Clinical Implications of Plasma N-acetyl-seryl-aspartyl-lysyl-proline Level in Stable Kidney Transplant Recipients.

BACKGROUND: N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is a natural inhibitor of pluripotent hematopoietic stem cell proliferation and is normally found in human plasma. Because AcSDKP is partially eliminated in urine, accumulation of AcSDKP due to chronic renal failure may cause anemia. However, the status of plasma AcSDKP level in stable kidney transplant recipients is unknown although some recipients develop anemia after kidney transplantation. In this study, we investigated the relationship between plasma AcSDKP-like immunoreactive substance (IS) level and clinical characteristics associated with renal anemia in stable kidney transplant recipients. METHODS: Forty Japanese kidney transplant recipients who underwent transplantation more than 90 days prior to the study were included. Morning blood samples were collected and plasma AcSDKP-IS levels were measured using an enzyme immunoassay. RESULTS: A significant correlation was observed between plasma AcSDKP-IS level and creatinine clearance. On the other hand, no significant correlation was observed between plasma AcSDKP-IS level and prolyl oligopeptidase activity, angiotensin II, or erythropoietin level. A significant difference in plasma AcSDKP-IS level was observed between recipients with no renal anemia and those with renal anemia. CONCLUSIONS: These results suggest that plasma AcSDKP level may depend largely on renal function and suggest a possibility that accumulation of AcSDKP may be partially involved in the pathogenesis of renal anemia in stable kidney transplant recipients.

Significant decrease in plasma N-acetyl-seryl-aspartyl-lysyl-proline level in patients with end stage renal disease after kidney transplantation.

N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is an endogenous peptide released from its precursor (thymosin-ß4) by prolyl oligopeptidase. AcSDKP is a natural inhibitor of pluripotent hematopoietic stem cell proliferation and is normally found in human plasma. AcSDKP has been shown to be a potent angiogenic factor and to suppress renal fibroblast proliferation. Impairment of renal function has been suggested to have a significant impact on plasma AcSDKP level. The aim of this study was to assess whether improvement of renal function after kidney transplantation has an impact on plasma AcSDKP-like immunoreactive substance (IS) level. Fourteen patients with end stage renal disease (ESRD) who were scheduled to undergo the first kidney allograft transplantation were enrolled. Plasma AcSDKP-IS levels were measured before and 3, 7, 10, 14, 21, 30, 60 and 90 d after kidney transplantation. Plasma AcSDKP-IS level decreased significantly from day 3 after kidney transplantation compared to before kidney transplantation. Creatinine clearance increased significantly from day 7 after kidney transplantation. A significant negative correlation was observed between creatinine clearance and plasma AcSDKP-IS level from before transplantation to 90 d after kidney transplantation. Stepwise multiple regression analysis identified creatinine clearance as the only significant independent factor associated with plasma AcSDKP-IS levels. These results suggest that recovery of kidney function after kidney transplantation may lead to a decrease in plasma AcSDKP level in patients with ESRD, and that plasma AcSDKP level may depend largely on renal function.

[Case of primary cutaneous Aspergillus caldioustus infection caused by nerve block therapy].

We report a case of primary cutaneous Aspergillus caldioustus infection caused by nerve block therapy. A 67-year-old Japanese woman had been treated with oral predonisolon and tacrolimus for adult-onset Still disease and interstitial pneumonia. She presented with a 2-month-history of the lesions on the left back. A biopsy specimen from the skin lesion revealed granulomatous inflammation with hyphae. Culture of the pus and the skin specimen confirmed the diagnosis of cutaneous Aspergillus infection. The sequence of ß- tubulin gene was analyzed to confirm the mycological diagnosis and the causative agent was identified as A. caldioustus. The patient was treated with surgical removal of the lesions and oral 200 mg/day itraconazole but she died of infectious interstitial pneumonia due to Pneumocystis jiroveci and Cytomegalovirus infection Percutaneous infection may have been responsible for the incidence of localized infection. There was no evidence of systemic aspergillosis. A. caldioustus is an emerging opportunistic fungal pathogen in immunocompromised patients. Immunocompromised patients who have persistent traumatic atypical skin lesion need to be ruled out of such rare fungus infection. An opportunistic infection in Immunocompromised patients can be life-threatening and prompt treatment based on accurate diagnosis is important.

