A new systematization of histological analysis for the diagnosis of Hirschsprung's disease

Background: Hirschsprung's Disease (HD) is characterized by intestinal sub-occlusion and the absence of enteric ganglion cells. A rectal biopsy examination is performed to confirm the diagnosis. In a recent study, we demonstrated that the analysis of 60 sections of rectal mucosa and submucosa stained by H&E may ensure a 90% diagnostic accuracy. Although the need to analyze so many sections makes the process of reading the slides more time-consuming, this encouraged us to study their distribution in the healthy rectal submucosa, to simplify the diagnosis. Objectives: To develop a method that facilitates HD diagnosis by studying the distribution of ganglion cells in the submucosal plexus. Methods: Using the calretinin technique, we studied the distribution of plexuses in 60 fragments of rectal submucosa from 19 cadavers. After the study, the reading method created was used for diagnosis in 47 cases of suspected HD, using H&E staining. The accuracy was verified by comparing the results obtained with H&E to those obtained with the acetylcholinesterase technique, the golden standard in our laboratory. Results: The study of submucosal plexus distribution showed that just by examining the submucosal region every 20 µm, approximately, it is possible to locate a ganglionic plexus, and we have already been able to diagnose HD with 93% accuracy. Conclusion: The study of ganglion cell distribution enabled the creation of a simplified method for reading the slides. The method applied achieved good accuracy and it can be used as an alternative method in HD diagnosis

Eversão da vesícula urinária no coelho

No setor de criação de coelhos da DTAEP- F.M. USP foi verificada em uma coelha N.Z.W. adulta a eversão completa da vesícula urinária. Ao exame macroscópico, a superfície mucosa apresentou-se de coloração avermelhada e com formações papilomatosas de 3 a 7 cm irregularmente distribuídas na porção distal do órgão. Cortes histológicos da área papilomatosa demonstraram uma erosão da mucosa recoberta por crosta de fibrina e hemáceas em alguns pontos. Preparados histológicos em outras regiões apresentaram quadro semelhante, com áreas focais de infiltrado inflamatório mais intenso. Tais resultados permitiram concluir um quadro de cistite crônica inespecífica.A rare case of complete urinary bladder inversion was verified in a female adult new zeeland while rabbit. Under the macroscopical analysis, the mucosal lining had a red hue and irregularly distributed in the distal, portion of the organ papillomatous structures were verified. In this region, a mucosal erosion overlaid in some places by fibrin with erythrocytes scab was observed under a microscopical aspect. The same pattern as described above was observed in other regions of the bladder, where the focal areas had a more intensive inflammatory infiltrate. These results allowed to conclude a inespecific chronic cystitis aspect

RKIP Inhibition in Cervical Cancer Is Associated with Higher Tumor Aggressive Behavior and Resistance to Cisplatin Therapy

Cervical cancer is one of the most common cancers in women worldwide, being high-risk group the HPV infected, the leading etiological factor. The raf kinase inhibitory protein (RKIP) has been associated with tumor progression and metastasis in several human neoplasms, however its role on cervical cancer is unclear. In the present study, 259 uterine cervix tissues, including cervicitis, cervical intraepithelial lesions and carcinomas, were analyzed for RKIP expression by immunohistochemistry. We found that RKIP expression was significantly decreased during malignant progression, being highly expressed in non-neoplastic tissues (54% of the samples; 73/135), and expressed at low levels in the cervix invasive carcinomas (∼15% (19/124). Following in vitro downregulation of RKIP, we observed a viability and proliferative advantage of RKIP-inhibited cells over time, which was associated with an altered cell cycle distribution and higher colony number in a colony formation assay. An in vitro wound healing assay showed that RKIP abrogation is associated with increased migratory capability. RKIP downregulation was also associated with an increased vascularization of the tumors in vivo using a CAM assay. Furthermore, RKIP inhibition induced cervical cancer cells apoptotic resistance to cisplatin treatment. In conclusion, we described that RKIP protein is significantly depleted during the malignant progression of cervical tumors. Despite the lack of association with patient clinical outcome, we demonstrate, in vitro and in vivo, that loss of RKIP expression can be one of the factors that are behind the aggressiveness, malignant progression and chemotherapy resistance of cervical cancer.</p

O impacto do diagnóstico citológico de atipiasi indeterminadas no sistema público de saúde

