Epigenetic Silencing of Eyes Absent 4 Gene by Acute Myeloid Leukemia 1-Eight-twenty-one Oncoprotein Contributes to Leukemogenesis in t(8;21) Acute Myeloid Leukemia / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The acute myeloid leukemia 1 (AML1)-eight-twenty-one (ETO) fusion protein generated by the t(8;21)(q22;q22) translocation is considered to display a crucial role in leukemogenesis in AML. By focusing on the anti-leukemia effects of eyes absent 4 (EYA4) gene on AML cells, we investigated the biologic and molecular mechanism associated with AML1-ETO expressed in t(8;21) AML.</p><p><b>METHODS</b>Qualitative polymerase chain reaction (PCR), quantitative reverse transcription PCR (RT-PCR), and Western blotting analysis were used to observe the mRNA and protein expression levels of EYA4 in cell lines. Different plasmids (including mutant plasmids) of dual luciferase reporter vector were built to study the binding status of AML1-ETO to the promoter region of EYA4. Chromatin immunoprecipitation assay was used to study the epigenetic silencing mechanism of EYA4. Bisulfite sequencing was applied to detect the methylation status in EYA4 promoter region. The influence of EYA4 gene in the cell proliferation, apoptosis, and cell clone-forming ability was detected by the technique of Cell Counting Kit-8, flow cytometry, and clonogenic assay.</p><p><b>RESULTS</b>EYA4 gene was hypermethylated in AML1-ETO+ patients and its expression was down-regulated by 6-fold in Kasumi-1 and SKNO-1 cells, compared to HL-60 and SKNO-1-siA/E cells, respectively. We demonstrated that AML1-ETO triggered the epigenetic silencing of EYA4 gene by binding at AML1-binding sites and recruiting histone deacetylase 1 and DNA methyltransferases. Enhanced EYA4 expression levels inhibited cellular proliferation and suppressed cell colony formation in AML1-ETO+ cell lines. We also found EYA4 transfection increased apoptosis of Kasumi-1 and SKNO-1 cells by 1.6-fold and 1.4-fold compared to negative control, respectively.</p><p><b>CONCLUSIONS</b>Our study identified EYA4 gene as targets for AML1-ETO and indicated it as a novel tumor suppressor gene. In addition, we provided evidence that EYA4 gene might be a novel therapeutic target and a potential candidate for treating AML1-ETO+ t (8;21) AML.</p>

Association of rotavirus viroplasms with microtubules through NSP2 and NSP5

Rotavirus replication and virus assembly take place in electrodense spherical structures known as viroplasms whose main components are the viral proteins NSP2 and NSP5. The viroplasms are produced since early times after infection and seem to grow by stepwise addition of viral proteins and by fusion, however, the mechanism of viropIasms formation is unknown. In this study we found that the viroplasms surface colocalized with microtubules, and seem to be caged by a microtubule network. Moreover inhibition of microtubule assembly with nocodazole interfered with viroplasms growth in rotavirus infected cells. We searched for a physical link between viroplasms and microtubules by co-immunoprecipitation assays, and we found that the proteins NSP2 and NSP5 were co-immunoprecipitated with anti-tubulin in rotavirus infected cells and also when they were transiently co-expressed or individually expressed. These results indicate that a functional microtubule network is needed for viroplasm growth presumably due to the association of viroplasms with microtubules via NSP2 and NSP5.

The application of latent class analysis for diagnostic test validation of chronic Trypanosoma cruzi infection in blood donors

The main strategy to prevent transfusion-associated Chagas disease is the identification of T. cruzi-infected blood donors by serological screening tests, however there is no perfect serological gold standard. We evaluated an enzyme immunoassay (EIA), an indirect hemaglutination (IHA), and an indirect immunofluorescence (IIF) test for detecting T. cruzi antibodies in Brazilian blood donors. The results were submitted to latent class analysis, and a radioimmunopreciptation (RIPA) test was performed on repeatedly positive samples. Among 1951 donors, 11 (0.56) were positive by EIA, 6 (0.31) by IHA and 16 (0.82) by IIF. Six samples were positive with all tests, while 4 reacted with EIA and IIF. The RIPA was positive in 6 (75.0), 7 (66.6), and 4 (54.0) samples reacting by the EIA, IHA and IIF tests, respectively. The latent class model detected a high sensitivity rate (100) for the EIA and IIF, and a specificity rate of 99.95 and 99.69 for the EIA and IIF tests, respectively. The probability of being case according to the model was 99.92 when both EIA and IIF were positive, and 100 for the association of EIA, IIF, and IHA.

