach for the identification of commonly occurring proteins as well as proteins with restricted expression patterns to a specific cell line.

Study of the efficacy of a monoclonal antibody biotin-avidin system for the diagnosis of schistosomiasis japonica / 中华预防医学杂志

<p><b>OBJECTIVE</b>To explore the diagnostic efficiency of circulating antigen using the TM5.28 mAB-biotin-avidin system for the detection of schistosomiasis japonica.</p><p><b>METHODS</b>A mAb-biotin-avidin system was set up using a TM5.28 mAB which was prepared against a gut associated antigen of Schistosoma japonicum. Detection was performed on the sera from 50 acute schistosomiasis patients, 224 chronic patients, 49 advanced patients and 46 schistosomiasis patients who were followed up at 6 months and 12 months post treatment. In addition, 19 cases of clonorchiasis, 31 cases of paragonimiasis, 23 cases of hepatitis B and 100 healthy individuals were also included.</p><p><b>RESULTS</b>The system showed sensitivity of 83.1% and specificity of 94.0% when applied to detect chronic schistosomiasis and healthy persons respectively, while 94.0% to acute schistosomiasis. The Youden's index of the system was 0.771. The rate of cross-reaction to paragonimiasis, clonorchiasis and hepatitis B was 12.9%, 15.8% and 13.0% respectively. The rates of negative turning were 43.9% and 62.1% respectively in chronic schistosomiasis at the 6 month and 12 month intervals after treatment. Geometric mean of the OD values also decreased from 0.172 before treatment to 0.081 at 6 months and 0.068 at 12 months after treatment with a reduction rate of 60.30%. The detection rate in the heavy infected population reached a maximum of 90.0%. This was similar in moderate and light infected populations, i.e., 83.9% and 82.1%, respectively.</p><p><b>CONCLUSION</b>The TM5.28 mAb-biotin-avidin system showed a relatively high efficiency in the diagnosis of schistosomiasis and a high negative turning rate after treatment. It is, therefore, a valuable tool for the estimation of prevalence in endemic populations, as well as individual diagnosis and for assessing the effect of chemotherapy.</p>

Diagnóstico histoquímico e imunoistoquímico da enteropatia proliferativa (Lawsonia intracellularis) em suínos / Detection of swine proliferative enteropathy (Lawsonia intracellularis) in slaughtered pigs by histochemical and immunohistochemical techniques

Estudou-se a eficácia do diagnóstico macroscópico de lesões da enteropatia proliferativa suína (EPS) usando-se fragmentos de íleo de 663 suínos, coletados em abatedouros localizados em três municípios do Rio Grande do Sul. As amostras foram processadas por métodos histológicos rotineiros e coradas por uma técnica desenvolvida pela combinacão das coloracões Warthin-Starry, alcian blue e hematoxilina-eosina para deteccão simultânea de Lawsonia intracellularis e lesões associadas com EPS. Lâminas suspeitas de EPS foram submetidas à técnica de imunoistoquímica, utilizando anticorpo policlonal anti-Lawsonia intracellularis na diluicão de 1:15.000 pelo método avidina-biotina. A coloracão combinada detectou 11 casos positivos, e a imunoistoquímica, nove casos adicionais. Entre as 643 amostras consideradas negativas, 12 apresentaram desaparecimento de células caliciformes e proliferacão adenomatosa características de EPS, mas ausência de bactérias intracelulares. A eficiência do exame macroscópico para diagnóstico de EPS foi medida pela associacão entre os resultados das avaliacões macroscópicas e histológicas realizadas em 219 amostras. Embora 51 delas tenham sido consideradas macroscopicamente positivas, apenas quatro foram confirmadas pela presenca de bactérias intracelulares associadas com lesões características de EPS. Não se observou associacão entre as alteracões macroscópicas e histológicas de EPS.

Estudo do estado de diferenciação da célula mioepitelial nas neoplasias de glândula salivar / Study of the myoepithelial cell differentiation state in salivary gland tumors

A célula mioepitelial (CM) nos tumores de glândula salivar apresenta-se em diferentes estágios de diferenciação. Sabe-se que em glândula normal ela expressa actina músculo específica (AME) e Citoqueratina (CK) 14. Por outro lado, é conhecida a participação dos componentes de matriz extracelular, dentre eles a laminina (LN), na morfogênese e citodiferenciação das estruturas glandulares. Em vista do exposto, nos propusemos a estudar os diferentes estágios de diferenciação da CM através da expressão da AME, da CK 14, bem como a participação da LN neste processo. Para tanto, utilizamos tumores onde se postulam a participação da CM: adenoma pleomórfico, mioepitelioma, adenoma de células basais e carcinoma adenóide cístico e submetemos os espécimes ao método imunohistoquímico da avidina-biotina. Nossos resultados mostraram que a presença da AME foi rara, assim como a CK 14 que só esteve presente em CM de estruturas ductiformes bem formadas. Já a LN esteve presente junto à CM, independentemente da expressão de CK 14 e de AME, e no estroma tanto de tumores diferenciados, como indiferenciados. Em conclusão, é possível identificar diferentes estágios de diferenciação mioepitelial através da expressão da CK 14 e da AME, mas parece não existir uma correlação da LN com a diferenciação da CM tumoral, pois ou essa ou sua precursora continua a secretar LN, mesmo que imperfeitamente após estímulo oncogênico

Padronização da técnica imunoenzimática do ELISA de captura, no sistema avidina-biotina para a identificação de sangue ingerido por Lutzomyia (Lutzomyia) longipalpis (Lutz & Neiva, 1912) / Enzyme-linked Immunosorbent Assay biotin/avidin method standardization, for identification of Lutzomyia (Lutzomyia) longipalpis bloodmeals (Lutz & Neiva, 1912)

