In vivo and in vitro study of co-expression of LMP1 and Cripto-1 in nasopharyngeal carcinoma / Estudo in vivo e in vitro da coexpressão de LMP1 e Cripto-1 em carcinoma nasofaríngeo

Abstract Introduction: Nasopharyngeal carcinoma, an epithelial-derived malignant tumor which because of its anatomical location and atypical early symptoms, when diagnosed invasion and metastasis often have occurred. This requires a better understanding of the development mechanism, identifying diagnostic markers, and developing new treatment strategies. Objective: To study the relationship of LMP1 and Cripto-1 in nasopharyngeal carcinoma. Methods: The expression of LMP1 and Cripto-1 in specimens obtained from nasopharyngeal carcinoma patients (n = 42) and nasopharyngitis patients (n = 22) were examined. The expression of LMP1 and Cripto-1 in LMP1-negative and LMP1-positive (CNE1-LMP1) cells were also examined. Results: The expression of LMP1 and Cripto-1 was significantly higher in nasopharyngeal carcinoma than in nasopharyngitis (p < 0.05). Their expression in nasopharyngeal carcinoma with metastasis were significantly higher than that without metastasis (p < 0.05), which was correlated with TNM staging (p < 0.05). High Cripto-1 expression and high proliferation rate were seen in CNE1-LMP1 cells. Conclusions: The expression of LMP1 and Cripto-1 in nasopharyngeal carcinoma is positively related. Their co-expression might contribute to the proliferation and metastasis of nasopharyngeal carcinoma.

Resumo Introdução: O carcinoma nasofaríngeo é um tumor maligno derivado do epitélio de localização anatômica recôndita e sintomas iniciais atípicos; quando diagnosticado, frequentemente invasão e metástases já ocorreram. Isso requer uma melhor compreensão do seu mecanismo de desenvolvimento, identificação dos marcadores diagnósticos e desenvolvimento de novas estratégias de tratamento. Objetivo: Estudar a relação de LMP1 e Cripto-1 no carcinoma nasofaríngeo. Método: A expressão de LMP1 e Cripto-1 em espécimes obtidos de pacientes com carcinoma de nasofaringe (n = 42) e pacientes com nasofaringite (n = 22) foi analisada. A expressão de LMP1 e Cripto-1 em células LMP1-negativas e LMP1-positivas (CNE1-LMP1) também foi analisada. Resultados: A expressão de LMP1 e Cripto-1 foi significantemente maior na presença de carcinoma nasofaríngeo do que na nasofaringite (p < 0,05). Sua expressão em carcinomas com metástase foi significantemente maior do que em casos sem metástase (p < 0,05), o que se correlacionou com o estadiamento TNM (p < 0,05). Uma alta expressão de Cripto-1 e alta taxa de proliferação foram observadas nas células CNE1-LMP1. Conclusões: A expressão de LMP1 e Cripto-1 é positivamente relacionada com carcinoma nasofaríngeo. Sua coexpressão pode ser atribuída à proliferação e metástase do tumor.

Pp65 antigenemia and cytomegalovirus diagnosis in patients with lupus nephritis: report of a series / Antigenemia pp65 e diagnóstico de citomegalovírus em pacientes com nefrite lúpica: série de casos

ABSTRACT Introduction: In contrast to organ transplantation, few studies correlate the monitoring of pp65 antigenemia with a diagnosis of cytomegalovirus (CMV) in patients with systemic lupus erythematosus (SLE). Objective: To highlight the importance of CMV outside transplantation, we monitored pp65 antigenemia in a series of SLE patients. Methods: From March 2015 to March 2016, SLE patients presenting kidney involvement, fever, and an unclear infection at hospital admission were monitored through pp65 antigenemia. The pp65 antigenemia assay, revealed by immunofluorescence, was correlated with clinical and laboratory findings. Results: We included 19 patients with a suspected unclear infection. A positivity for pp65 antigenemia was found in seven patients (36.8%). The mean age was 33.5 ± 11.2 years, 16 (84%) were females, and 16 (84%) were black. Lymphopenia, anemia, and higher scores of SLEDAI were significantly more common in pp65-positive patients. Five patients received antiviral therapy with ganciclovir. Although receiving specific CMV treatment, one patient died because of suspected CMV disease. Conclusions: Pp65 antigenemia might be relevant in SLE patients, and studies with a greater number of patients are needed in order to establish sensitivity and specificity of pp65 antigenemia in different clinical contexts of SLE patients.

