Evaluación preliminar de la actividad antiviral de Ageratina havanensis frente al virus dengue-2 utilizando ensayos inmunoenzimáticos

RESUMEN Introducción: Se presenta la evaluación preliminar de la actividad antiviral de extractos de Ageratina havanensis (etanólico de tallo, AH-T-EtOH; butanólico de hoja, AH-H-ButOH y acetato de etilo de hoja, AH-H-AcEtO) utilizando dos sistemas inmunoenzimáticos, un ELISA celular (ELISAc) y la técnica de inmunoperoxidasa. Objetivo: Evaluar la actividad antiviral de los extractos de Ageratina havanensis utilizando dos sistemas inmunoenzimáticos. Métodos: Se normalizó y empleó un ELISAc y la técnica de inmunoperoxidasa en evaluar de forma preliminar la actividad antiviral de tres extractos de Ageratina havanensis a diferentes concentraciones y tiempos de adición mediante la detección de la expresión de la proteína E del virus dengue. Resultados: El ELISAc logró como parámetros óptimos: línea celular Vero, antígeno viral en cultivo de células, tiempo de expresión de la proteína E de 96 h, compuesto fijador metanol-acetona y bloqueador leche descremada al 1 %. La menor expresión de la proteína E (mayor inhibición sobre la replicación viral), fue al adicionar el extracto AH-T-EtOH a su mayor concentración y 1 h antes de inocular el virus dengue-2 cepa A15 (VDEN-2 A15). El extracto AH-H-ButOH a concentraciones de 125 µg/mL y 250 µg/mL presentó una ligera limitación de expresión de E 1 h después de la inoculación viral. El extracto AH-H-AcEtO a las concentraciones empleadas, no mostró difererencia añadido antes y después de la inoculación. El ensayo de inmunoperoxidasa exhibió resultados análogos para los extractos. Conclusiones: La mayor inhibición de la replicación viral fue obtenida con el extracto AH-H-ButOH, a su mayor concentración y ambos tiempos de adición. Los ensayos inmunoenzimáticos aplicados son herramientas útiles para evaluar extractos de Ageratina havanensis con posible actividad antiviral.

ABSTRACT Introduction: A preliminary evaluation is presented of the antiviral activity of Ageratina havanensis extracts (stem ethanolic, AH-T-EtOH; leaf butanolic, AH-H-ButOH; and leaf ethyl acetate, AH-H-AcEtO) using two enzyme immunoassays, cellular ELISA (C-ELISA) and immunoperoxidase technique. Objective: Evaluate the antiviral activity of Ageratine havanensis extracts using two enzyme immunoassays. Methods: C-ELISA and immunoperoxidase technique were standardized for use in the preliminary evaluation of the antiviral activity of three Ageratina havanensis extracts at different concentrations and addition times through detection of the expression of the E protein of dengue virus. Results: C-ELISA achieved the following optimal parameters: Vero cell line, viral antigen in cell culture, protein E expression time 96 h, methanol-acetone fixation compound and 1% skimmed milk blocker. The smallest expression of protein E (greatest inhibition over viral replication) was achieved at addition of extract AH-T-EtOH at its highest concentration and 1 h before inoculating dengue-2 virus strain A15 (VDEN-2 A15). Extract AH-H-ButOH at concentrations of 125 µg/ml and 250 µg/ml presented a slight limitation in the expression of E 1 h after viral inoculation. Extract AH-H-AcEtO at the concentrations used did not show any difference when added before and after inoculation. The immunoperoxidase assay exhibited similar results for the extracts. Conclusions: The greatest inhibition of viral replication was obtained with extract AH-H-ButOH at its highest concentration and at both addition times. The enzyme immunoassays applied are useful tools to evaluate Ageratine havanensis extracts with potential antiviral activity.

Comparative evaluation of immunoperoxidase versus immunofluorescent techniques in interpretation of kidney biopsies.

