Effect of 0, 15% sodium hialuronate and 0, 5% carboxymethylcellulose on tear film breakup time in healthy dogs and in dogs with keratoconjunctivitis sicca / Efeito do hialuronato de sódio a 0, 15% e da carboximetilcelulose sódica a 0, 5% no tempo de ruptura do filme lacrimal de cães saudáveis e com ceratoconjuntivite seca

This study aimed to evaluate the effect of the 0.15% sodium hyaluronate (SH) and of 0.5% carboxymethylcellulose (CMC) on tear film breakup time (TFBUT) in 10 healthy dogs and in 32 eyes of dogs with keratoconjunctivis sicca (KCS). In addition, the goblet cell density (GCD) of this population was quantified. TFBUT was assessed at baseline and at different time points following the instillation of SH and CMC. KCS was graded as mild, moderate, and severe. GCD were quantified from conjunctival biopsies. The number of GCD differed significantly between patients with mild and moderate KCS (P<0.01). TFBUT of healthy dogs increased only for 1 minute after treatment with SH (P<0.01). Regarding baseline and treatments, SH significantly increased TFBUT for up to 30 minutes on the ocular surface, in comparison to CMC, in all categories of KCS (P<0.01). TFBUT and GCD correlated positively when the healthy and diseased eyes were grouped (r=0.41, P=0.006). It can be concluded that in dogs with KCS, SH lasts longer periods on the ocular surface than CMC, but such agents does not increase TFBUT in healthy dogs. Additionally, tear film stability tends to reduce in a linear fashion from the mild to severe form of KCS.(AU)

Objetivou-se avaliar os efeitos do hialuronato de sódio a 0,15% (HS) e da carboximetilcelulose a 0,5% (CMC) no teste de ruptura do filme lacrimal (TRFL) em 10 cães saudáveis e em 32 olhos de cães com ceratoconjuntivite seca (CCS). Ademais, quantificou-se a densidade de células caliciformes (DCC) deles. Mensurou-se o TRFL em momentos distintos antes e após a instilação do HS e da CMC. Graduou-se a CCS em leve, moderada e severa. Quantificou-se a DCC a partir de biópsias conjuntivais. A DCC diferiu apenas entre pacientes com CCS leve e severa (P<0,01). Em cães saudáveis, o TRFL se elevou apenas após um minuto do tratamento com HS (P<0,01). Relativamente ao período basal e entre os tratamentos, o HS elevou o TRFL de forma mais eficaz e permaneceu por até 30 minutos na superfície ocular, comparativamente à CMC, em todas as categorias de CCS (P<0,01). Ao se agruparem os olhos saudáveis e os com CCS, o TRFL se correlacionou com a DCC (r=0.41, P=0.006). Conclui-se que o HS permanece por maior tempo na superfície ocular que a CMC em cães com CCS, mas que tais substâncias não elevam o TRFL em cães saudáveis. Ademais, a estabilidade do filme lacrimal tende a se reduzir de modo linear da forma leve até à severa da CCS.(AU)

Effect of 0, 15% sodium hialuronate and 0, 5% carboxymethylcellulose on tear film breakup time in healthy dogs and in dogs with keratoconjunctivitis sicca / Efeito do hialuronato de sódio a 0, 15% e da carboximetilcelulose sódica a 0, 5% no tempo de ruptura do filme lacrimal de cães saudáveis e com ceratoconjuntivite seca

This study aimed to evaluate the effect of the 0.15% sodium hyaluronate (SH) and of 0.5% carboxymethylcellulose (CMC) on tear film breakup time (TFBUT) in 10 healthy dogs and in 32 eyes of dogs with keratoconjunctivis sicca (KCS). In addition, the goblet cell density (GCD) of this population was quantified. TFBUT was assessed at baseline and at different time points following the instillation of SH and CMC. KCS was graded as mild, moderate, and severe. GCD were quantified from conjunctival biopsies. The number of GCD differed significantly between patients with mild and moderate KCS (P<0.01). TFBUT of healthy dogs increased only for 1 minute after treatment with SH (P<0.01). Regarding baseline and treatments, SH significantly increased TFBUT for up to 30 minutes on the ocular surface, in comparison to CMC, in all categories of KCS (P<0.01). TFBUT and GCD correlated positively when the healthy and diseased eyes were grouped (r=0.41, P=0.006). It can be concluded that in dogs with KCS, SH lasts longer periods on the ocular surface than CMC, but such agents does not increase TFBUT in healthy dogs. Additionally, tear film stability tends to reduce in a linear fashion from the mild to severe form of KCS.(AU)