RESUMO Introdução: Diferentemente do transplante de órgãos, poucos estudos correlacionam o monitoramento da antigenemia pp65 com o diagnóstico de citomegalovírus (CMV) em pacientes com lúpus eritematoso sistêmico (LES). Objetivo: De modo a destacar a importância do CMV para além do transplante, monitorizamos a antigenemia pp65 em uma série de pacientes com LES. Métodos: De março de 2015 a março de 2016, pacientes com LES que apresentaram acometimento renal, febre e infecção indeterminada na internação foram monitorados através da antigenemia pp65. O ensaio de antigenemia, revelada por imunofluorescência, foi correlacionado com achado clínicos e laboratoriais. Resultados: Foram incluídos 19 pacientes com suspeita de infecção indeterminada. Positividade para antigenemia pp65 foi encontrada em sete pacientes (36,8%). A idade média foi de 33,5 ± 11,2 anos; 16 (84%) eram do sexo feminino e 16 (84%) eram negros. Linfopenia, anemia e escore de SLEDAI mais elevado foram significativamente mais comuns em pacientes pp65 positivos. Cinco pacientes receberam terapia antiviral com ganciclovir. Apesar de receber tratamento específico para CMV, um paciente com suspeita de doença por CMV veio a óbito. Conclusões: Antigenemia pp65 pode ser relevante em pacientes com LES, e estudos com maior número de pacientes são necessários para estabelecer a sensibilidade e a especificidade da antigenemia pp65 em diferentes contextos clínicos envolvendo pacientes com LES.

Imunogenicidade da proteína M recombinante de Streptococcus equi subsp. equi coadministrada com um adjuvante molecular / Immunogenicity of recombinant M protein of Streptococcus equi subsp. equi co administered with a molecular adjuvant

The strangles is an infectious disease that affects horses from all ages and causes important economic losses in the equine-related business. The aim of this work was to evaluate the immunogenicity of the recombinant M protein from Streptococcus equi (rSeM) co-administered with the recombinant heat-labile enterotoxin B subunit from Escherichia coli (rLTB) in mice and horses. A total of 72 female Balb-c mice were divided into eight groups and 18 horses were divided into six groups. The animals were inoculated by intramuscular (IM) or intranasal (IN) routes with different treatments of rSeM, rLTB and/or Al(OH)3. The results obtained in both species, independent of administration routes, demonstrated that rSeM + rLTB had higher levels of specific serum immunoglobulins, however, in mucosal immunity the increase was not identified. Thus, the use of rSeM as vaccine antigen and rLTB as adjuvant can be a potential tool in the control of equine strangles.(AU)

Clinical correlates of pp65 antigenemia monitoring in the first months of post kidney transplant in patients undergoing universal prophylaxis or preemptive therapy

Abstract Introduction: Human cytomegalovirus is a major cause of morbidity in kidney transplant patients. Objectives: We aimed to study viral replication and serological response in the first months post kidney transplant in patients undergoing universal prophylaxis or preemptive therapy and correlate the findings with the clinical course of Human cytomegalovirus infection. Patients and methods: Independent from the clinical strategy adopted for managing Human cytomegalovirus infection, prophylaxis versus preemptive therapy, the pp65 antigenemia assay and serological response were assessed on the day of transplantation, and then weekly during the first three months of post-transplant. Results: From the 32 transplant recipients, 16 were positive for pp65 antigenemia, with a similar incidence rate in each group. There were no positive results in the first three weeks of monitoring; the positivity rate peaked at week eight. There was a trend for a higher and earlier frequency of positivity in the universal prophylaxis group in which the course of the Human cytomegalovirus infection was also more severe. Despite the differences in clinical picture and in the initial immunosuppressant schedule, the serological response was similar in both groups. Conclusion: Routine monitoring during the first three post-transplant months has a positive impact on the early detection of Human cytomegalovirus viral replication allowing for timely treatment in order to reduce morbidity of the disease. The strategy of universal therapy employing intravenous ganciclovir was associated to a worse clinical course of the Human cytomegalovirus infection suggesting that the use of >10 cells/2 × 105 leukocytes as a cut-off in this setting may be inappropriate.