Análise da expressão imuno-histoquímica de c-erbB-2 e EGFR em carcinoma epidermóide de esôfago / Immunohistochemical expression of c-erbB-2 and EGFR in esophageal squamous cell carcinoma

INTRODUÇÃO: O carcinoma epidermóide de esôfago (CEE) possui alta incidência em nosso país, com altas taxas de mortalidade. A família dos receptores do fator epitelial de crescimento (EGFR) é composta por quatro membros, e muitos estudos têm sido direcionados para a expressão de EGFR e c-erbB-2, com implicações terapêuticas. OBJETIVO: Investigar as expressões imuno-histoquímicas de EGFR e c-erbB-2 e correlacioná-las a aspectos clinicopatológicos em casos de CEE. MATERIAL E MÉTODOS: Para esse estudo, dados clinicopatológicos de 613 CEE foram revistos. A imunoistoquímica foi feita utilizando anticorpo policlonal para c-erbB-2 e monoclonal para EGFR em 597 e 585 casos, respectivamente. Os casos representados por peças cirúrgicas foram distribuídos em três blocos de parafina de tissue microarray (TMA), inseridos em duplicata; aqueles com biópsias foram analisados em corte convencional. Todos foram classificados de acordo com intensidade e padrão de marcação de membrana das células tumorais. RESULTADOS: As expressões de c-erbB-2 e EGFR foram observadas em 42,4 por cento e 77,6 por cento dos casos, respectivamente. Observou-se correlação estatisticamente significativa entre as expressões de c-erbB-2 (p = 0,04) e EGFR (p = 0,01) e grau histológico. Ambos os marcadores foram significativamente mais expressos em casos bem/moderadamente diferenciados do que nos pouco diferenciados/indiferenciados. Embora não tenha sido significativa, houve uma tendência de associação entre superexpressão de c-erbB-2 e sítio do tumor, em que casos positivos ocorreram com mais freqüência no terço médio do esôfago. Nenhuma correlação significativa foi verificada entre essas proteínas e sobrevida global. CONCLUSÃO: Os resultados podem sugerir um papel primordial para essas proteínas na diferenciação tumoral em CEE.

INTRODUCTION: Esophageal squamous cell carcinoma (ESCC) is highly prevalent in Brazil, and responsible for high mortality index. The epidermal growth factor receptor (EGFR) family has four members and much attention has been focused on the expressions of EGFR and c-erbB-2 with therapeutic implications. OBJECTIVE: The aim of the present study was to investigate immunohistochemical expressions of c-erbB-2 and EGFR in ESCC, and correlate them with clinicopathological data. MATERIAL AND METHODS: Medical records of 613 patients with ESCC were reviewed. Immunohistochemistry was carried out using polyclonal c-erbB-2 and monoclonal EGFR in 597 and 585 cases, respectively. Cases represented by surgical resections were performed in three tissue microarray (TMA) paraffin blocks spotted in duplicate; those with small biopsies without surgical resections were performed individually. All cases were scored according to intensity and pattern of membrane staining of tumor cells. RESULTS: The expressions of c-erbB-2 and EGFR were observed in 42.4 percent and 77.6 percent of the cases, respectively. A significant correlation was found between c-erbB-2 (p = 0.04) and EGFR (p = 0.01) expressions and histological grade. Both markers were significantly more expressed in well/moderate differentiated ESCC than in poorly differentiated ESCC. Although it was not significant, there was a tendency of association between c-erbB-2 overexpression and tumor location, in which positive cases occurred more frequently in the middle third of the esophagus. There was no correlation with overall survival. CONCLUSION: The results may suggest a role for these markers in tumoral differentiation in ESCC.

O arranjo em matriz de amostras teciduais (tissue microarray): larga escala e baixo custo ao alcance do patologista / Tissue microarrays: high throughput and low cost avaiable for pathologists

O arranjo em matriz de amostras teciduais, ou tissue microarray (TMA), é uma técnica descrita em 1998 por Kononen et al. com ampla aceitação pela literatura mundial. Com um conceito extremamente simples, trata-se de agrupar um grande número de amostras teciduais em um único bloco de parafina, permitindo o estudo de expressão de marcadores moleculares em larga escala com grande aproveitamento do material arquivado, do tempo e dos custos. Discutimos as vantagens e limitações do método, as estratégias e técnica de construção, as aplicações e dificuldades encontradas para a patologia investigativa nos últimos cinco anos de uso no Hospital do Câncer A. C. Camargo.

Tissue microarrays (TMA) is a worldwide well accepted technique described in 1998 by Kononen et al. It uses an extremely simple concept of ordering hundreds of samples in just one paraffin block to evaluate protein expression in large cohorts with great advantages on costs, time and sample saving. We discuss the technique, its advantages and limitations, strategies to construct the receptor block, its usefulness and difficulties experienced in the last five years at Hospital do Cancer A.C. Camargo.