Objetivou-se avaliar os efeitos do hialuronato de sódio a 0,15% (HS) e da carboximetilcelulose a 0,5% (CMC) no teste de ruptura do filme lacrimal (TRFL) em 10 cães saudáveis e em 32 olhos de cães com ceratoconjuntivite seca (CCS). Ademais, quantificou-se a densidade de células caliciformes (DCC) deles. Mensurou-se o TRFL em momentos distintos antes e após a instilação do HS e da CMC. Graduou-se a CCS em leve, moderada e severa. Quantificou-se a DCC a partir de biópsias conjuntivais. A DCC diferiu apenas entre pacientes com CCS leve e severa (P<0,01). Em cães saudáveis, o TRFL se elevou apenas após um minuto do tratamento com HS (P<0,01). Relativamente ao período basal e entre os tratamentos, o HS elevou o TRFL de forma mais eficaz e permaneceu por até 30 minutos na superfície ocular, comparativamente à CMC, em todas as categorias de CCS (P<0,01). Ao se agruparem os olhos saudáveis e os com CCS, o TRFL se correlacionou com a DCC (r=0.41, P=0.006). Conclui-se que o HS permanece por maior tempo na superfície ocular que a CMC em cães com CCS, mas que tais substâncias não elevam o TRFL em cães saudáveis. Ademais, a estabilidade do filme lacrimal tende a se reduzir de modo linear da forma leve até à severa da CCS.(AU)

Evaluation of the effectiveness of sodium hyaluronate, sesame oil, honey, and silver nanoparticles in preventing postoperative surgical adhesion formation. An experimental study

Purpose: To evaluate the effectiveness of sodium hyaluronate, sesame oil, honey, and silver nanoparticles in preventing of postoperative surgical adhesion formation. Methods: Forty male Wistar rats were randomly assigned into five groups with eight rats in each group including control, hyaluronate, sesame, honey and silver groups. After two weeks the animals underwent laparotomy and were evaluated by two different blinded surgeons for severity of adhesions based on the two different classification scoring systems including Nair classification and cumulative adhesion scoring scale. Results: The scores of severity of adhesions in the hyaluronate and sesame groups were significantly lower than the control group based on the Nair classification (both P-values = 0.02), however based on the cumulative adhesion scoring scale just the score of severity of adhesions in the hyaluronate group was significantly lower than the control group (P-value = 0.02). In the hyaluronate group the severity of adhesions was decreased by 48% based on the cumulative adhesion scoring scale. Conclusions: Sodium hyaluronate and sesame oil may have a significant effect in preventing postoperative surgical adhesion formation.(AU)

ALDH1 and CD44 immunoexpression as prognostic indicators of invasion and metastasis in oral squamous cell carcinoma / Immunoexpressão de ALDH1 e CD44 como indicador de prognóstico de invasão e metástase em carcinoma epidermoide de boca

Oral squamous cell carcinoma (OSCC) is one of the most common cancer in the head and neck and results in high morbidity and mortality annually, being the worst prognosis related to the presence of metastasis in cervical lymph nodes. Metastasis has been associated with a subpopulation of tumor cells, called cancer stem cells (CSCs), which consists of a small population with stem-like cells properties, higher rate of migration and metastatic potential compared to other ordinary tumor cells from the tumor bulk. The aim of present study was to evaluate the immunoexpression of the CSC markers ALDH1 and CD44 in primary sites of OSCC and corresponding metastatic lymph nodes, by means of immunohistochemistry. The immunolabeling was further correlated with clinicopathological data. Archived Formalin-fixed, Paraffin-embedded tumor tissue specimens (n=50) and corresponding metastatic lymph nodes (n=25) were obtained from 50 patients with OSCC after surgical treatment. CD44 and ALDH1 immunostaining were semi-quantitatively scored according to the proportion and intensity of positive cells within the invasive front and metastatic cervical lymph nodes as a whole. The percentage of ALDH1 and CD44 positive tumor cells as well as immunostaining intensity was graded and a combined score, ranging from 0 to 9 (ALDH1) or 0 to 12 (CD44), was obtained by multiplying both parameters. Next, combined scores were dichotomized into a final score classified as low (ALDH1≤ 2; CD44≤ 4) or high (ALDH1> 2; CD44> 4) immunoexpression. ALDH1 and CD44 immunoexpression was detected in both primary and metastatic tumor sites, although with different immunolabeling pattern. ALDH1-positive tumor cells consisted of scattered patches and no immunoexpression was observed within keratin pearls. Conversely, CD44 immunopositivity was more homogeneous and widely distributed, with higher labeling in peripheral areas of the tumor islands within the tumor invasion front. Although not statistically significant, the means of ALDH1high (p= 0.0985) and CD44high (p= 0.1632; Mann- Whitney post-test) immunoexpression were higher in metastatic lymph nodes compared to primary tumors. ALDH1high was positively associated (p= 0.0184) with perivascular invasion, while CD44high was positively associated (p= 0.0186; Fisher's Exact Test) with metastasis (N+). Five-year survival rates tended to be lower in patients with ALDH1high immunoexpression compared to ALDH1low, although with no statistical significance (p= 0.1303). In summary, the present study revealed that CD44 is highly labeled in tumor cell from metastatic sites, being associated with lymph node metastasis, while ALDH1 high immunostaining was associated with perivascular invasion. Altogether, it suggests that immunoexpression of CD44 and ALDH1 links the cancer stem cell phenotype with OSCC invasion and metastasis.(AU)