Background: Immunofluorescence (IF) on frozen sections has been considered to be the gold standard for evaluation of kidney biopsy specimens. Immunohistochemistry (IHC) method can also be used for this purpose with advantages of being applicable on paraffin embedded tissue, providing permanent sections, and not requiring a specialized microscope for interpretation. Our aim was to evaluate IHC as an alternative to IF in the diagnostic assessment of kidney biopsy specimens. Methods: One hundred kidney biopsy specimens were subjected to both IF and IHC staining for immunoglobulins (Ig), IgG, IgA, IgM and complement components c3 and c1q. IF staining was done on frozen sections. IHC staining was performed on paraffin‑embedded tissue following proteolytic antigen retrieval. The sections were evaluated, and the results of IHC were compared with IF. Results: Concordant observations were 98%, 87%, 89%, 83%, and 89% for IgA, IgM, IgG, C3 and C1q, respectively. The sensitivity of IHC method for Igs was found to be high (92%, 86.5%, and 95.1%, respectively for IgA, IgM, and IgG). 91% cases showed concordance of the intensity of the deposits while 100% cases showed a concordance of the pattern. Statistically, there was no significant difference in outcomes between IF and IHC for IgA, IgM, and IgG. However, statistically significant difference was found in the results for complement proteins. Conclusion: In this study, it is documented that IHC is, with few exceptions, equal to IF for the detection of Igs. Standardized immunoperoxidase method on the paraffin embedded, formalin fixed needle kidney biopsies could successfully replace the IF method in the diagnosis of glomerulonephritis.

Expressions of pro-apoptotic protein Smac and HtrA2 in children with acute leukemia and its clinical significance / 中华实用儿科临床杂志

Objective To investigate the expression and clinical significance of Smac and HtrA2 in children with acute leukemia(AL).Methods Bone marrow samples were obtained from 77 children with AL (including 32 newly diagnosed children,33 complete remission children and 12 relapsed children)and the control group of 15 children without malignant blood disease.The expressions of Smac and HtrA2 protein were measured by streptavidin/peroxidase immunoperoxidase technique(SP) in all children.SPSS 13.0 software was applied to analyze the statistical data.Results Protein Smac was detected only in some samples,but HtrA2 was detected in all samples.The levels of Smac and HtrA2 protein in newly diagnosed AL children were both higher than those of the complete remission children (x2 =17.38,F =2.36,all P ＜ 0.05) and normal controls (x2 =12.89,F =5.26,all P ＜ 0.05),there was a statistical significance,but compared with those in the relapsed children,the difference had no statistical significance (x2 =1.18,F =1.57,all P ＞ 0.05).The levels of Smac and HtrA2 protein in complete remission children were both higher than those of the normal controls,and the difference had no statistical sigmficance(x2 =1.20,F =2.23,all P ＞ 0.05).In the newly diagnosed children,the levels of Smac and HtrA2 protein in children with acute lymphocytic leukemia(ALL) were higher than those of the acute myeloid leukemia(AML),but the differences had no statistical significance(x2 =0.113,t =1.024,all P ＞ 0.05).In newly diagnosed AL children,the complete remission(CR) rate of the negative expression of Smac(Smac-,90.9％) and the low expression of HtrA2(HtrA2low,84.6％) in the level of protein were higher than those of the positive expression of Smac(Smac +,47.6％) and the high expression of HtrA2 (HtrA2high,47.4％),and there was statistical significance respectively(x2 =5.772,4.596,all P ＜ 0.05).The CR rate of Smac-HtrA2low group (100％) was higher than that of Smac+ HtrA2high group(30.8％)in the children with AL,and the statistical data were of great significance(x =9.692,P ＜0.01).The protein level of Smac in newly diagnosed AL children was correlatedwith HtrA2 (r =0.979,P ＜ 0.001).Conclusions Pro-apoptotic protein Smac and HtrA2 may be involved in and af-fected each other in the pathogenesis and progression in AL,but levels of Smac and HtrA2 protein may be not correlatedwith the types of AL.In newly diagnosed AL children,the high expression of protein Smac and HtrA2 predicts poorprognosis.