Differential cardiac gene expression during cardiopulmonary bypass: ischemia-independent upregulation of proinflammatory genes.

OBJECTIVE: Cardiac surgery with cardiopulmonary bypass induces both systemic and local inflammatory responses implicated in the pathogenesis of myocardial dysfunction. Multifactorial perioperative sources of myocardial injury complicate understanding of the molecular mechanisms involved. By using microarray technology, this study examines myocardial gene expression responses to cardiopulmonary bypass in the absence of cardioplegic arrest and ischemia-reperfusion injury. METHODS: We used a unique rat model of cardiopulmonary bypass in which sternotomy, direct operations on the heart, aortic crossclamping, and cardioplegic arrest were not performed. Hearts from 6 animals randomized to either 90 minutes of cardiopulmonary bypass or sham control animals were used to perform cDNA microarray analyses of 2343 genes. Real-time quantitative polymerase chain reaction was used to confirm the microarray results for a subset of genes. RESULTS: Compared with sham-operated control animals, myocardium from animals undergoing cardiopulmonary bypass revealed 42 differentially expressed genes. Upregulated genes include the transcription activator nuclear factor kappaB, adhesion molecules (vascular cell adhesion molecule 1 and P-selectin), and interleukin 6 receptor subunits; downregulated genes include transforming growth factor beta receptor 2, tissue inhibitor of metalloproteinase 3, and mitogen-activated protein kinase 1. Distinct proinflammatory gene cascades were confirmed by means of category overrepresentation analysis. CONCLUSIONS: This study represents an initial report on the use of microarray technology to elucidate cardiac transcriptional programs in response to cardiopulmonary bypass-specific injury in vivo. These preliminary findings, combined with future functional genomic studies superimposing ischemia and reperfusion and other inflammatory stimuli, should improve our understanding of the molecular regulatory networks involved in myocardial responses to injury and aid in the development of novel cardioprotective and perfusion strategies.

A análise da expressão de c-erbB-2, EGFR, VEGF e ciclinas em carcinoma epidermóide do esôfago / Study of c-erbB-2, EGFR, VEGF and cyclins expression in Esophageal Squamous Cell Carcinoma