O carcinoma epidermóide de boca (CEB) é uma das neoplasias mais comuns da região de cabeça e pescoço e resulta em alta morbidade e mortalidade anualmente, estando o pior prognóstico relacionado à presença de metástase em linfonodos cervicais. O processo de metástase tem sido associado a uma subpopulação de células tumorais, chamadas células-tronco de câncer (CSC, do inglês Cancer stem cells), que consistem em uma pequena população de células com propriedades de células-tronco, incluindo maior taxa de migração e potencial metastático em comparação com outras células tumorais. O objetivo do presente estudo foi avaliar os marcadores candidatos de CSCs ALDH1 e CD44 em tumores primários de CEB e metástases linfonodais correspondentes, por meio de imuno-histoquímica. A imunomarcação foi posteriormente correlacionada com dados clínico-patológicos. Foram obtidas amostras de tecido tumoral parafinado fixado em formalina (n = 50) e os linfonodos metastáticos correspondentes (n = 25) de 50 pacientes com CEB submetidos somente ao tratamento cirúrgico. Os marcadores CD44 e ALDH1 foram analisados de forma semi-quantitativa de acordo com a proporção e intensidade de células positivas no fronte de invasão e em linfonodos cervicais metastáticos como um todo. A porcentagem de células tumorais ALDH1 e CD44 positivas, bem como a intensidade da imunomarcação, foi classificada em um escore combinado obtido pela multiplicação de ambos os parâmetros, variando de 0 a 9 (ALDH1) ou 0 a 12 (CD44). Em seguida, as pontuações combinadas foram dicotomizadas em um escore final classificado como baixo (do inglês low) (ALDH1 ≤ 2; CD44 ≤ 4) ou alto (do inglês high) (ALDH1> 2; CD44> 4). A imunoexpressão de ALDH1 e CD44 foi detectada tanto em tumores primários quanto em linfonodos cervicais metastáticos, embora com padrão diferente de imunomarcação. Células tumorais ALDH1-positivas foram identificadas como focais e dispersas ao longo do fronte de invasão, sem imunomarcação nas pérolas córneas. Em contraste, a imunopositividade para CD44 foi mais homogênea e amplamente distribuída, com maior imunomarcação em áreas periféricas das ilhotas tumorais presentes no fronte de invasão. Embora não estatisticamente significativa, as médias da imunoexpressão ALDH1high (p = 0.0985) e CD44high (p = 0.1632, pós-teste de Mann-Whitney) foram maiores em linfonodos metastáticos em comparação com tumores primários. ALDH1high foi positivamente associado com invasão perivascular (p = 0.0184), enquanto CD44high foi com metástase (N+) (p = 0.0186; teste exato de Fisher). As taxas de sobrevida global em 5 anos tenderam a ser mais baixas em pacientes com imunoexpressão elevada de ALDH1 em comparação com ALDH1low, embora sem significância estatística (p = 0.1303). Em resumo, o presente estudo revelou que a elevada imunomarcação de CD44 está significativamente associada com metástases linfonodais, enquanto que a elevada imunomarcação de ALDH1 está associada com invasão perivascular. Em conjunto, sugerimos que a imunoexpressão de CD44 e ALDH1 esteja relacionada com o fenótipo de células tronco de câncer que tem capacidade de invasão e metástase em CEB.(AU)

Valor prognóstico da imunoexpressão de podoplanina e de CD44v6 na recidiva locorregional dos pacientes com câncer de lábio / Prognostic value of podoplanin and CD44v6 immunoexpressions on locoregional recurrence of the patients with lip cancer

O objetivo deste estudo consistiu em avaliar a expressão imuno-histoquímica da podoplanina e do CD44v6 pelas células malignas, verificando a associação destas proteínas com as variáveis clínicas, microscópicas, com o índice histopatológico de malignidade e com a sobrevivência livre de doença de 91 pacientes portadores de carcinomas espinocelulares (CEC) de lábio inferior, tratados no Centro de Tratamento e Pesquisa do Hospital do Câncer A.C.Camargo, São Paulo. Os tumores foram corados, separadamente, com os anticorpos anti-podoplanina e anti-CD44v6, sendo avaliada a imunoexpressão destas proteínas pelas células neoplásicas, no front de invasão tumoral, por meio de um método semi-quantitativo de escores. A associação da expressão da podoplanina e do CD44v6 com as variáveis demográficas, clínicas e microscópicas foi feita pelo teste do qui-quadrado ou exato de Fisher. As taxas de sobrevivência livre de doença, acumuladas em cinco e dez anos, foram calculadas pelo teste de Kaplan-Meier e a influência das variáveis clínicas e microscópicas no prognóstico avaliadas pelo modelo de regressão de Cox. A correlação entre a podoplanina e o CD44v6 foi analisada pelo teste de Spearman. Em todos os testes estatísticos utilizou-se um nível de significância de 5%. Os resultados mostraram uma predominância da forte expressão membranosa e citoplasmática da podoplanina pelas células malignas. Verificou-se uma associação significativa da podoplanina citoplasmática com a recidiva locorregional (p=0,028) e da podoplanina membranosa com o índice histopatológico de malignidade tumoral (p=0,026). O CD44v6 foi fortemente expresso pelas células neoplásicas de 95,4% dos CECs e significativamente, associado com o estadiamento clínico T (p=0,034). Não houve correlação entre a podoplanina e o CD44v6 nos CECs de lábio inferior. A forte expressão de podoplanina membranosa (p=0,016) e citoplasmática (p=0,030) pelas células malignas foi fator de prognóstico favorável independente na sobrevivência livre de doença. Concluímos que a podoplanina e o CD44v6 são fortemente expressos pelas células neoplásicas e que a forte imunoexpressão membranosa e citoplasmática da podoplanina pode auxiliar na identificação do risco de recidiva locorregional nos pacientes portadores de carcinoma espinocelular de lábio inferior.(AU)