Potentially pathogenic mycoplasmas in the external ear canal of clinically normal cattle in Southeast Brazil: first report / Micoplasmas potencialmente patogênicos no canal auditivo de bovinos clinicamente sadios no Sul do Brasil: primeiro relato

Mycoplasmas were searched in the ear canal flushing of 60 bovine in Brazil. The prevalence obtained was 80 percent. The percentages of typified species were 12.5 percent, for M. alkalenses; 2.1 percent, M. arginini; 8.35 percent, M. bovirhinis; 2.1 percent, M. bovis; 25.0 percent, M. conjunctivae; 14.6 percent, M. mycoides subsp. mycoides LC and 10.4 percent M. capricolum.

Foram pesquisados micoplasmas no conduto auditivo de 60 bovinos no Brasil. A prevalência obtida foi de 80 por cento. A porcentagem das espécies tipificadas foi de M. alkalenses, 12,5 por cento; M. arginini, 2,1 por cento; M. bovirhinis, 8,35 por cento; M. bovis, 2,1 por cento; M. conjunctivae, 25,0 por cento; M. mycoides subsp. mycoides LC, 14,6 por cento e M. capricolum, 10,4 por cento.

Comparison of immunoperoxidase and immunofluorescence assays for pp65 cytomegalovirus antigen in immunocompromised patients

We compared the pp65 antigen detection by an in house method (immunoperoxidase assay) and by a commercial kit (immunofluorescence assay) available for cytomegalovirus infection diagnosis in immunocompromised patients. Sixty-four blood samples were analyzed in duplicate for both techniques. Eight-six percent of the samples had concordant qualitative results. The discordant results occurred more frequently in samples with low quantity of positive cells. There were no significant differences with qualitative and quantitative results of the methods.

Identificação da Leptospira interrogans sorovar pomona em camundongos geneticamente selecionados, para a alta e baixa produção de anticorpos, através da técnica de imunoperoxidase em tecido renal e isolamento bacteriano em meio de Fletcher / Identification of Leptospira interrogans serovar pomona in mice genetically selected for high and low antibody production, by means of the immunoperoxidase technique in renal tissue and bacterial isolation in Fletcher medium

O presente trabalho teve por objetivo identificar a presença da Leptospira interrogans sorovar pomona em camundongos geneticamente selecionados para a alta e baixa resposta a anticorpos. Todos os animais foram submetidos ao isolamento bacteriano, imunohistoquímica (imunoperoxidase) em cortes de tecido renal e coloração através da hematoxilina-eosina. A técnica de imunoperoxidase apresentou-se pouco mais sensível em relação ao cultivo, entretanto, ambas foram bons parâmetros de identificação do agente. Presença de lesões renais mais intensas ocorreram em períodos em que houve maior número de bactérias isoladas em meio de cultivo. Camundongos da linhagem HIV-A conseguiram eliminar as leptospiras com maior eficiência e rapidez em relação as linhagem LIV-A, entretanto o estudo demonstrou que ambas linhagens da seleção IV-A foram eficientes em controlar o processo infeccioso.

The present work had the objective of identifying the presence of Leptospira interrogans serovar pomona in mice that had been genetically selected for high and low response to antibodies. All the animals were subjected to bacterial isolation, immunohistochemical analysis (immunoperoxidase) in renal tissue sections and hematoxylin-eosin staining. The immunoperoxidase technique was little more sensitive than culturing, but both were good parameters for agent identification. More severe renal lesions were present at times when there were greater numbers of bacteria isolated in culture medium. Mice of the lineage HIV-A were able to eliminate the Leptospira more efficiently and faster than the lineage LIV-A could. However, the study demonstrated that both lineages of the IV-A selection were efficient in controlling the infectious process.

Padronização da técnica de imunoperoxidase para detecção do vírus da diarréia bovina a vírus em cultura de células: Standardization of immunoperoxidase test to detection bovine viral diarrhea virus in cell culture

The aim of this study was to standardize the immunoperoxidase in cell monolayer assay (IPMA) for the etiological diagnosis of bovine viral diarrhea (BVD). The method was standardized in monolayer of primary bovine fetal lung culture inoculated with cytophatic and non-cytophatic classical strains of BVD virus and tested using samples that were considered suspected in the classical technique of viral isolation. The IPMA successfully identified BVD virus and presented better results when heat was used for fixation, BSA 4% solution in PBS was used for blocking and AEC chromogen was used for revelation. Both monoclonal and polycloral antibodies gave good results when used as primary antibodies.