Interferência de anticorpos em testes de funçäo tireoidiana: relato de caso / Antibodies interference in thyroid function tests: repost of case

Na avaliaçäo da funçäo tireoideana utiliza-se como método diagnóstico as medidas de TSH, T4 e T3 totais e livres. Entretanto, em alguns casos acorre a ligaçäo näo específica com reagentes dos ensaios que väo, desta forma, interferir com as medidas destes hormônios. Estas interferências iräo resultar em concentraçöes séricas anormais de hormônios tireoideanos, näo consistentes com a avaliaçäo clínica e demais exames laboratoriais destes pacientes. Auto-anticorpos anti-hormônio tireoideano säo a classe de fatores que mais frequentemente interferem com vários ensaios. Relatamos o caso de uma paciente de 62 anos, com queixas de ansiedade e palpitaçöes e exame físico normal. na avaliaçäo laboratorial detectamos níveis séricos persistentemente elevados de T3 total, com níveis séricos normais de TSH e T4 total. A presença de anticorpos anti-T3 foi confirmada por radioimunoprecipitaçäo. Resultados que parecem ser inconscientes ou imcompatíveis com os demais exames laboratoriais, na presença ou näo de sintomas em geral inespecíficos, devem levantar a suspeita da presença de fatores interferentes no ensaio. Desta forma, evita-se o diagnóstico errôneo de disfunçäo tireoideana e, consequentemente, um tratamento desnecessário e até mesmo deletério.

Antinuclear Antibody Production in Patients with Malaria Infection / 대한임상병리학회지

BACKGROUND: Infection can activate the immune system and may trigger the production of autoantibodies. It has been reported that malaria infection triggers the production of various autoantibodies. Therefore, we investigated the pattern and significance of antinuclear antibodies (ANA) found in patients with malaria infection. METHODS: Our study group included 36 patients who were diagnosed with malaria infection at Mokdong Hospital from July 1998 to July 2001. We performed antinuclear antibody test using indirect immunofluorescence method (Quantafluor, Sanofi Diagnostics Pasteur Inc., USA), extractable nuclear antigen test (ENA) using double immunodiffusion method (Nova Gel, Inova Diagnostics Inc., USA), anti-double stranded DNA Ab test (anti-ds DNA Ab) using Farr assay (DPC anti-DNA, Diagnostic products Corporation, USA), and anti-single stranded DNA Ab test (anti-ssDNA Ab) using enzyme immunoassay method (QUANTA, Lite ssDNA, Inova Diagnostics Inc., USA). RESULTS: Among the 36 patients, 32 patients (88.9%) showed ANA positivity and 27 patients (75.0%) showed cytoskeleton or speckled pattern of ANA. Anti-ssDNA Ab was found in 3 of 20 patients; however, anti-dsDNA Ab and ENA were not found in all patients. Patients who had ANA showed higher levels of IgG, IgM and IgA, compared with those patients who did not have ANA. Follow up (11-37 month) of the 13 patients with ANA positivity revealed no symptoms associated with autoimmune disorder. CONCLUSIONS: Malaria infection may develop ANA, especially cytoskeleton or speckled pattern. The follow up of patients with ANA positivity showed no symptoms associated with any autoimmune disorder, but further evaluation would be necessary to reveal the relationship between malaria infection and development of autoimmune disorder.

Capacidad de predecir una recaída de lupus eritematoso generalizado, mediante anticuerpos anti-DNA de hebra doble, por la técnica de Farr / Predictive capacity of double stranded anti DNA antibodies for relapses in patients with inactive systemic lupus erythematosus using Farr technique