Swim training and the genetic expression of adipokines in monosodium glutamate-treated obese rats

Objective The aim of this study was to evaluate the genetic expression of adipokines in the adipocytes of monosodium glutamate (MSG)-treated obese rats submitted to physical activity.Materials and methods Obesity was induced by neonatal MSG administration. Exercised rats (MSG and control) were subjected to swim training for 30 min for 10 weeks, whereas their respective controls remained sedentary. Total RNA was obtained from sections of the mesenteric adipose tissue of the rats. mRNA levels of adiponectin (Adipoq), tumor necrosis factor alpha (Tnf), peroxisome proliferator-activated receptor alpha (Ppara), and peroxisome proliferator-activated receptor gamma (Pparg) adipokines were quantified by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR).Results In the exercise-trained control group, the expression of Adipoq increased compared to the sedentary control, which was not observed in the MSG-obese rats. Increased levels of Tnf in MSG-obese rats were not reversed by the swim training. The expression of Ppara was higher in sedentary MSG-obese rats compared to the sedentary control. Swimming increased this adipokine expression in the exercise-trained control rats compared to the sedentary ones. mRNA levels of Pparg were higher in the sedentary MSG-rats compared to the sedentary control; however, the exercise did not influenced its expression in the groups analyzed.Conclusions In conclusion, regular physical activity was not capable to correct the expression of proinflammatory adipokines in MSG-obese rat adipocytes.

Celulitis por Streptococcus equi: reporte de caso y revisión de la literatura / Streptococcus equi Cellulitis: Case Report and Literature Review

El Streptococcus equi es un coco gram positivo, perteneciente al grupo C de Lancefield, causa una enfermedad de gran relevancia en caballos, la gurma o adenitis equina (1-2); en humanos, estas infecciones son poco frecuentes, siendo más frecuentes las infecciones de piel y tejidos blandos, faringitis, neumonía, síndrome tóxico similar al shock y endocarditis. Cuando la infección está asociada a bacteriemia, la mortalidad reportada es del 25%.(3) Presentamos el caso de un hombre de 44 años que ingresa al servicio de urgencias de la Clínica universidad de la Sabana con un cuadro clínico de celulitis en mano derecha por Streptococcus equi .

Streptococcus equi is a gram-positive cocci, from group C of Lance 􀃀 eld. It causes an important disease in horses, strangles or equine adenitis (1-2). In humans, these infections are rare, and skin and soft tissue infections, pharyngitis, pneumonia, toxic shock-like syndrome and endocarditis are more frequently observed. When the infection is associated with bacteremia, the reported mortality is near 25% (3). We report the case of a 44-year old man who was admitted to the emergency department of the University of Sabana Clinic with cellulitis due to Streptococcus equi in his right hand.

Comparison of the performance of polymerase chain reaction and pp65 antigenemia for the detection of human cytomegalovirus in immunosuppressed patients / Comparação de métodos para detecção de infecção por citomegalovírus em pacientes imunossuprimidos

INTRODUCTION: Human cytomegalovirus (HCMV) is often reactive in latently infected immunosuppressed patients. Accordingly, HCMV remains one of the most common infections following solid organ and hemopoietic stem cell transplantations, resulting in significant morbidity, graft loss and occasional mortality. The early diagnosis of HCMV disease is important in immunosuppressed patients, since in these individuals, preemptive treatment is useful. The objective of this study was to compare the performance of the in-house qualitative polymerase chain reaction (PCR) and pp65 antigenemia to HCMV infection in immunosuppressed patients in the Hospital de Clínicas of Porto Alegre (HCPA). METHODS: A total of 216 blood samples collected between August 2006 and January 2007 were investigated. RESULTS: Among the samples analyzed, 81 (37.5 percent) were HCMV-positive by PCR, while 48 (22.2 percent) were positive for antigenemia. Considering antigenemia as the gold standard, sensitivity, specificity, positive predictive values and negative predictive values for PCR were 87.5 percent, 76.8 percent, 51.8 percent and 95.5 percent respectively. CONCLUSIONS: These results demonstrated that qualitative PCR has high sensitivity and negative predictive value (NPV). Consequently PCR is especially indicated for the initial diagnosis of HCMV infection. In the case of preemptive treatment strategy, identification of patients at high-risk for HCMV disease is fundamental and PCR can be useful tool.