Este estudo teve como objetivo a padronização do ensaio de imunoperoxidase em monocamada de células (IPM) para o diagnóstico etiológico da diarréia bovina a vírus (DBV). O teste foi padronizado em monocamada de cultivo primário de pulmão fetal bovino (PFB) inoculada com as amostras clássicas, citopatogênica (CP) e não citopatogênica (NCP), do vírus da DBV e testado em amostras biológicas suspeitas processadas no teste clássico de isolamento viral (IV). O método de IPM identificou o vírus da DBV, apresentando melhores resultados com a utilização do calor como agente fixador, a soroalbumina bovina a 4% em PBS como bloqueador e a revelação com o cromógeno 3-amino-9-etil-carbazol (AEC). Como anticorpos primários, tanto o anticorpo policlonal como o monoclonal forneceram bons resultados.

Significance of thyroglobulin in thyroid diseases detected by the immunoperoxidase method.

Thyroglobulin (TG) is a protein that synthesized from the thyroid follicular epithelium and can reflect the thyroid gland in origin together with the cellular activity. The aim of this study is to detect TG qualitatively and semiquantitatively by immunoperoxidase method in various thyroid lesions. The result shows that all of the nodular goitre (43 cases), primary diffuse hyperplasia (18 cases), adenoma 60 cases, papillary carcinoma (43 cases) and follicular carcinoma (22 cases) show cytoplasmic staining of TG in varying pattern and intensity, while only 3 out of the 7 anaplastic carcinoma show irregular cytoplasmic staining. These findings can be used in identifying the thyroid gland in origin in the metastatic lesion and the functional status of the lesion.

The Comparison of the Urine RBC Phase Contrast Microscopy and Immunoperoxidase Stain in Differentiation of Hematuria / 대한신장학회잡지

Differentiation of renal(RH) and non-renal(NRH) hematuria is important in the diagnosis and treatment of the patients with hematuria. Recently, urine RBC immunoperoxidase(IPx) staining method was developed, but there was no report on the usefulness of IPx in Korea. We validated the usefulness of IPx by comparing with the PCM. Both PCM and IPx were performed at the same time in 26 patients with RH confirmed by renal biopsy and 23 patients with NRH confirmed by radiologic and/or pathologic studies who were admitted to Chungbuk National University Hospital from January 1996 to December 1996. The age of RH and NRH group were 36.6+/-15.0 and 56.5+/-22.2 years. 35.7+/-30.4% of urine RBC were stained by IPx in RH group and only 1.6+/-4.4% were stained in NRH group(P<0.001). 23.4+/-29.9% of urine RBC by PCM were counted as dysmorphic RBC in RH group and 5.7+/-13.6% were counted in NRH group(P<0.05). At the cut-off value of 20%, the sensitivity and specificity of IPx were 57.7% and 100%. At the cut-off value of 30%, those of PCM were 30.9% and 95.7%, respectively. When comparing overall test performance by calculating AUCs of ROC(receiver operating characteristics) curve, IPx was better than PCM. IPx was better than PCM in localizing the origin of hematuria. The NRH might be excluded when IPx(+) cells are more than 20% of total urine RBC.

Estrogen and Progesterone receptors in Meningioma

We studied the status of estrogen(ER) and progesterone(PR) receptors in meningiomas removed from 32 patients, using immunoperoxidase(IP) assays. PR were detected in 72% of the cases & ER were detected in 31%. The possible correlation between age, sex, histological type, ploidy pattern and proliferation index values with steroid receptor activity were discussed. The date suggest that the majority of meningiomas contain high affinity receptors for progesterone, that estrogen receptors are present in only a few meningiomas.