Background: Patients with inactive systemic lupus erythematosus (SLE) and elevated high affinity double-stranded anti-DNA antibodies (anti-dsDNA), measured using Farr technique, would have a risk of relapse that fluctuates between 40 to 80 percent according to different series. Aim: To study the association between anti-dsDNA levels measured using Farr technique and disease activity and their predictive capacity for relapses. Material and methods: Anti-dsDNA antibodies were measured according to Farr method in 60 healthy subjects, 69 patients with other connective tissue diseases and in 120 patients with SLE. Farr positive were considered those individuals with anti-dsDNA levels over 10.4 IU/ml. Disease activity, assessed using MEX-SLEDAI score was related with anti-dsDNA levels in 101 patients. Forty seven patients with inactive disease were followed for 17ñ14 months. Results: Anti-dsDNA levels were 3ñ2.5 IU/ml (range 1-26) in subjects without LED, and 127ñ500 IU/ml (range 1-5280) in patients with LED. Sixty subjects had an active SLE and 43 (72 percent) were Farr positive; in 41 the disease was inactive and 13 (32 percent) were Farr positive (p <0.001), OR 5.45. Twelve of the 47 followed patients had a relapse and 10 (83 percent) were Farr positive. Of those that did not have a relapse, 13 (37 percent) were Farr positive (p< 0.02, RR 5.22). Six of 15 patients that were followed for more than on year (40 percent), were Farr positive. Conclusions: Elevated anti-dsDNA antibodies measured using Farr technique in patients with inactive generalised lupus erythematosus, predicted the risk of relapse. However less than half of patients with inactive disease and elevated Farr relapsed in a period of one year. The need to treat patients with inactive SLE and positive Farr should therefore be considered debatable

Integration of HPV and the Antibody Respones to HPV Proteins in Patients with Cervical Cancer / 대한암학회지

PURPOSE: HPV (human papillomavirus) are known as the major causative agent for development of cervical cancer. High-risk HPVs, especially HPV-16 /18 DNA, are often found to be integrated into the human genome in high grade CINs as well as cervial cancer. Investigation of the relationship between the genomic states of HPV genes and their antibody response against the HPV-16 Ll/L2 virus-like particles (VLPs) and the in vitro translated E6 and E7 proteins may help to explain the mechanism of HPV-related cervical carcinogenesis and host immune responses. MATERIALS AND METHODS: Cervical cancer tissues obtained from 41 patients with cervical cancer were studied by PCR, Southern blot hybridization and the antibody response against HPV-16 Ll/L2 VLPs and HPV-16 E6, E7 proteins of serum were tested by ELISA and radioimmunoprecipitation assay (RIPA), respectively. RESULTS: Integrated forms of the HPV-16 DNA were found in 23 of the 38 patients (60.5%). The HPV-16 positive cervial cancer patients had a significantly higher prevalence (39.5%; 15/38) of antibodies to HPV-16 Ll/L2 VLPs than 8.7% (2/28) of the the control group (p0.05). CONCLUSION: Integrated forms of HPV-16 DNA were prevalent in most patients with cervical cancer. Antibodies to HPV-16 Ll/L2 VLPs, in vitro translated HPV-16 E6 and E7 proteins appeared in the significantly larger proportions of the HPV-associated cervical cancer patients than in the controls. Antibodies to HPV-16 Ll/L2 VLPs were more detectable in the cervical cancer patients with episomal form of HPV-16 DNA than those who having only integrated forms of HPV-16. Antibody responses to HPV-16 E6 and E7 proteins were not influenced by the different viral states. More numbers of studies would be necessary to determine the relationship between the genomic states of HPV and the immune responses to their proteins by the such genomic and serologic parameters.

La microbiología en la transfusión sanguínea / Microbiology in blood transfusion

La familia de los Retrovirus se encuentra en el hombre y en los animales y son una causa considerable de mortalidad. Aunque se sabe que existen hace casi 100 años, su rol como agentes patógenos humano se recononoció hace aproximadamente 15 años. Su Genoma está compuesto por ARN. Los cuadro Retrovirus humanos: HIV-1, HIV-2, HTLV-1 y HTLV-II comparten cararterísticas comunes aunque se trata de entidades diferentes. Los ensayos que se utilizan para cada uno de estos virus son similares tanto en los principios como en las técnicas. En este primer apartado (I) del ensayo "Los Retrovirus" de detallará: etipatología, configuración antigénetica, respuesta inmune específica del huésped, clasificación de los métodos de diagnósticos sérico de HIV-1 y HIV-2 luego se completará el dictum mediante una lista parcial de los equipos comerciales de diagnóstico en el Anexo 1

Tuberculose peritoneal: Relato de um caso / Peritoneal tuberculosis: A case report