INTRODUÇÃO: O citomegalovírus humano (HCMV), causador de infecção latente, reativa com frequência em pacientes imunossuprimidos. Portanto, o HCMV permanece uma das infecções mais comuns após transplantes de órgãos sólidos e de células hematopoiéticas resultando em significativa morbidade, perda do enxerto e ocasional mortalidade. Assim, o diagnóstico precoce para uma terapia preventiva é de grande importância. Este estudo visa comparar o desempenho dos métodos PCR qualitativo in-house e antigenemia pp65 para o diagnóstico de infecção por CMV em pacientes imunossuprimidos do Hospital de Clínicas de Porto Alegre. MÉTODOS: O estudo foi realizado em 216 amostras de sangue total (EDTA) coletadas de 85 pacientes, entre agosto de 2006 e janeiro de 2007. RESULTADOS: Dentre as 216 amostras analisadas, 81 (37,5 por cento) amostras apresentaram resultados positivos na PCR, enquanto 48 (22,2 por cento) apresentaram resultados positivos na antigenemia. A sensibilidade, especificidade, valor preditivo positivo e valor preditivo negativo para a PCR, considerando antigenemia como padrão foram 87,5 por cento, 76,8 por cento, 51,8 por cento e 95,5 por cento, respectivamente. CONCLUSÕES: Estes resultados demonstraram que a PCR tem alta sensibilidade e valor preditivo negativo. Consequentemente PCR é especialmente indicada para o diagnóstico inicial de infecção por HCMV. No caso da estratégia de terapia preventiva, a identificação de pacientes com alto risco para a doença por HCMV é fundamental e a PCR pode ser uma ferramenta útil.

Comparison of a rapid cytomegalovirus pp65 antigenemia assay revealed by immunofluorescence to an in-house assay revealed by immunoperoxidase for diagnosis in solid organ transplant recipient patients

Cytomegalovirus (CMV) antigenemia is still one of the two major assays available for diagnosis and monitoring of CMV infections. A commercial rapid test recently available in Brazil for quantification of human cytomegalovirus pp65 antigenemia revealed by immunofluorescence technique was compared with the original in-house method revealed by immunoperoxidase in patients receiving solid organ transplants. Of 80 blood samples tested for CMV antigenemia, 34 (42.5 percent) were positive: commercial assay detected 33 (97 percent) and in-house assay detected 20 (58.8 percent) samples. The numbers of positive cells in the two assays were different, with a median of 4.5 and 12 positive cells obtained by in-house and commercial kit, respectively. Discrepancies between assays occurred in 15 specimens from patients with low-grade antigenemia (median 6 positive cells). The assay-time was reduced in approximately 50 percent compared to in-house methodology. In conclusion, besides comparable results obtained for both assays, the commercial antigenemia assay provides more rapid and sensitive results.

Evaluation of neutralization patterns of the five unique Argentine equine arteritis virus field strains reported / Evaluación de los patrones de neutralización de las únicas cinco cepas argentinas descritas de arteritis viral equina

Equine viral arteritis (EVA) is a contagious viral disease that frequently causes mild or subclinical infections in adult horses. Only one EAV serotype has been described. However, there are differences in antigenicity, pathogenicity and neutralization characteristics of virus field strains. The interaction of two viral proteins, GP5 and M, is critical for infectivity and amino acid changes in the GP5 sequences have an effect on the neutralizing phenotype, regardless the effects of other viral proteins. The objective of the present study was to evaluate the neutralization phenotypes of the 5 unique Argentine EAV strains reported and to compare them with the neutralization phenotypes of the EAV-UCD reference strain, with special emphasis on the analysis of M and GP5 proteins. The strains had a similar neutralization phenotype pattern when anti-EAV serum, derived from EAV seropositive horses, was used in the analysis. Meanwhile, low titers were observed when equine polyclonal anti-EAV reference sera were used in the assay. Argentine strains have almost the same amino acid substitutions, with the exception of LP01 strain, that mainly involves the first variable region V1, especially in neutralization sites B and C. However, they are fairly different from the EAV-UCD strain. Nevertheless, the nucleotide and amino acid differences observed among the Argentine strains LP02/R, LP02/C, LP02/P and LP-LT-ARG did not show any variations in the neutralization phenotype.