As alterações citológicas de significado indeterminado representam uma importante limitação diagnóstica nos programas de escrutíneo de lesões cérvico-vaginais. A introdução de métodos biomoleculares, como o sistema de captura híbrida para detecção de HPV de alto risco contribui para a otimização da conduta clínica dessas pacientes, indicando colposcopia com precisão. Objetivo: avaliar o significado de lesões de significado indeterminado com relação à infecção pelo HPV, com o uso do teste de DNA para HPV com o método da captura de híbridos II. Métodos: foram estudadas amostras de 236 casos consecutivos examinados no laboratório da DIGENE-BRASIL, de pacientes com diagnóstico citológico prévio de ASCUS. As amostras foram submetidas ao teste de captura híbrida para identificação de DNA-HPV de alto e baixo riscos. Resultados: dos 236 casos analisados, 183 (77,5%) foram negativos para o teste de captura híbrida, seis (2,6%) foram positivos para HPV de baixo risco e 47 (19,9%) foram positivos para HPV de alto risco. Conclusão : as amostras positivas para HPV de baixo risco representam uma pequena e não- onsiderável minoria de casos, provavelmente, transientes. Cerca de 20% dos casos foram positivos para HPV de alto risco e deverão ser encaminhados à colposcopia e biopsia, se necessário. Esses casos representam um grande potencial de progressão para lesões cervicais.In order to optimize the morphological analysis of the cases with uncertain diagnosis, we critically analyzed the cases with Atypia of Squamous Cells of Undertemined Significance (ASCUS) in cytological samples of uterine cervix collected in conventional smears (CS) and liquidbased preparations (LBC) an to correlate the findings with Hybrid Capture II (HC2) assay and biopsy. Objective: to evaluate the meanig of undetermined cytological atypia in relation to HPV infection detected by hybrid capture II test. Methods: 97 cases taken from women examined at Perola Byignton Hospital, São Paulo, Brazil, during the year of 2002. The conventional smears were taken previously than LBC. The residual sample was placed in liquid-medium and LBC preparation with DNA-Citoliq system was performed. If at least one of the paired samples were classified as ASCUS, the pair was submitted to a guided revision in order to evaluate the type of alteration taken in account to categorized ASCUS. Results: from 97 cases studied, 14 were categorized as ASCUS by the two methods simultaneously. The others had different classification under or hyper estimated. Six cases diagnosed as squamous intraepithelial lesion (SIL) by CS were ASCUS by LBC; in contrary, 19 ASCUS by CS were SIL by LBC. Eleven ASCUS by CS were diagnosed as negative by LBC, but CS categorized 47 LBC ASCUS as negative. From the morphological parameters nuclear enlargement and coarse chromatin were regarded as ASCUS. From 68 ASCUS by LBC, 36 were HC2 positive for high risk HPV (hr-HPV) : ten of them with biopsy proven lesion. From 42 CS ASCUS, 23 were hr-HPV positive, but only 7 with histological lesion. Conclusion: our results reinforced the hypothesis that ASCUS is poorly reproducible by morphological examination by CS or LBC preparations. To add HC2 as adjunct method to ASCUS cytology can improve the routine diagnosed of the uncertain atypies

The association of p16INK4A and fragile histidine triad gene expression and cervical lesions

Objective. This cross-sectional study was intended to assess the association between immunohistochemical analysis of p16INK4A and fragile histidine triad (FHIT) and the presence of precancerous cervical lesions. Materials and Methods. Women seen at Pe´ rola Byington Hospital, São Paulo, Brazil, with histologically confirmed cervicitis (n = 31), cervical intraepithelial neoplasia (CIN) 1 (n = 30), CIN 2,3 (n = 30), and cervical cancer (n = 7) had also cervical material collected for liquid-based cytology, human papillomavirus Hybrid Capture 2 (HC2) test, and p16 and FHIT immunohistochemical reactions. Results. p16 and FHIT reactions were scored as the following: G1%, 1% to 5%, 95% to 25%, and 925%. Receiver operating curve analysis was used to select p16 and FHIT score cutoffs for further categorical analyses. All but one of the 37 CIN 2,3/cancer cases had a p16 score of greater than 1% to 5%. Among the 61 cervicitis/CIN 1 cases, 46 (75%) had a p16 score lower than 1% to 5%. In contrast, no association of FHIT expression and severity of cervical lesions could be demonstrated in this data set. Receiver operating curve analyses suggested the score of 1% to 5% for p16 as the cutoff that best discriminates CIN 2,3/cancer from cervicitis/CIN 1. No cutoff for FHIT scores could be suggested with data set. Conclusions. p16, but not FHIT expression, has the potential to be used as complementary diagnostic tool to investigate human papillomavirusYinduced cervical lesions, if these results are confirmed in larger studies.(undefined

Immunocytochemical expression of p16 INK4A and Ki-67 in cytologically negative and equivocal pap smears positive for oncogenic human papillomavirus