The aim of this study was evalute the podoplanin and CD44v6 immunohistochemical expression by malignant cells and its association with the clinical and microscopic variables, tumor histopathological grading and disease-free survival of 91 patients with lip squamous cell carcinomas (SCC), submitted to surgical treatment at Research and Treatment Center of the Cancer Hospital A.C. Camargo, São Paulo. The tumors were stained separately, with the antibodies anti-podoplanin and anti-CD44v6, and the immunoexpression of these proteins, by the neoplastic cells in the invasion front, was evaluated by a semi-quantitative scores method. Chi-square test or Fishers exact test was used to analyze the association of podoplanin and CD44v6 expression with demographic, clinical, and microscopic variables. Disease-free survival in five and ten years, were calculated by the Kaplan-Meier method and the influence of clinical and microscopic variables on prognosis were evaluated by the Cox regression model. The correlation between podoplanin and CD44v6 expression was analyzed by Spearman's test and a significance level of 5% was used in all statistical tests. The results showed a predominance of strong membranous and cytoplasmic podoplanin expression by malignant cells. An association between cytoplasmic podoplanin and locorregional recurrence (p=0,028) and membranous podoplanin with tumor histopathological grading (p=0,026). CD44v6 was strongly expressed in 95.4% of the SCCs neoplastic cells and significantly associated with the clinical staging T (p=0,034). There was no correlation between podoplanin and CD44v6 expression in the lower lip SCC. The strong expression of membranous (p=0.016) and cytoplasmic (p=0.030) podoplanin by malignant cells was a favorable independent prognostic factor in disease-free survival. Concluding, the podoplanin and CD44v6 are strongly expressed by neoplastic cells and the strong membranous and cytoplasmic immunoexpression of podoplanin can help the identification of locoregional recurrence risk in patients with squamous cell carcinoma of the lower lip.(AU)

Cells involved in extracellular matrix remodeling after acute myocardial infarction.

OBJECTIVE: Evaluate the effects of VEGF165 gene transfer in the process of remodeling of the extracellular matrix after an acute myocardial infarct. METHODS: Wistar rats were submitted to myocardial infarction, after the ligation of the left descending artery, and the left ventricle ejection fraction was used to classify the infarcts into large and small. The animals were divided into groups of ten, according to the size of infarcted area (large or small), and received or not VEGF165 treatment. Evaluation of different markers was performed using immunohistochemistry and digital quantification. The primary antibodies used in the analysis were anti-fibronectin, anti-vimentin, anti-CD44, anti-E-cadherin, anti-CD24, anti-alpha-1-actin, and anti-PCNA. The results were expressed as mean and standard error, and analyzed by ANOVA, considering statistically significant if p≤0.05. RESULTS: There was a significant increase in the expression of undifferentiated cell markers, such as fibronectin (protein present in the extracellular matrix) and CD44 (glycoprotein present in the endothelial cells). However, there was decreased expression of vimentin and PCNA, indicating a possible decrease in the process of cell proliferation after treatment with VEGF165. Markers of differentiated cells, E-cadherin (adhesion protein between myocardial cells), CD24 (protein present in the blood vessels), and alpha-1-actin (specific myocyte marker), showed higher expression in the groups submitted to gene therapy, compared to non-treated group. The value obtained by the relation between alpha-1-actin and vimentin was approximately three times higher in the groups treated with VEGF165, suggesting greater tissue differentiation. CONCLUSION: The results demonstrated the important role of myocytes in the process of tissue remodeling, confirming that VEGF165 seems to provide a protective effect in the treatment of acute myocardial infarct.

The endometrium of cycling cows contains populations of putative mesenchymal progenitor cells.