La arteritis viral equina (AVE) ocasiona infecciones, en su mayoría subclínicas, pero puede causar abortos y enfermedad respiratoria. Si bien se ha descrito un solo serotipo de AVE, existen diferencias en cuanto a la antigenicidad, patogenicidad y patrones de neutralización en las cepas de campo. Los ORF5 y ORF6 del virus codifican las proteínas de envoltura GP5 y M; la interacción entre estas proteínas es crítica para la infectividad. Los cambios en las secuencias de aminoácidos en la proteína GP5, especialmente en la región V1, afectan el fenotipo neutralizante, sin tener en cuenta variaciones aminoacídicas de otras proteínas virales. En este estudio evaluamos los fenotipos neutralizantes de las 5 únicas cepas de arteritis viral equina aisladas en Argentina y los comparamos con los de la cepa de referencia EAV-UCD por virus neutralización cruzada y análisis de secuencias aminoacídicas de las proteínas M y GP5. Las cepas argentinas presentaron un patrón de neutralización similar cuando se utilizaron sueros positivos del banco de sueros, mientras que fueron neutralizadas en menor medida por los sueros policlonales de referencia anti-AVE. A excepción de la cepa LP01, las cepas argentinas tienen casi las mismas sustituciones aminoacídicas en la primera región variable V1 de la proteína GP5, específicamente en los sitios neutralizantes B y C, pero difieren en gran medida respecto de la cepa de referencia EAV-UCD. Las diferencias encontradas en los aislamientos LP02/R, LP02/C, LP02/P y LT-LP-ARG no se reflejaron en variaciones en el fenotipo neutralizante.

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.

Detecção do vírus Epstein-Barr em tonsilites recorrentes / Detection of Epstein-Barr virus in recurrent tonsillitis

As tonsilites recorrentes têm sido objeto de muitos estudos. Eventos considerados na predisposição e causa incluem a utilização errônea de antibióticos em crises agudas, alterações da microflora, mudanças estruturais nas criptas epiteliais tonsilares e infecções virais. A infecção pelo vírus Epstein-Barr (EBV) ocorre freqüentemente na infância persistindo em linfócitos de tonsilas, podendo causar tonsilites recorrentes. Pouco se conhece sobre a persistência e reativação do EBV em pacientes imunocompetentes. Alguns métodos como a hibridização in situ, a reação em cadeia da polimerase (PCR) e a imuno-histoquímica têm sido utilizados no estudo da patogenia do vírus. OBJETIVO: Para caracterizar a associação do vírus Epstein-Barr com tonsilites recorrentes examinamos a presença do EBV pela PCR e por imuno-histoquímica usando como alvo a proteína viral LMP-1. FORMA DE ESTUDO: Estudo transversal com análise de prevalência amostral. MATERIAL E MÉTODOS: Foram selecionados 24 blocos parafinados de tonsilas, provenientes do Serviço de Anatomia Patológica, removidas de crianças de 2 a 12 anos com diagnóstico de tonsilite recorrente. Resultados: O genoma do EBV foi detectado em 13 (54,1%) e a LMP-1 em 9 (37,5%) dos casos. CONCLUSÃO: As tonsilas das crianças podem ser colonizadas pelo EBV e este pode estar associado à patogenia das tonsilites recorrentes.

Recurrent tonsillitis has been the subject of frequent investigation. Misuse of antibiotic therapy in acute tonsillitis, changes to the tonsillar microflora, structural changes to the tonsillar crypts, and viral infections have been listed as predisposing or causal factors for recurrent tonsillitis. Epstein-Barr virus (EBV) infection usually occurs in early childhood and may persist in tonsillar lymphocytes, thus leading to the onset of recurrent tonsillitis. Little is known about the persistence and reactivation of EBV strains in immunocompetent patients. Methods such as in situ hybridization, polymerase chain reaction (PCR), and immunochemistry have been used to study the pathogenesis of the EBV. AIM: this study aims to characterize the association between EBV and recurrent tonsillitis by investigating the presence of EBV through PCR and immunohistochemistry, using viral protein LMP-1 as a target. STUDY DESIGN: this is a cross-sectional study with analysis of sample prevalence. MATERIALS AND METHOD: twenty-four paraffin-embedded tonsil specimens from the Pathology Service were selected. The specimens were removed from children aged between 2 and 12 years diagnosed with recurrent tonsillitis. RESULTS: EBV genome was detected in 13 (54.1%) specimens, whereas viral protein LMP-1 was found in 9 (37.5%) specimens. CONCLUSION: children's tonsils can be colonized by EBV and such colonies may be associated with the pathogenesis of recurrent tonsillitis.