This study was designed to analyze the cross-sectional comparison of the p16 INK4A and Ki-67 immunocytochemical expression in negative and equivocal (atyp-ical squamous cells of undetermined significance (ASC-US)) liquid-based cytology (LBC) samples testing positive for high-risk human papillomavirus (HPV) types with HC2 assay or polymerase-chain reaction (PCR). A series of 199 consecutive LBC speci-mens derived from the same number of women participating in the ongoing Latin American Screening Study at Leonor Mendes de Barros Hospital, São Paulo, were analyzed using immunocytochemistry for expression of p16 INK4A and Ki-67 in negative and equivocal LBC samples testing positive for high-risk HPV types with hybrid capture II test (HC2) or PCR. All patients with at least one test positive (cytology, PCR, and/or HC2) were followed each 6 months for 3 years. The follow-up procedure consisted of visual examination, colposcopic inspection, cytology, and HC2 assay. Among the neg-ative cytologic samples, 101 were HPV-positive and 55 HPV-negative. Of the HPV-pos-itive group, 59 of 101 cases (58.4%) were positive for both p16 and Ki67 immunostaining, and 17 of 101 (16.8%) were negative for both. The proportion of Ki-67-positivity increased almost in parallel with the increasing grade of p16-positivity (p = 0.0001 for linear trend). In the HPV-negative group, both markers were negative in 41 of 55 cases (74.5%), and no statistical relationship was observed between the two markers (Pearson, p = 0.595). HPV-positive ASC-US samples demonstrated a simulta-neous positive immunoreaction for p16 and Ki67 in 11 of 16 cases (68.7%), whereas 3 (18.7%) were concurrently negative. The relationship between the two markers was of borderline significance (Pearson, p = 0.053), but no linear relationship was found be-tween the graded p16 and Ki-67 expression (p = 0.065 for linear trend). In the HPV-negative ASC-US group, there was no statistical association between the graded p16 and Ki-67 positivity (Pearson, p = 0.281). After 36 months of follow-up of the ASC-US patients, 6 women still displayed ASC-US smear, of which 4 of 6 were HPV-positive and expressed both p16 and Ki-67 markers. Two of 43 ASC-US smears had high-grade squamous intraepithelial lesions diagnosed (4.6%), and 1 had low-grade squamous intraepithelial lesion (2.3%). All of those were positive for HPV, p16 and Ki-67. Patients with ASC-US diagnosis and positive high-risk HPV status and positive for p16 INK4A Ki67 should be carefully observed to exclude occurrence of a squamous intraepithelial lesion. The combination of these two markers can be a useful implement for manage-ment of women with equivocal cytology.European Commission (EC) - INCO-DEV Programme - Project #ICA4-CT-2001-10013

Screening for cervical Cancer in high-risk populations: DNA pap test or hybrid capture II test alone?

This study was designed to evaluate whether Hybrid Capture II (HC2) test alone refer women to colposcopy as appropriately as DNA Papanicolaou (Pap) test, in the context of a high-risk group of women using the recently validated DNACitoliq LBC system. Women with suspected cervical disease were included in this crosssectional study at a tertiary center in São Paulo, Brazil, for further workup. All women had cervical material collected for LBC and HC2 for high-risk human papillomavirus (hrHPV)-DNA test. Irrespective of cytology and HC2 results, colposcopy, and cervical biopsy when applicable, was systematically performed. All tests were performed blindly. Sensitivity, specificity, positive and negative predictive values, and overall accuracy of both methods were computed in relation to histology. A total of 1,080 women were included: 36.4% (393/1080) had ACUS+, 10.2% (110/1080) were high-grade squamous intraepithelial lesions (HSIL) or cancer. Mean age was 33.5 years. All women underwent colposcopy, and cervical biopsies were performed in 38.4% (415/1080): 33% (137/415) of the biopsies were negative, 14.4% (155/415) were low-grade squamous intraepithelial lesions (LSIL), 10.7% (116/415) were HSIL, and 0.6% (7/415) were cancer. HC2 sensitivity to diagnose biopsy-proven HSIL was 100%. Because all HSIL cases had a positive HC2 test, sensitivity could not be improved by adding LBC. Specificity and positive and negative predictive values of DNA Pap were not significantly different from HC2 test alone when considering LSIL+ histology as ‘‘gold standard’’ and HSIL+ histology. As a screening strategy for women with high-risk for cervical cancer, DNA Pap test does not seem to add substantially to HC2 alone in terms of appropriately referring to colposcopy

RKIP Inhibition in cervical cancer Is associated with higher tumor aggressive behavior and resistance to cisplatin therapy