Endometrial stem cells have been identified in humans, mice and pigs. This study was designed to determine whether the uterine endometrium of cycling cows contains such cells, to identify markers of stemness and ultimately to isolate putative stem/progenitor cell and evaluate their capability to differentiate into mesodermal derivatives. Uteri from healthy cows in the early (days 1-5) and late luteal phases (days 13-18) of the oestrous cycle were collected. Total RNA and proteins were isolated and searched for gene markers of embryonic (OCT4, NANOG, SOX2) and mesenchymal (CD44, STAT3, CD-117) stem cells and for protein markers (Oct4, Sox2, Cd44) in Western blots or immunostaining of paraffin-embedded tissue. Primary cell cultures were isolated; characterized in terms of morphology, colony formation and gene/protein expression; and induced osteogenic and chondrogenic differentiation. We identified expression of embryonic (OCT4 and SOX2, but not NANOG) and mesenchymal (STAT3, CD44 and c-KIT) gene markers in the endometrium of cycling cows and the encoded proteins (Oct4, Sox2 and Cd44) in both stages of the oestrous cycle. Derived cell lines displayed essentially the same gene expression pattern; however, at the protein level, Oct4 was not detected. No clear influence of the stage of the oestrous cycle was found. Cell lines from late luteal phase displayed osteogenic and chondrogenic differentiation potential upon chemical stimulation. In this research, we demonstrated the presence of mesenchymal progenitor cell populations of apparently mesenchymal origin in the endometrium of cycling cows, in both the early and late phases of the oestrous cycle. The cells isolated from the late luteal phase were more acquiescent to differentiate into mesodermal derivatives than cells in the early luteal phase. Our findings might have implications for the understanding of uterine stem cell biology in cows and other farm animal species.

Spatial distribution of cancer stem cells in head and neck squamous cell carcinomas.

BACKGROUND: CD44 and aldehyde dehydrogenase 1 (ALDH1) are considered putative markers of highly tumorigenic cells (i.e., cancer stem-like cells) in head and neck squamous cell carcinomas. This small subset of cells is believed to be the primary responsible for tumor initiation and progression. The objectives of this study were (i) to evaluate the patterns of CD44 and ALDH1 expression in the tumor center and in the invasive front, as well as in adjacent non-tumor epithelium, and (ii) to correlate these findings with clinical parameters. MATERIALS AND METHODS: The sample comprised 44 patients with primary head and neck squamous cell carcinomas. Hematoxylin and eosin (HE) staining was used for histopathological tumor grading and for morphological analysis of adjacent non-tumor epithelium. Semiquantitative analysis was performed in histological sections immunostained for CD44 and ALDH1. RESULTS: ALDH1 immunostaining in the invasive front showed positive association with tumor size, regional metastasis, tumor histopathological grading, and disease progression. Moreover, expression of this marker in both tumor invasive front and adjacent non-tumor epithelium was related with more aggressive tumors. CD44 immunostaining was heterogeneous in all areas evaluated and did not show association with clinical data. CONCLUSION: Collectively, these data suggest that ALDH1 immunostaining in the invasive front and in adjacent non-tumor epithelium may help identify tumors with a more aggressive behavior, potentially contributing to improving treatment customization and the monitoring of patients with head and neck cancer.

Combined preoperative CEA and CD44v6 improves prognostic value in patients with stage I and stage II colorectal cancer.

AIM: Combination of biomarkers may improve diagnosis and have better prognostic value than single markers. The purpose of this study was to investigate whether combined CEA and CD44v6 improves prognostic value in stage I and stage II (stage I/II) colorectal cancer (CRC). METHODS: Preoperative serum CEA level and the expression of CD44v6 in CRC tissues were examined by electrochemiluminescence immunoassay and immunohistochemistry, respectively. The association of CEA and CD44v6 with clinicopathological features and their possible prognostic values was analyzed. RESULTS: The preoperative elevated serum CEA level and positive CD44v6 expression were detected in 30.1 % (52/173) serum samples and 60.5 % (101/167) CRC tissues, respectively. Patients with an elevated-CEA level or a CD44v6-negative tumor had a worse disease-specific survival (DSS) than those with a normal-CEA level or CD44v6-positive tumor (P = 0.024, P = 0.012, respectively). Moreover, CD44v6 expression could be used in discriminating patients from good to poor prognosis in normal-CEA subgroup (P = 0.043), but not in elevated-CEA subgroup (P = 0.563). Multivariate analysis revealed that combined CEA and CD44v6 was an independent prognostic factor for patients with stage I/II CRC (P = 0.023). However, serum CEA level only retained a borderline significance for correlation with a worse DSS (P = 0.059), and CD44v6 expression alone was not an independent prognostic factor for DSS in multivariate analysis (P = 0.123). CONCLUSION: These results suggested that combined CEA/CD44v6 had better prognostic value than CEA or CD44v6 alone for patients with stage I/II CRC.

Extranodal nasal NK/T-cell lymphoma: a rare oral presentation and FASN, CD44 and GLUT-1 expression.

Extranodal natural killer (NK)/T-cell lymphoma is an aggressive malignant tumor with distinctive clinicopathological features, characterized by vascular invasion and destruction, prominent necrosis, cytotoxic lymphocyte phenotype and a strong association with Epstein-Barr virus. Here is reported an extranodal nasal NK/T-cell lymphoma case, involving the maxillary sinus, floor of the orbit, and interestingly extending to the oral cavity through the alveolar bone and buccal mucosa, preserving the palate, leading to a primary misdiagnosis of aggressive periodontal disease. Moreover, this work investigated for the first time the immunohistochemical expression of fatty acid synthase (FASN) and glucose transporter 1 (GLUT-1) proteins in this neoplasia. FASN showed strong cytoplasmatic expression in the neoplastic cells, whereas GLUT-1 and CD44 were negative. These findings suggest that the expression of FASN and the loss of CD44 might be involved in the pathogenesis of the extranodal nasal NK/T-cell lymphoma, and that GLUT-1 may not participate in the survival adaptation of the tumor cells to the hypoxic environment. Further studies with larger series are required to confirm these initial results.