Genetic variations of EBV-LMP1 from nasopharyngeal carcinoma biopsies: potential loss of T cell epitopes

To find Epstein-Barr virus (EBV) strains with genetic variations of EBV latent membrane protein 1 (EBV-LMP1) from nasopharyngeal carcinoma (NPC), the full-length DNA of LMP1 genes from 21 NPC biopsies obtained in Hunan province in southern China was amplified and sequenced. Our sequences were compared to those previously reported by the Clustal V method. Results showed that all 21 sequences displayed two amino acid changes most frequently in LMP1 of CD4+ T cell epitopes at codons 144 (F arrow right I, 21/21) and 212 (G arrow right S, 19/21) or (G arrow right N, 2/21). We also show that type A EBV strain is prevalent in the cases of NPC from Hunan province with a 30-bp 18/21 deletion, and we highlight that this deletion resulted in loss of one of the CD4+ T cell-restricted epitopes. The other 3 sequences without this deletion all had a change at codon 344 (G arrow right D). Furthermore, in the major epitope sequence of CD8+ T cells restricted by HLA-A2, all 21 sequences showed changes at codons 126 (L arrow right F) and 129 (M arrow right I). Our study discovered that one of the 21 sequence variations harbored a new change at codon 131 (W arrow right C), and 5/21 specimens showed another novel change at codon 115 (G arrow right A) in the major epitope sequence of CD8+ T cells restricted by HLA-A2. Our study suggests that these sequence variations of NPC-derived LMP1 may lead to a potential escape from host cell immune recognition, protecting latent EBV infection and causing an increase in tumorigenicity.

Detection of free circulating Epstein-Barr virus DNA in plasma of patients with HodgkinÆs disease / Detecção do DNA livre circulante do vírus Epstein-Barr no plasma de pacientes com doença de Hodgkin

CONTEXT AND OBJECTIVE: Free circulating Epstein-Barr virus (EBV) DNA is often present in the plasma of HodgkinÆs disease patients. The aim here was to evaluate the prevalence of this finding, its correlation with the immunohistochemical expression of LMP-1 (latent membrane protein 1) and the influence of other clinical factors. DESIGN AND SETTING: Prospective study in two public tertiary institutions: Hematology Service, Universidade Federal do Rio de Janeiro, and Oncology Service, Instituto Nacional do Câncer, Rio de Janeiro. METHODS: A cohort of 30 patients with newly diagnosed HodgkinÆs disease was studied. The control group consisted of 13 healthy adult volunteers. EBV DNA was determined by conventional polymerase chain reaction (PCR). RESULTS: The median age was 28 years, and 16 patients were women. Advanced disease was present in 19 patients, and six were HIV-positive. EBV DNA was present in the plasma of 13 patients and one control (43 percent versus 8 percent, p = 0.03). EBV DNA prevalence was higher in HIV-positive patients (100 percent versus 29 percent, p = 0.0007) and those with advanced disease (63 percent versus 9 percent, p = 0.006). Among HIV-negative patients alone, EBV DNA prevalence remained higher in those with advanced disease. EBV DNA was found in 10/11 patients with LMP-1 expression in the lymph nodes, and in 3/19 without LMP-1 expression (kappa coefficient = 0.72). CONCLUSION: EBV DNA was present in 91 percent of patients with EBV-associated HodgkinÆs disease, and in all patients with HIV-associated HodgkinÆs disease. EBV DNA prevalence was higher in patients with advanced disease, irrespective of HIV status.

CONTEXTO E OBJETIVO: O DNA do vírus Epstein-Barr (EBV) está freqüentemente presente no sangue periférico de pacientes com doença de Hodgkin. O objetivo deste estudo foi avaliar a prevalência deste achado, e correlacioná-lo com a expressão imunoistoquímica da LMP-1 (latent membrane protein 1) e a presença de outros fatores clínicos. TIPO DE ESTUDO E LOCAL: Estudo prospectivo realizado no Serviço de Hematologia da Universidade Federal do Rio de Janeiro e no Serviço de Oncologia do Instituto Nacional do Câncer, Rio de Janeiro, Brasil. MÉTODOS: Trinta pacientes com doença de Hodgkin recém-diagnosticada foram estudados, assim como um grupo controle composto por 13 indivíduos saudáveis. O DNA do EBV no plasma foi determinado pela reação em cadeia da polimerase (PCR) convencional. RESULTADOS: A idade mediana foi 28 anos e 16 pacientes eram do sexo feminino. A doença disseminada esteve presente em 19 pacientes e seis eram HIV+. O DNA do EBV foi detectado no plasma de 13 pacientes e um controle (43 por cento versus 8 por cento, p = 0,03). A prevalência do DNA do EBV foi maior nos pacientes HIV+ (100 por cento versus 29 por cento, p = 0,0007) e naqueles com doença disseminada (63 por cento versus 9 por cento, p = 0,006). Quando somente os pacientes HIV-negativos foram analisados, a prevalência do DNA do EBV permaneceu maior nos pacientes com doença disseminada. A prevalência do DNA do EBV variou de acordo com o subtipo histológico: foi de 32 por cento nos pacientes com esclerose nodular e de 100 por cento nos pacientes com celularidade mista e depleção linfocítica (p = 0,02). O DNA do EBV foi encontrado em 10/11 pacientes com a expressão da LMP-1 em linfonodos, e em 3/19 pacientes sem a expressão da LMP-1 (coeficiente de kappa = 0,72). CONCLUSÕES: O DNA circulante do EBV foi detectado no plasma de 91 por cento dos pacientes com doença de Hodgkin associada ao EBV, e em todos os pacientes com doença de Hodgkin associada ao HIV. A prevalência do DNA circulante do EBV foi detectado no plasma de 91% dos pacientes com doença de Hodgkin associada ao EBV, e em todos os pacientes com doença de Hodgkin associada ao HIV. A prevalência do DNA circulante do EBV foi maior nos pacientes com doença avançada, independentemente do status para o HIV.