A identificação de sangue ingerido pelos insetos é um importante parâmetro para elucidar aspectos ligados à transmissão de zoonoses, dentre elas, as leishmanioses. Dos métodos empregados para esclarecer a atração de vetores por animais que possam atuar como reservatórios dessas parasitoses, destacam-se os imunológicos. O estudo teve como objetivo, padronizar a técnica imunoenzimática de captura e titular amostras de sangue ingerido em fêmeas de flebotomíneos ingurgitadas de Lutzomyia longipalpis criadas em laboratório e alimentadas experimentalmente em rato. Em vista da alta sensibilidade, favorecida pelo sistema avidina-biotina, foi possível a realização de pelo menos noventa testes, de cada uma das amostras em duplicata, e constatar a presença de sangue para todas as amostras com períodos de 12 e 24 horas pós-ingestão, observando-se diferença significativa entre os respectivos títulos.

Immunohistochemical Expression of the alpha- and gamma-Catenin in the Fetal Skin Development / 대한피부과학회지

BACKGROUND: Catenins are the associated protein with E-cadherin in the formation of adhesion complexes in normal and tumor cells related with epithelial differentiation and development of organ formation as well as in the tumor spread. The present study was aimed to find the distribution of alpha- and gamma-catenins in fetal skin development. OBJECTIVE: The purpose of this study was to observe the distribution of above two adhesion related proteins in the fetal skin during development, and to find its relationship by expression and their distribution pattern. METHODS: Skin was obtained from the scalp, chest, and sole of 21 human fetuses, ranging from 13 to 37 weeks of gestational age. Immunohistochemical staining was performed by the avidin biotin peroxidase complex method on paraffin embedded tissue using the anti-human monoclonal antibody against the human alpha- and gamma-catenins. RESULTS: alpha- and gamma-catenins were expressed strongly in basal cells of the epidermis and germ cells of skin adnexa, such as hair and eccrine glands at 13th week, followed by decreased basal cell expression. Increase in the suprabasal epithelium and differentiated adnexal epithelium, such as outer root sheath cells and eccrine ducts and glands at 18th week, and adult pattern in 23th week of gestation. Both showed similar distribution pattern in skin though gamma-catenin appeared two or three weeks later. alpha- and gamma-catenins are expressed not only in the epithelium of the skin, but also in the mesenchymal cells such as endothelial cells and fibroblasts. Though both catenins are more strongly expressed in the membrane portion, cytoplasmic expression is also noted. CONCLUSION: Both alpha- and gamma-catenin showed basically the same expression distribution pattern in the fetal skin developmental stage, suggesting that both adhesion molecules are highly related to each other in function and development of epidermis and adnexae of the skin in fetal stage.

Application of microwave irradiation for the retrieval of masked antigenic sites to react with vimentin

To improve immunoreactivity of the tumor marker vimentin in tissues over-fixed by formalin, using microwave radiation. A total of 10 patients were studied; five cases with thyroid papillary carcinoma, 3 cases with rhabdomyosarcoma and two cases with salivary adenoma. Two serial sections of 5 micro m thickness were cut off from paraffin blocks from the previously menttioned cases. Section were placed on 10 different pairs of slides coated with an adhesive [Vecta-bond, made by Vector Laboratories] to avoid tissue removal due to microwaving steps. One of each pair of slides was incubated in 0.01 M citrate buffer [pH 6] then heated by microwave radiation, while the other slide was left untreated. Both slides were immunostained manually for vimentin using an Avidin Biotin Complex detection kit. Result: The results showed that heating improved the detection of a specific positive reaction, while weak positive and negative results were seen in the untreated slides. Heating of over-fixed slides helped in breaking cross-reacting bonds of proteins and unmasked the antigenic sites that are needed for antigen-antibody reaction. Untreated slides still had masked antigenic sites thus giving faintly stained to negative results. It is recommented to apply heat by microwave for 8-10 minutes on formalin-fixed tissues before immunostaining for vimentin to obtain optimal results

Immunohistochemical Expression of Placental Form of Glutathione S-transferase (GST-pi) in Fetal Skin / 대한피부과학회지

BACKGROUND: Glutathione S-transferases(GST) are a family of multi-functional enzymes involved in cellular detoxification and excretion of a variety of exogenous and endogenous toxic or carcinogenic compounds. The GST family has been divided into three classes, alpha, mu, and pi, based on substrate specificity and sequence homology. GST-pi is an acidic type and predominant in skin, small intestine, breast, lung and prostate. The overexpression of GST-pi associated with skin tumor and tumor-like lesion suggests that GST-pi is a major detoxifying enzyme in skin tumors. OBJECTIVE: The purpose of this study was to observe the expression and the distribution pattern of GST-pi in the human fetal skin. METHODS: Skin was obtained from the scalp, chest, and sole of 49 human fetuses, ranging from 8th to 40th weeks of gestational age. Immunohistochemical staining was performed using avidin biotin peroxidase complex method on paraffin embedded tissue using antirabbit polyclonal antibody against the human GST-pi. RESULTS: GST-pi was expressed in intermediated layer of epidermis at 8th week, and gradually increased in strength of expression stronger in suprabasal layer. In hair unit, GST-pi was expressed in sebaceous gland, bulge, hair matrix cell and outer root sheath cell from 15th week. In eccrine gland, also GST-pi was expressed in central differentiated cells of intradermal eccrine duct from 18th week, and in terminal duct and acini from 26th week of fetal age. CONCLUSION: GST-pi was expressed from the 8th week of gestation suggesting that GST-pi plays an important role in detoxification for the protection of the skin in fetal stage from the various toxic agent.