Introdução: O carcinoma epidermóide do esôfago (CEE) é o tipo histológico mais comum de câncer do órgão, sendo a sexta neoplasia em freqüência em nosso país. Geralmente, o CEE está associado a um prognóstico ruim, por se tratar de um tumor em estadio avançado no momento do diagnóstico, apresentando alta taxa de mortalidade. As alterações moleculares do CEE são pouco estudadas e os trabalhos existentes na literatura mostram resultados controversos e casuística reduzida. O objetivo desse estudo foi analisar expressão de proteínas envolvidas na regulação da proliferação celular em CEE e sua correlação com variáveis clínico-patológicas e sobrevida dos pacientes. Métodos: Foi realizada imunoistoquímica para análise de expressão de c-erbB-2, EGFR, VEGF e ciclinas A, B1 e D1 em 613 casos de CEE. Os casos representados por peças cirúrgicas foram distribuídos em três blocos de parafina de Tissue Microarray, em duplicata; os casos com biópsias foram analisados em corte convencional. Os casos foram classificados de acordo com a freqüência, intensidade e padrão de marcação das células, dependendo do anticorpo avaliado. As análises estatísticas de correlação entre as variáveis consideradas foram realizadas pelos métodos de χ2 ou teste exato de Fisher. Para as análises de sobrevida global, o cálculo da estimativa de sobrevida cumulativa foi realizado pela técnica de Kaplan-Meier e as comparações entre as curvas de sobrevida, em relação as variáveis estudadas, pelo teste de Log-rank. A análise multivariada foi feita pelo modelo de riscos proporcionais de Cox. Foi considerado o nível de significância de 5%. Resultados: Nossos resultados mostraram correlação estatisticamente significativa entre a expressão de cerbB-2 (p=0,04) e EGFR (p=0,01) e grau histológico. Ambos os marcadores foram significativamente mais expressos em casos bem/moderadamente diferenciados do que nos pouco diferenciados/ indiferenciados. Embora não tenha sido significativo, houve uma tendência de associação entre a superexpressão de c-erbB-2 e sítio do tumor, em que casos positivos ocorreram com mais freqüência no terço médio do esôfago. Nenhuma associação significativa foi verificada entre a expressão dessas proteínas e sobrevida global. Em relação a ciclina D1, foi observada correlação significativa entre a superexpressão dessa proteína e cor (p=0,026), sendo mais expressa em pacientes de cor branca. Foi observada também uma tendência à significância para correlação entre a superexpressão dessa proteína e grau de diferenciação (p=0,070), em que a ciclina D1 esteve mais expressa em casos bem/moderadamente diferenciados. Quanto a ciclina B1, foi verificado que casos negativos para essa proteína foram significativamente mais freqüentes em tumores que atingiram níveis mais profundos de infiltração (p=0,033) e com presença de invasão linfática (p=0,006). Não foi observada nenhuma correlação significativa entre a superexpressão de VEGF e ciclina A e aspectos clínico-patológicos. As análises de sobrevida global mostraram a existência de associação estatisticamente significativa entre a superexpressão de ciclinas A (p<0,0001), B1 (p=0,014) e D1 (p<0,0001) e VEGF (p=0,002) e menores estimativas de sobrevida cumulativa. Foi observada correlação significativa entre a coexpressão da maioria das proteínas estudadas, exceto entre as expressões de c-erbB-2/ciclina A, c-erbB-2/ciclina B1, c-erbB-2/VEGF, EGFR/ciclina A e EGFR/VEGF. Em relação as variáveis clínico-patológicas, gênero (p=0,044), cor (p=0,0005) e pT (p=0,021) mostraram-se como indicadores de pior prognóstico. Pela análise multivariada, pT (p=0,030) e gênero (p=0,004) permaneceram como fatores prognósticos independentes. No entanto, quando desconsideramos pT da análise multivariada, ciclina D1 (p<0,0001), cor (p=0,003) e ciclina A (p=0,023) persistiram como marcadores prognósticos independentes. Conclusão: No presente trabalho, verificamos correlação significativa entre a superexpressão de c-erbB-2, de EGFR e de ciclina D1 e grau histológico, conferindo um comportamento menos agressivo aos tumores, sugerindo que esses marcadores podem estar envolvidos na indução de outra via de sinalização, como diferenciação celular. Casos negativos para expressão de ciclina B1 foram mais freqüentes entre os tumores com invasão linfática e níveis mais profundos de infiltração, não permitindo esclarecer sua relação com a progressão tumoral nesse tumor. Não foi constatada nenhuma correlação com significância estatística entre a superexpressão de VEGF e de ciclina A e as variáveis clínicopatológicas avaliadas. As variáveis gênero, cor, pT e expressão de VEGF, ciclinas A, B1 e D1 foram consideradas indicadores de pior prognóstico pela análise univariada. No entanto, na análise multivariada, apenas pT e gênero permaneceram como marcadores prognósticos, mas quando pT foi desconsiderado dessa análises, ciclina D1, cor e ciclina A permaneceram como fatores prognósticos independentes em CEE.