Arsenic trioxide reduces the invasive and metastatic properties of nasopharyngeal carcinoma cells in vitro

Nasopharyngeal carcinoma (NPC) is notorious for the metastases, which are in close association with Epstein-Barr virus-encoded latent membrane protein 1 (LMP1). Arsenic trioxide (As2O3) has been shown to induce apoptosis and differentiation in NPC xenografts. Then, can it repress the cancer cells' metastasis potential? To elucidate this issue, the present study was performed. LMP1-negative cell line HNE1 and LMP1-positive cell line HNE1-LMP1 were used as in vitro model. Cells (1 x 10(5)/mL) were cultured with or without 3 æM As2O3 for 48 h. Then the survival cells were collected to investigate their potential of colony formation, attachment, invasion, and migration. Both confocal immunofluorescence staining and Western blot were used to detect the changes of LMP1 expression. The changes of MMP-9 were examined by RT-PCR assay and Western blot. The results were as follow: i) the colony formation inhibition rate (75.41 ± 3.9 percent in HNE1-LMP1 cells vs 37.89 ± 4.9 percent in HNE1 cells), the rate of attachment (HNE1-LMP1 vs HNE1: 56.40 ± 3.5 vs 65.87 ± 5.9 percent), the invasion inhibitory rate (HNE1-LMP1 vs HNE1: 56.50 ± 3.7 and 27.91 ± 2.1 percent), and the migration inhibitory rate (HNE1-LMP1 vs HNE1: 48.70 ± 3.9 vs 29.19 ± 6.27 percent) were all significantly different between the two cell lines (P < 0.01). ii) LMP1 was down-regulated in As2O3-treated HNE1-LMP1 cells. iii) The reduction of MMP-9 was found in As2O3-treated groups, more evident in HNE1-LMP1 cells. Thus, we conclude that As2O3 can reduce metastasis potential of NPC cells, involving inhibition of MMP-9 expression. LMP1 were also reduced in this process and seemed to enhance anti-metastasis activity of As2O3.

Búsqueda de resistencia a amantadina en cepas de virus influenza A aisladas en Santiago de Chile, entre los años 2001 y 2002 / Search of amantadine-resistance in influenza A strains isolated in Santiago, Chile, 2001-2002

Amantadina es un fármaco eficaz para el tratamiento y prevención de influenza A. Su mecanismo de acción es inhibir la proteína M2. Su uso por períodos prolongados puede generar resistencia, la cual ocurre por mutaciones en el gen que codifica para la proteína M2. La mutación más frecuentemente encontrada se ubica en la posición 31. El uso de técnicas de biología molecular permite detectar estas mutaciones. Los objetivos fueron determinar la existencia de resistencia a amantadina en cepas de virus influenza A aisladas entre los años 2001 y 2002 en un laboratorio de virología en Santiago de Chile, y validar un nuevo método de biología molecular para reconocer cepas resistentes. Para ello se utilizó metodología de RPC y análisis de tamaño de fragmentos de restricción. En 31 cepas procesadas no se observó la presencia de cambios en la posición 31. Estos hallazgos sugieren que la resistencia a amantadina es muy baja o está ausente en nuestro medio. Esto podría explicarse por un limitado uso de este fármaco en esta población. El método descrito puede servir de base para un monitoreo prospectivo de resistencia, que pueda ser de utilidad al médico clínico.