Os autores descrevem um caso de tuberculose peritoneal solucionado através de prova terapêutica. Tratava-se de menina de nove anos com ascite cuja punçäo mostrou líquido amarelo com elevaçäo de proteínas e predomínio de linfócitos. A evoluçäo foi favorável

Púrpura trombocitopênica aloimune neonatal: caracterizaçäo de aloanticorpo plaquetário anti-HPA-1A (Anti-PIA1) por radioimunoprecipitaçäo indireta / Alloimmune neonatal thrombocytopenic purpura: characterization of platelet alloantibody anti-HPA-1A (Anti-PIA1) by indirect radioimmunoprecipitation

A púrpura trombocitopênica alo-imune neonatal (PTAN) é uma doença grave na qual a hemorragia cerebral pode ser fatal ou levar a seqüelas cerebrais permanentes. Similarmente, à doença hemolítica do recém-nascido (RN), a PTAN ocorre devido a aloimunizaçäo materna por um alo-antigeno incompatível presente nas plaquetas fetais. A apresentaçäo clínica é de púrpura generalizada acompanhada de hemorragia gastrointestinal, urinária, e/ou intracranial. O alo-antigeno HPA-1 (PL(A)) é responsável por cerca de 70-80 por cento dos casos de PTAN. Nesse estudo, os autores descrevem o caso de uma gestante que deu a luz a um RN com plaquetopenia acentuada devido a trombocitopenia aloimune. A contagem plaquetária da mae e do RN era 173 x 10(9)/L e 15 x 10(9)/L, respectivamente. O estudo sorológico realizado com a técnica de radioimunoprecipitaçäo indireta demonstrou forte reatividade do soro materno com o complexo glicoprotético GPIIb/IIIa em plaquetas PI(A1)-positivas de doador normal. Assim, o soro materno continha forte atividade anti-HPA-1a (anti-PI(A1)). O RN foi tratado com corticosteróides e gamaglobulina intravenosa. Após o tratamento, o RN teve alta hospitalar com contagem plaquetária elevada para 70 x 10(9)/L.

HTLV-I associated myelopathy in patients from Brazil and Iran

Foram analisadas amostras de líquidocefalorraquidiano (LCR) e plasma de 20 pacientes com mielopatia associada ao HTLV-I (HAM/TSP) provenientes do Brasil e Irä e como controle, 16 de indivíduos brasileiros soronegativos para HTLV-I acometidos por outras doenças neurológicas (esclerose múltipla, epilepsia idiopática e mielopatia de etiologia desconhecida). Observou-se no grupo de pacientes com HAM/TSP: 1) reaçäo inflamatória no SNC em todos os casos; 2) 95 por cento (19/20) mostravam bandas oligoclonais refletindo sintese intrafecal de IgG no SNC; 3) 85 por cento (7/20) apresentavam síntese local de anticorpos para HTLV-I; 4) 35 por cento (tinham síntese mensurável de imunoglobulinas no SNC. Os parâmetros do LCR dos pacientes com HAM/TSP foram comparados aos dados clínicos (idade de início, duraçäo da doença e grau de incapacidade). Os dados apresentados neste estudo indicam a importancia da análise do LCR para diagnóstico de HAM/TSP. Entretanto nenhuma associaçäo entre a gravidade da doença e os achados do LCR foi demonstrada

HTLV-I antibody study in normal individuals and unselected hospital patients in Malaysia.

A serological investigation for human T cell leukemia virus I (HTLV-I) infection was carried out at the University Hospital, Kuala Lumpur. A total of 626 sera from a non-patient population and 1,038 sera from unselected in-patients were screened for HTLV-I antibodies using an enzyme-linked immunosorbent assay (ELISA). 27/1664 (1.6%) were found to be reactive. However, on Western blotting, only 2 sera were confirmed positive, both showing reactions for the major core (p19 and p24) and the envelope (gp46) proteins. Both of the serum samples were from unselected hospital patients. Most of the remaining sera which were reactive on screening showed indeterminate results on Western blotting. These were further tested by radioimmunoprecipitation assay (RIPA) and none of these sera gave a positive reaction. Therefore, only 2/1038 (0.19%) unselected patients could be confirmed to have antibodies to HTLV-I. None of the normal individuals screened showed a positive Western blot result. Our data indicate that HTLV-I infection is present in our population, but at a low prevalence rate.