Detection of Treponema pallidum in tissue: a comparative study of the avidin-biotin-peroxidase complex, indirect immunoperoxidase, FTA-ABS complement techniques and the darkfield method

With 37 formalin-fixed, paraffin embedded specimens from the lesions of 30 patients with primary, secondary or gastric syphilis, we performed avidin-biotin-peroxidase complex (ABC), indirect immunoperoxidase (IIP) and FTA-ABS complement techniques. Darkfield examination was done in 17 skin lesions. The immunoperoxidase technique, especially the ABC technique, revealed higher reactivity than the FTA-ABS complement technique and darkfield examination in detecting Treponema pallidum in tissues. Furthermore, the ABC technique produced less intense nonspecific background staining than the IIP technique. Histologically, most of the treponemes were located in the upper dermis, epidermis and vessel walls in the order named, and rarely in the lower dermis of the syphilitic skin lesions.

Solitary myeloma with massive extracellular crystalline structures: a case report

We describe a case of solitary myeloma showing cystic change filled with massive crystalline structures in a 54-year-old woman. A bone X-ray showed a solitary cystic osteolytic lesion in the right iliac bone. Serum and urine protein electrophoresis showed no demonstrable M-protein, and bone-marrow aspirates did not show any myeloma cells. Histologic examination of the tumor revealed aggregation of plasma cells with massive extracellular infiltration of the rhomboid-shaped crystalline structures. In immunoperoxidase staining, both these crystalline structures and the cytoplasms of the myeloma cells demonstrated a positive reaction for lambda light chain. By electron microscope, the large extracellular crystalline structures were observed, and we found unique rhomboid or rectangular-shaped crystalline structures in the cytoplasms of the myeloma cells.

Localization of Keratinolytic Proteinase in Skin Tissue Sections of Guinea Pigs with Microsporum canis Infection by Immunoperoxidase Technique in Electron Microscopy

An immunoperoxdase technique in electron microscopy was used to investigate the ultrastructural site of keratinolytic proteinase (KPase) of M crosporum cans in sections of skin from guinea pigs infected with the same organism. Ultrastructurally, the KPase was present only in the cell walls of the invading dermatophytes as a continuous deposition of the electron-dense reaction product on the inner and outer aspects of the cell wall of the fungal hyphae without deposition in the keratin surrounding the invading hyphae. Our results suggest that the KPase may not play an absolute role in the invasion of dermatophytes into keratinized tissue in vivo.

Detection of Treponema pallidum in Tissues by Indirect Immunoperoxidase Technique and FTA - ABS Complement Technique / 대한피부과학회지

Using the indirect immunoperoxidase and FTA ABS complement techniques, 36 skin specimen: from 30 patients with primary and secondary syphilis and a gastric mucosal specimen from a patient with secondary syphilis which were confirmed by elinical history, physical examination, VDRL, FTA ABS, TPHA, and 19s(IgM)-FTA test, were examined. The follwing results were obtained. l. Of the 37 specimens, 33(89%) were positive in the indirect immunoperoxidase techique and 26 of the 37 specimens(70g) were positive in the FTA ABS complement technique. Of the 17 specimens, 12(71%) were positive in the darkfield examination. 2. The ratio of agreement of the results between the indirect immunoperoxidase and FTA ABS complement techniques was 81%, The ratio of agreement of the results between the indirect immunoperoxidase technique and darkfield examination was 82%. 3. Most, of the treponemes were located in the upper dermis, epidermis, and blood vessel walls in the order named, and rarely in the lower dermis of the syphilitic skin lesion. There was no remarkable difference in histologic clistribution of treponemes between the clinical stages and types of syphilitic skin lesions. From the results, the indirect immunoperoxidase technique is considered a superior method than the FTA ABS complement technique and darkfield examination for detecting the treponemes in the suspected syphilitic lesions.