Epstein-Barr virus in biopsies from patients with Hodgkin and non-Hodgkin lymphoma at the University of Puerto Rico immunohistochemistry laboratory

OBJECTIVES: We aimed to determine the Epstein-Barr Virus (EBV) presence rate in our laboratory's lymphoma tissue biopsies for comparison with that reported in literature. BACKGROUND: The presence of EBV has been established in Hodgkin lymphoma (HL), endemic Burkitt Lymphoma and some non-Hodgkin lymphomas (NHL). It has been linked to geographic, ethnic and socioeconomic factors, with a lower rate in developed countries. METHODS: We used the immunoperoxidase technique to determine the rate of the EBV LMP-1 in eighty-seven biopsies diagnosed as lymphoma. Tissue slides were stained using the Ventana Automated Slide Stainer with the DAKO EBV LMP-1 primary antibody and the results were analyzed with the SYSTAT program. RESULTS: We found an LMP-1 positive rate of 50 per cent for 22 cases of HL and 35 per cent for 63 cases of NHL. Among HL, 5 were children and 16 were adults, with LMP-1 positive rates of 60 per cent and 50 per cent respectively. Among NHL, 3 were children and 59 were adults, with equal LMP-1 positive rates of 33 per cent. The sex LMP-1 positive rates for HL were 42 per cent for 12 males and 60 per cent for 10 females. Among NHL, the sex LMP-1 positive rates were 39 per cent for 38 males and 28 per cent for 27 females. NHL was further subdivided into subtypes and LMP-1 primary antibody positive rates were reported. CONCLUSIONS: We found a similar presence rate of EBV in the HL biopsies to that of developed countries, but a similar presence rate of EBV in NHL biopsies to that of developing countries.

Efectividad de la terapia anticipada con ganciclovir en receptores de trasplante renal de alto riesgo (R-/D+) para desarrollo de enfermedad por citomegalovirus / Effectiveness of preemptive therapy with ganciclovir in recipients of renal transplants at high risk (R-/D+) for the development of cytomegalovirus disease

Current management of renal transplant recipients who are CMV seronegative (R-) and receive an organ from a seropositive donor (D+) is controversial. These patients are at high risk for CMV disease and are usually treated with ganciclovir prophylaxis at variable dose and duration. An alternative to this approach is to administer ganciclovir only to those patients who are identified by virological markers to be at the highest risk to develop the disease (preemptive therapy). This prospective trial was conducted to asses the value of preemptive therapy to prevent CMV disease in R-/D+ kidney transplant recipients on triple drug immunosuppression without antilymphocyte induction. Sixteen adults receiving their first kidney transplant were enrolled and followed with pp65 antigenemia assay performed biweekly for the first 16 postransplant weeks, and then monthly to complete 12 months. Ganciclovir (5 mg/kg/day i.v., for 15 days) was administered as preemptive therapy upon detection of one or more antigen-positive cells per 150 x 10(3) peripheral blood leucocytes examined. For those receiving preemptive therapy, pp65 antigenemia was also repeated after completion of the regimen. CMV antigenemia was detected in 7/16 patients. At mean follow-up of 9 months (4-12 m) none of the 16 patients developed CMV disease. CMV serology (IgM) became positive in all patients after the first antigenemia result. The last follow-up mean serum creatinine (SCr) level was similar in both groups (1.35 mg/dL). In CMV R-/D+, the use of preemptive therapy guided by pp65 antigenemia is effective in preventing CMV disease. By using this strategy, 9 of 16 patients were spared ganciclovir prophylaxis with no effect on rejection or CMV disease. The clinical benefit and cost/effectiveness of this strategy should be evaluated against universal prophylaxis in these high-risk patients.

Western blot detection of infectious bursal disease virus infection

In order to evaluate the use of a Western blot methodology for the diagnosis of infectious bursal disease virus (IBDV) infection, chickens were experimentally infected with IBDV strains and tested for the presence of viral antigens and antibodies by a blocking Western blot test (bWB). The viral proteins obtained from the bursa of Fabricius (BF) were transferred to a nitrocellulose membrane after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the chicken sera obtained by heart puncture were used for the detection of these proteins. In order to eliminate nonspecific reactions, we used a rabbit anti-chicken serum (blocking tool). By the use of the bWB test, two distinct viral proteins of 43-kDa (VP2) and 32-kDa (VP3) were detected. We suggest the use of this methodology for the detection of IBDV infection in animals suspected of having IBDV reinfection and a chronic subclinical form of the disease. With the use of the rabbit anti-chicken sera for blocking, this method is practical, sensitive and less time consuming.