CD44/CD24 immunophenotypes on clinicopathologic features of salivary glands malignant neoplasms.

BACKGROUND: Salivary glands malignant neoplasms (SGMNs) account for 3-6% of head and neck cancers and 0.3% of all cancers. Tumor cells that express CD44 and CD24 exhibit a stem-cell-like behavior. CD44 is the binding site for hyaluronic acid, and CD24 is a receptor that interacts with P-selectin to induce metastasis and tumor progression. The present study aims to evaluate the expression of CD44 and CD24 on SGMNs and correlated these data with several clinicopathologic features. METHODS: Immunohistochemical stains for CD44 and CD24 were performed on tissue microarrays containing SGMN samples from 69 patients. The CD44, CD24 and CD44/CD24 expression phenotypes were correlated to patient clinicopathologic features and outcome. RESULTS: CD44 expression was associated with the primary site of neoplasm (p = 0.046). CD24 was associated with clinical stage III/IV (p = 0.008), T stage (p = 0,27) and lymph node (p = 0,001). The CD44/CD24 profiles were associated with the primary site of injury (p = 0.005), lymph node (p = 0.011) and T stage (p = 0.023). Univariate analysis showed a significant relationship between clinical staging and disease- free survival (p = 0.009), and the overall survival presents relation with male gender (p = 0.011) and metastasis (p = 0.027). CONCLUSION: In summary, our investigation confirms that the clinical stage, in accordance with the literature, is the main prognostic factor for SGMN. Additionally, we have presented some evidence that the analysis of isolated CD44 and CD24 immunoexpression or the two combined markers could give prognostic information associated to clinicopathologic features in SGMN. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1284611098470676.

Immunohistochemical expression of CD44s in renal cell carcinoma lacks independent prognostic significance.

PURPOSE: To analyze the immunohistochemical expression of the standard isoform of CD44 (CD44s) adhesion molecule in clear cell renal cell carcinoma (CCRCC) and its impact on clinical outcomes. MATERIALS AND METHODS: Ninety-nine consecutive patients treated surgically for RCC between 1992 and 2009 were selected. A single pathologist reviewed all cases to effect a uniform reclassification and determine the most representative tumor areas for construction of a tissue microarray. The same pathologist, who was blinded to the outcome of the cases, semi-quantitatively scored the staining intensity of CD44s in all specimens. The counting was done using the H-Score algorithm. RESULTS: Of the 99 immunostained RCC specimens, 57(57.7 %) showed low expression, and 42(42.4 %) showed high expression levels of CD44s. The expression of CD44s was directly associated with tumor size (p = 0.03), clinical stage (p = 0.02) and Fuhrman grade (p = 0.02). Disease specifi c survival (DSS) rates for patients whose specimens expressed low and high levels of CD44s was 88.1 % and 67.5 %, respectively (p = 0.009). Progression free survival (PFS) rates in patients with low and high expression of CD44s were 78.8 % and 61.7 %, respectively (p = 0.05). Classical features such as the presence of metastasis and clinical stage remained isolated predictors of survival. CONCLUSIONS: Immunohistochemical expression of CD44s was associated with important clinical variables such as stage and Fuhrman grade. However, it was not an independent predictor of survival. Therefore, we believe it has a limited role as a prognostic marker in patients with CCRCC.

Prognostic significance of CD24 and claudin-7 immunoexpression in ductal invasive breast cancer.

This study aimed to identify the CD24 and CD44 immunophenotypes within invasive ductal breast carcinoma (IDC) subgroups defined by immunohistochesmistry markers and to determine its influence on prognosis as well as its association with the expression of Ki-67, cytokeratins (CK5 and CK18) and claudin-7. Immunohistochemical expression of CD44 and CD24 alone or in combination was investigated in 95 IDC cases arranged in a tissue microarray (TMA). The association with subgroups defined as luminal A and B; HER2 rich and triple negative, or with the other markers and prognosis was analyzed. CD44+/CD24- and CD44-/CD24+ were respectively present in 8.4% and 16.8% of the tumors, a lack of both proteins was detected in 6.3%, while CD44+/CD24+ was observed in 45.3% of the tumors. Although there was no significant correlation between subgroups and different phenotypes, the CD44+/CD24- phenotype was more common in the basal subgroups but absent in HER2 tumors, whereas luminal tumors are enriched in CD44-/CD24+ and CD44+/CD24+ cells. The frequency of CD44+/CD24- or CD44-/CD24+ was not associated with clinical characteristics or biological markers. There was also no significant association of these phenotypes with the event free (DFS) and overall survival (OS). Single CD44+ was evident in 57.9% of the tumors and was marginally associated to grading and not to any other tumor characteristics as well as OS and DFS. CD24+ was positive in 74.7% of the tumors, showing a significant association with estrogen receptor, progesterone receptor and Ki-67 and a marginal association with CK18 and claudin-7. Expression of claudin-7 and Ki-67 did not associate with the cancer subgroups, while a positive association between CK18 and the luminal subgroups was found (P=0.03). CK5, CK18 and Ki-67 expression had no influence in OS or DFS. Single CD24+ (P=0.07) and claudin-7 positivity (P=0.05) were associated with reduced time of recurrence, suggesting a contribution of these markers to aggressiveness of breast cancer.