Cervical cancer is one of the most common cancers in women worldwide, being high-risk group the HPV infected, the leading etiological factor. The raf kinase inhibitory protein (RKIP) has been associated with tumor progression and metastasis in several human neoplasms, however its role on cervical cancer is unclear. In the present study, 259 uterine cervix tissues, including cervicitis, cervical intraepithelial lesions and carcinomas, were analyzed for RKIP expression by immunohistochemistry. We found that RKIP expression was significantly decreased during malignant progression, being highly expressed in non-neoplastic tissues (54% of the samples; 73/135), and expressed at low levels in the cervix invasive carcinomas (,15% (19/124). Following in vitro downregulation of RKIP, we observed a viability and proliferative advantage of RKIP-inhibited cells over time, which was associated with an altered cell cycle distribution and higher colony number in a colony formation assay. An in vitro wound healing assay showed that RKIP abrogation is associated with increased migratory capability. RKIP downregulation was also associated with an increased vascularization of the tumors in vivo using a CAM assay. Furthermore, RKIP inhibition induced cervical cancer cells apoptotic resistance to cisplatin treatment. In conclusion, we described that RKIP protein is significantly depleted during the malignant progression of cervical tumors. Despite the lack of association with patient clinical outcome, we demonstrate, in vitro and in vivo, that loss of RKIP expression can be one of the factors that are behind the aggressiveness, malignant progression and chemotherapy resistance of cervical cancer.This work was partially supported by the Portuguese Fundacao para a Ciencia e Tecnologia (grant PTDC/SAU-TOX/114549/2009). Olga Martinho and Sara Granja were recipients of PhD fellowships (SFRH/BD/36463/2007 and SFRH/BD/51062/2010, respectively), and Filipe Pinto and Vera Miranda-Goncalves were recipients of research fellowships (UMINHO/BI/016/2011 and SFRH/BI/33503/2008, respectively), both from FCT, Portugal. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study

HPV16 Oncoproteins Induce MMPs/RECK-TIMP-2 Imbalance in Primary Keratinocytes: Possible Implications in Cervical Carcinogenesis

Cervical cancer is the third most common cancer in women worldwide. Persistent infection with high-risk HPV types, principally HPV16 and 18 is the main risk factor for the development of this malignancy. However, the onset of invasive tumor occurs many years after initial exposure in a minority of infected women. This suggests that other factors beyond viral infection are necessary for tumor establishment and progression. Tumor progression is characterized by an increase in secretion and activation of matrix metalloproteinases (MMPs) produced by either the tumor cells themselves or tumor-associated fibroblasts or macrophages. Increased MMPs expression, including MMP-2, MMP-9 and MT1-MMP, has been observed during cervical carcinoma progression. These proteins have been associated with degradation of ECM components, tumor invasion, metastasis and recurrence. However, few studies have evaluated the interplay between HPV infection and the expression and activity of MMPs and their regulators in cervical cancer. We analyzed the effect of HPV16 oncoproteins on the expression and activity of MMP-2, MMP-9, MT1-MMP, and their inhibitors TIMP-2 and RECK in cultures of human keratinocytes. We observed that E7 expression is associated with increased pro-MMP-9 activity in the epithelial component of organotypic cultures, while E6 and E7 oncoproteins co-expression down-regulates RECK and TIMP-2 levels in organotypic and monolayers cultures. Finally, a study conducted in human cervical tissues showed a decrease in RECK expression levels in precancer and cancer lesions. Our results indicate that HPV oncoproteins promote MMPs/RECK-TIMP-2 imbalance which may be involved in HPV-associated lesions outcome

Does Arterialization of Portal Vein Have Any Effects in Large-for-Size Liver Transplantation? Hemodynamic, Histological, and Biomolecular Experimental Studies

Background: In pediatric liver transplantation, the optimal size of the transplanted liver ranges between 0.8% and 4.0% of the recipient’s weight. Sometimes, the graft weight exceeds this upper limit, characterizing the large-for-size condition potentially associated with reduced blood flow and worsening of ischemia-reperfusion injury. Therefore, it would be beneficial to increase the portal flow through arterialization of the portal vein. Materials and methods: Fifteen pigs underwent large-for-size liver transplants. They were divided into two groups: control (CTRL 6 animals – conventional technique) and arterialization – a shunt was established between the portal vein and the splenic artery (ART 9 animals). Hemodynamic, biochemical, histological, and molecular variables were compared. Results: Arterialization resulted in a significant increase in portal vein pressure but no changes in other hemodynamic variables, as shown in the analysis of variance. It was observed lower ALT values (p = 0.007), with no differences regarding the values of blood pH and lactate (p = 0.54 and p = 0.699 respectively) or histological variables (edema, steatosis, inflammation, necrosis, and IRI – p = 1.0, p = 0.943, p = 0.174, p = 0.832, p = 0.662, respectively). The molecular studies showed significantly increased expression of IL6 after 3 hours of reperfusion (p = 0.048) and decreased expression of ICAM immediately after reperfusion (p = 0.03). The regression analysis suggested a positive influence of portal flow and pressure on biochemical parameters. Conclusion: Arterialization of the portal vein showed no histological, biochemical, or molecular benefits in large-for-size transplantation