Background: Esophageal squamous cell carcinoma (ESCC) is the most commom histologic type of cancer in this organ, being the sixth most common tumor in Brazil. ESCC is associated with poor prognosis, because it is usually a advanced stage disease at the moment of diagnostic, showing high mortality rates. There are few studies involving molecular alterations in ESCC and the reports show controversial results and small number of cases. The aim of this study was to analyze expression of proteins involved in cellular proliferation regulation in ESCC and the correlation with clinicopathological variables and patient survival. Methods: Immunohistochemistry was performed to analyze the c-erbB-2, EGFR, VEGF and cyclins A, B1 and D1 expression in 613 cases of ESCC. Cases represented by surgical resections were performed in three tissue microarray paraffin blocks spotted in duplicate and cases with small biopsies were performed in conventional slides. The cases were scored according the frequency, intensity and staining pattern of tumoral cells, depending on the antibody evaluated. Statistical analysis between the variables considered in this study were performed using chi-square or Fisher's exact tests. For the overall survival analysis, the cumulative survival rates was calculated by Kaplan-Meier method and the comparison between the survival curves, according the variables studied, by the Log-rank test. Multivariate analysis was performed using Cox regression method. A p value <0,05 was considered statistically significant. Results: Our results showed significant correlation between c-erbB-2 (p=0,04) and EGFR (p=0,01) expression and histological grade. Both markers were significantly more expressed in well/moderatelly differentiated than in poorly differentiated/undifferentiated cases. Despite it were not statistically significant, there was a tendency to association between c-erbB-2 overexpression and tumor site, which positive cases were more frequently in the middle third of the esophagus. No significant association was verified between these proteins expression and overall survival. It was observed a tendecy to significance for the correlation between cyclin D1 overexpression and histological grade (p=0,070), in which this protein was more expressed in well/moderately differentiated cases. Regarding cyclin B1, it was verified that negative cases for this protein was significantly more frequent in deeper infiltration lesions (p=0,033) and in cases with lymphatic invasion (p=0,006). It was not observed any significant correlation between VEGF and cyclin A overexpression and clinicopathological aspects. Overall survival analysis showed statistically significant association between cyclins A (p<0,0001), B1 (p=0,014), D1 (p<0,0001) and VEGF (p=0,002) and lower cumulative survival rates. It was verified significant correlation between the majority of proteins studied, except c-erbB-2/cyclin A, c-erbB-2/cyclin B1, c-erbB-2/VEGF, EGFR/cyclin A and EGFR/VEGF. Regarding the clinicopathological features, gender (p=0,044), ethnia (p=0,0005) and pT (p=0,021) were considered worse prognostic factors. Multivariate analysis showed that pT (p=0,030) and gender (=0,004) persisted as independent prognostic factors. However, when T is not considered in the multivariate analysis, cyclin D1 (<0,0001), ethnia (p=0,003) and cyclin A (p=0,023) remain as independent prognostic markers. Conclusion: In the present work, we verified significant correlation between c-erbB-2, EGFR and cyclin D1 overexpression and histological grade, giving a less agressive behaviour to the tumors, suggesting that these markers can be involved in induction of other signaling pathway, as cellular differentiation. Negative cases for cyclin B1 expression occurred more frequent in cases with lymhtic invasion and deeper infiltration, not being possible elucidate cyclin B1 relation with tumoral progression in ESCC. It was not verified any correlation with statistically significance between VEGF and cyclin A overexpression and the clinicopathological features considered. The variables gender, ethnia, pT and VEGF, cyclins A, B1 nd D1 expression were considered worse prognostic factores by univariate analysis. However, in multivariate analysis only pT and gender remained as prognostic markers, but when pT was not considered in this analysis, cyclin D1, ethnia and cyclin A persisted as independent prognostic factores in ESCC.

Differential cerebral gene expression during cardiopulmonary bypass in the rat: evidence for apoptosis?

UNLABELLED: Cardiopulmonary bypass (CPB) is associated with a spectrum of cerebral injuries. The molecular changes in the brain that might contribute to these injuries are not clearly known. We sought to determine whether the expression of apoptotic genes is increased after CPB in the rat. Rats (n = 7) were subjected to 90 min of normothermic CPB. A group of sham-operated rats (n = 7) served as non-CPB controls. After a 3-h post-CPB period of recovery, their brains were removed, homogenized, and processed for messenger RNA (mRNA) extraction. By using a ribonuclease protection assay, the ratios of both pro- and antiapoptotic mRNA (bcl-x, bcl-2, bax, caspase 2, and caspase 3) to the housekeeping glyceraldehyde phosphate dehydrogenase (GAPDH) gene were determined. Additionally, Western immunoblotting was performed to detect the presence of activated caspase 3, a protein central in the apoptotic process. Compared with the non-CPB controls, the CPB group had significantly increased levels of apoptotic/GAPDH mRNA ratios (bcl-x, 0.414 +/- 0.152 CPB versus 0.251 +/- 0.051 non-CPB, P = 0.048; caspase 2, 0.030 +/- 0.014 CPB versus 0.018 +/- 0.005 non-CPB, P = 0.048; bax, 0.106 +/- 0.035 CPB versus 0.066 +/- 0.009 non-CPB, P = 0.009; bcl-2, 0.011 +/- 0.006 CPB versus 0.006 +/- 0.002 non-CPB, P = 0.035). However, no activated caspase 3 protein was detected in either group. Elucidating the molecular biological sequelae of CPB may aid in the understanding of the pathophysiology of cardiac surgery-associated cerebral injury and, in doing so, may be useful in identifying potential therapeutic targets for pharmacologic neuroprotection. IMPLICATIONS: Cardiopulmonary bypass (CPB) appears to induce transcription of pro- and antiapoptotic genes in the rat brain, but caspase-mediated apoptosis itself does not appear to be activated. Elucidating the molecular biological sequelae of CPB may aid in the understanding of the pathophysiology of cardiac surgery-associated cerebral injury and, in doing so, may be useful in identifying potential therapeutic targets for pharmacologic neuroprotection.