CD44+/CD24- cells and lymph node metastasis in stage I and II invasive ductal carcinoma of the breast.

The presence of tumor-initiating cells (CD44(+)/CD24(-)) in solid tumors has been reported as a possible cause of cancer metastasis and treatment failure. Nevertheless, little is know about the presence of CD44(+)/CD24(-) cells within the primary tumor and metastasis. The proportion of CD44(+)/CD24(-) cells was analyzed in 40 samples and in 10 lymph node metastases using flow cytometry phenotyping. Anti-human CD326 (EpCam; FITC), anti-human CD227 (MUC-1; FITC), anti-human CD44 (APC), and anti-human CD24 (PE), anti-ABCG2 (PE), and anti-CXCR4 (PeCy7) were used for phenotype analysis. The mean patient age was 60.5 years (range, 33-87 years); mean primary tumor size (pT) was 1.8 cm (0.5-3.5 cm). The Wilcoxon or Kruskal-Wallis test was used for univariate analyses. Logistic regression was used for multivariate analysis. The median percentage of CD44(+)/CD24(-) cells within primary invasive ductal carcinomas (IDC) was 2.7% (range, 0.2-71.2). In lymph node metastases, we observed a mean of 6.1% (range, 0.07-53.7). The percentage of CD44(+)/CD24(-) cells in IDCs was not associated with age, pT, tumor grade and HER2. We observed a significantly enrichment of CD44(+)/CD24(-) and ABCG2(+) cells in ESA(+) cell population in patients with positive lymph nodes (P = 0.02 and P = 0.04, respectively). Our data suggest that metastatic dissemination is associated with an increase in tumor-initiating cells in stage I and II breast cancer.

Differential expression of HIF-1α in CD44+CD24-/low breast ductal carcinomas.

BACKGROUND: Cancer stem cell (CSC) hypothesis postulates that tumors are maintained by a self-renewing CSC population that is also capable of differentiating into non-self-renewing cell populations that constitute the bulk of tumor. Stem cells renewal and differentiation can be directly influenced by the oxygen levels of determined tissues, probably by the reduction of oxidative DNA damage in hypoxic regions, thus leading to a friendlier microenvironment, regarding to clonal expansion and for resistance to chemotherapeutic regimens. Furthermore, there have been strong data indicating a pivotal role of hypoxic niche in cancer stem cells development. There are evidence that hypoxia could drive the maintenance of CSC, via HIF-1α expression, but it still to be determined whether hypoxia markers are expressed in breast tumors presenting CD44+CD24-/low immunophenotype. METHODS: Immunohistochemical analysis of CD44+CD24-/low expression and its relationship with hypoxia markers and clinical outcome were evaluated in 253 samples of breast ductal carcinomas. Double-immunolabeling was performed using EnVision Doublestain System (Dako, Carpinteria, CA, USA). Slides were then scanned into high-resolution images using Aperio ScanScope XT and then, visualized in the software Image Scope (Aperio, Vista, CA, USA). RESULTS: In univariate analysis, CD44+CD24-/low expression showed association with death due to breast cancer (p = 0.035). Breast tumors expressing CD44+CD24-/low immunophenotype showed relationship with HIF-1α (p = 0.039) and negativity for HER-2 (p = 0.013). CONCLUSION: Considering that there are strong evidences that the fraction of a tumour considered to be cancer stem cells is plastic depending upon microenvironmental signals, our findings provide further evidence that hypoxia might be related to the worse prognosis found in CD44+CD24-/low positive breast tumors.

The interaction between aromatase, metalloproteinase 2,9 and CD44 in breast cancer.

OBJECTIVE: This study intends to verify the expression levels and correlation of aromatase, matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9) and CD44 in ductal carcinoma in situ (DCIS) and infiltrating ductal carcinoma (IDC) when both are found in the same breast. METHODS: One hundred and ten cases were evaluated by tissue microarray (TMA) and immunohistochemically screened with anti-aromatase polyclonal antibodies, anti-MMP-2 monoclonal antibodies, anti-MMP-9 polyclonal antibodies and anti-CD44 monoclonal antibodies. RESULTS: Aromatase was expressed in IDC and DCIS in 63 (57.3%) and 60 (67%) of the cases respectively; MMP-2 was similarly expressed in IDC and DCIS in 15 (13.60%) cases; MMP-9 was positively expressed in IDC and DCIS in 83 (75.50%) and 82 (74.50%) cases, respectively; CD44 was positively expressed in IDC and DCIS in 49 (44.50%) and 48 (42.60%) of the cases, respectively; all of them were highly correlated (p<0,001). The correlation analysis found positive, statistically significant correlation, in IDC between aromatase and MMP-2 (p<0.001) and between aromatase and MMP-9 (p=0.034). Positive correlation between aromatase and MMP-2 (p<0.001) and between MMP-9 and CD44 (p=0.030) were found in DCIS. CONCLUSION: These results allow us to conclude that aromatase through local estrogen synthesis in breast tissue plays an important role in breast carcinogenesis, mainly influencing MMP-2 and MMP-9 which are important participants in tumor cell invasion and dependence of their connection to CD44 for action.