STUDIES ON ANTIGEMC HOMOLOGY BETWEEN SCHISTOSOMA JAPONICUM AND ONCOMELANIA HUPENSIS HUPENSIS BY INDIRECT IMMUNOPEROXIDASE TECHNIQUE / 中国寄生虫学与寄生虫病杂志

Adult Schistosoma japonicum worms and Oncomelania hupensis hupensis tissue sections were employed as antigens in the studies of anti-O.h.hupensis rabbit serum and anti-S.japonicum adult worm rabbit serum by indirect immunaperoxidase assay.The result indicated that the most prominent peroxidase reaction was presented in the adult worm sections and the head-foot and liver sections of O.h,hupensis.These suggested the possible presence of antigenic homology between Schistosoma japonicum and Oncomelania hupensis hupensis.(Figs.1-7)

PRATICAL VALUE OF THREE TYPES OF ANTI- KERATIN ANTIBODIES AND IMMUNO-PEROXIDASE TECHNIQUE IN ROUTINE HISTOPATHOLOGICAL STUDY OF SKIN / 重庆医科大学学报

This paper reports the use of three different types of anti-keratin antibodies and immuno-peroxidase technique in the staining of various skin tumors paraffin-embedded. The results showed that three types of anti-keratin antibodies (Cam5.2, AF2 and PK)didn't stain none-pithelial tumors and,expressed their inherent different staining patterns for epithelial tumors derived f from different tissues. Therefore, three types of. anti-keratin antibodies may be helpful for diagnosis, differential diagnosis and classification of complex skin tumors in routine histopathology of skin

Factor VIII Related Antigen in Granuloma Pyogenicum / 대한피부과학회지

Using Avidin-Biotin immunoperaxidase method, we investigate the distribution patterns of factor VIII related antigen in various areas of skin lesions of 14 granuloma pyogenicum. and in the skin lesions of 5 capillary hemangioma and 5 normal skin. The results are summarized as follows: 1. Factor VIII related antigen was detected in 9 of 14 cases of granuloma pyogenicum, in 4 of 5 cases of capillary hemangioma, and in 5 of 5 cases of normal skin. 2. In the lesions of granuloma pyogenicum the areas with prominent capillary endothelial cell proliferation had less positivity than the vascular areas. And the areas with relatively large vesels in the lesions of granuloma pyogenicum had similar positivity as capillary hemangioma. 3. The lesions of granuloma pyogenicum and capillary hemangioma had less positivity than the vessels of normal derrnis.

T Cells in Cutaneous Lesions of Psoriasis / 대한피부과학회지

Anti-T cell monoclonal antibodies(OKT series: OKT4a OKT8A, OKT1 1) ir,amunoperoxidase technique studies were performed for the presence of T cells and their subpopulations in cutaneous lesions of early papules and late plaques from 10 patients with psoriasis. In this semiquantitative examination, T cells reactive to OKT11 antiserum constituted about 70 percents of total leukocytes found in the dermis, both in early papular lesions and late plaque lesions. The number of helper/inducer T cells was about equal to the number of suppressor/cytotoxic T cells in early papular lesions, but helper/inducer T cells were predominant in plaque lesions. Although in situ functions of T cell and their subpopulations can not precisely be deduced, this different proportions of the helper/inducer and suppressor/cytotoxic T cells found in this study might be related to the immunopathologic mechanisms pertainmg to the persistence and the expansion of psoriatic lesions.

A STUDY ON INDIRECT PEROXIDASE ANTIBODY TEST OF DETECTING IgM ANTIBODIES TO EPIDEMIC HEMORRHAGIC FEVER / 西安交通大学学报(医学版)

The immunoporoxidase staining technics (IPST) was used in 37 patients with epidemic hemorrhagic fever (EHF) for detecting the specific IgM antibodies to EHF and compared with the method of IFA-IgG. The sera obtained at the early stage of the disease were ana- lyzed. In two cases, the results were negative by using the method of IPA-IgM antibodies. In 35 cases, the results were significantly higher than those by means of the method of IFA-IgG. But, the sera obtained at the late stage showed no difference between the two methods. The inhibition test and 2-ME tolerance tcst showed that the antibodics detected by the IPST technics were specific IgM ones, which could be found in the patient's serum on the second day of onset. The results showed that the IPA-IgM method is sensitive and highspecific and the results can be read under the light microscope. Thus, this method is convenient for the country doctors to use.