Trypanosoma cruzi: Immunological predictors of benznidazole efficacy during experimental infection.

C3H/HeN male mice were infected with a lethal population of Trypanosoma cruzi and treated with benznidazole (Bz). Parasitemia, body weight and survival rate were registered during the therapy with significant improvement for T. cruzi-infected Bz-treated animals. Besides, flow cytometry resulted a useful method to discriminate between cured animals from those not cured by monitoring IgG(1) bound to live trypomastigotes levels. At the end of Bz therapy, the LT splenocyte compartment was studied for activation/memory cell surface markers (CD(69)(+) and CD(44)(+)). Cytofluorometric analysis showed that T. cruzi-infected untreated mice increased their activated LT numbers and this effect was completely abolished only in cured mice at the end of Bz administration. The same behavior was observed for the memory LT subpopulation correlating to an effector memory (CD(62L)(-)) displayed by T. cruzi infection. Bz treatment was able to modulate the immunological response by reducing the deleterious effect of the acute phase in all T. cruzi-infected mice.

Detection of hyaluronic acid receptor in human vocal folds by immunohistochemistry.

UNLABELLED: Hyaluronic acid receptor is a glycoprotein of the plasmatic membrane, and the CD44 is its representative, expressed in many cell types where it has the task of cell adhesion. AIM: the goal of the present experimental study is to investigate the possibility of using immunohistochemistry to identify the distribution of hyaluronic acid along the vocal fold. MATERIALS AND METHODS: We resected the normal vocal folds from a normal 23 year-old male black individual. The slides were analyzed by means of a histomorphometric study, comparing the color intensity in the superficial, middle and deep layers of the lamina propria. In the silanized slides we used immunohistochemistry, and evaluated the slides under light microscopy with 40x magnification, and the color changed to brown when there was a reaction with the receptor for hyaluronic acid. RESULTS: Immunohistochemical findings showed the presence of hyaluronic acid receptors in the epithelium covering the vocal fold, being more concentrated in the central region of the vocal fold. CONCLUSION: immunohistochemistry, used to assess the distribution of hyaluronic acid receptors in the central portion of the vocal fold, proved it to be present in the vocal fold epithelium and it prevailed in its middle third.

CD44 Presence in inflamed pulp tissue.

Inflammation of pulp tissue appears as a consequence of caries progression. Its main characteristic is inflamed infiltrate whose cells contain lymphocytes. CD44 is a widely expressed adhesion molecule present in several body cells such as leukocytes and parenchymatous cells, including endothelial cells, epithelial cells, and unstriated muscle cells. It interacts with hyaluronic acid, collagen, laminin, and fibronectin, and there are data that indicate an important role in the migration of leukocytes from the bloodstream toward inflammation areas. This project, which applied the immunologic assay method of agglutination inhibition of the CD44-hyaluronate system, evaluated the presence of CD44 in inflamed pulp tissue in both asymptomatic and symptomatic processes, as well as in healthy pulp tissue. The results demonstrated significant differences between both groups of pulp inflammatory processes with strong presence of the receptor. Moreover, healthy pulp had low to nondetectable levels of CD44. These results suggest that the expression of the CD44 molecule is higher during the initiation or maintenance of inflammatory processes.

Involvement of CD44 in leukocyte recruitment to the rat testis in experimental autoimmune orchitis.

Experimental autoimmune orchitis (EAO) is characterized by an interstitial mononuclear cell infiltrate and a severe lesion of the seminiferous tubules with germ cells that undergo apoptosis and sloughing. The aim of this study was to determine the role of CD44 in testicular leukocyte recruitment in EAO. The biological functions of CD44 have been attributed to the generation of a functionally active hyaluronan-binding phenotype. Orchitis was induced in Sprague-Dawley adult rats by active immunization with an emulsion of testicular homogenate and complete Freund's adjuvant using Bordetella pertussis as co-adjuvant. Control rats (C) injected with saline and adjuvants and normal (N) untreated rats were also studied. CD44 expression was analyzed by flow cytometry in peripheral blood mononuclear cells (PBMC) and lymph node cells isolated from rats at different times after the first immunization. We observed an increase in the mean fluorescence intensity of both samples in the C and experimental (E) groups only after the immunization period. A significant decrease in percentage of CD44+PBMC and in mean fluorescence intensity was observed in rats with orchitis compared with the C group. By in vitro hyaluronic acid-binding assay we demonstrated that the percentage of PBMC adhesion was higher in the E group compared with the C and N groups. By immunohistochemistry, we observed a significant increase in the number of CD44+cells in the testicular interstitium of rats with severe orchitis compared with the N and C groups. These results suggested that the CD44 molecule is involved in the homing of lymphomonocytes into the testes of rats with autoimmune orchitis.