Applicability of eucalyptus wood waste extracts for the histological staining of connective tissue.

Biological staining of tissue is a crucial procedure in histotechnology. Rudimentary methods for section preparation have often used stains from natural products, although use of synthetic dyes has become the contemporary standard. Artificial dyes increase the operating costs of a laboratory as well as increase the environmental and personnel risks during manufacturing and usage. These considerations have stimulated research to find alternative natural stains from the wide diversity of plant species. The present study investigated the effect of Eucalyptus sp. (Myrtaceae) wood waste extract on histological staining of animal tissues, using a series of pigment concentrations, pH conditions, and temperatures. Eucalyptus wooden slivers were dried, milled, and 1 g, 2 g, and 4 g of the fine powder was subjected to 50% ethanol extraction for 2 days. Staining tests were then performed on formalin fixed paraffin embedded (FFPE) sections. Increasing acetic acid concentrations (1%, 2% and 4%) were added to the extracts and compared to an acid-free extract. Staining was performed at both ambient room temperature (RT) and 60°C. Connective tissue acidophilic components were well-contrasted and a hematoxylin counterstain demonstrated distinct structural differences between matrix and cell nuclei. Therefore, the present findings demonstrate the potential utility of the eucalyptus wood extracts application as a natural stain alternative for routine histology.

Bioceramic cement in the filling of bone defects in rats.

PURPOSE: To evaluate PBS®MCIMMO cement in the filling of bone defects. METHODS: Thirty-six adult male Wistar rats were divided into three groups of twelve individuals each (group 1, group 2 and group 3). In all groups, a bone failure in the femur was induced, 2.0 mm wide and 7.0 mm deep. In group 1, the PBS®MCIMMO cement was applied to the bone defect produced and a titanium implant (CONNECTION®) 1.5 mm thick and 6 mm long was installed. In group 2, only the PBS® CIMMO cement was installed. In group 3, only bone failure was performed. Kruskal Wallis tests were performed to compare the mean area among the three groups. RESULTS: In all comparisons, significance was observed for group 2 (p = 0.0014-0.0026). CONCLUSION: The PBS®CIMMO cement induced bone neoformation, and integration between the newly formed bone, cement, and implant was observed.

Bioceramic cement in the filling of bone defects in rats

Abstract Purpose To evaluate PBS®MCIMMO cement in the filling of bone defects. Methods Thirty-six adult male Wistar rats were divided into three groups of twelve individuals each (group 1, group 2 and group 3). In all groups, a bone failure in the femur was induced, 2.0 mm wide and 7.0 mm deep. In group 1, the PBS®MCIMMO cement was applied to the bone defect produced and a titanium implant (CONNECTION®) 1.5 mm thick and 6 mm long was installed. In group 2, only the PBS® CIMMO cement was installed. In group 3, only bone failure was performed. Kruskal Wallis tests were performed to compare the mean area among the three groups. Results In all comparisons, significance was observed for group 2 (p = 0.0014-0.0026). Conclusion The PBS®CIMMO cement induced bone neoformation, and integration between the newly formed bone, cement, and implant was observed.

Experimental model of glucocorticoid-induced insulin resistance.

PURPOSE:: To evaluate metabolic effects in experimental model of glucocorticoid-induced insulin resistance. METHODS:: Twenty Wistar male rats were randomly divided into two groups, which were treated with intraperitoneally injected dexamethasone 1mg/Kg/day for ten days consecutively (Group D; n=10) and placebo (Group C; n=10). The variables analyzed were: from the first to the 10th day - body weight (before and after treatment); food and water daily consumption; on the 10th day - glycemia, insulinemia, HOMA-beta and HOMA-IR. The blood samples for laboratory analysis were obtained by intracardiac puncture. Also on the 10th day liver fragments were taken for analyzing glycogen and fattty. RESULTS:: Group D animals compared to group C had: weight reduction (g), (D=226.5±24.7 vs C=295.0±25.4; p=0.001); increased glycemia (mmol/l) (D=19.5±2.1 vs C=14.2±3.1; p=0.0001); diminished insulinemia (mU/l) (D=0.2±0.1 vs C=2.0±0.4; p=0.0001); reduced HOMA-ß (D=0.2±0.1 vs C=4.2±1.7; p=0.0002); diminished HOMA-IR (D=0.2±0.1 vs C=1.3±0.4; p=0.0002). Histological examination of the liver showed that 100% of group D and none of group C had moderate fatty. (p=0.2). CONCLUSION:: Animals treated with glucocorticoid, in this experimental model, expressed hyperglycemia, hypoinsulinism and decreased peripheral insulin sensitivity.

Experimental model of glucocorticoid-induced insulin resistance

ABSTRACT PURPOSE: To evaluate metabolic effects in experimental model of glucocorticoid-induced insulin resistance. METHODS: Twenty Wistar male rats were randomly divided into two groups, which were treated with intraperitoneally injected dexamethasone 1mg/Kg/day for ten days consecutively (Group D; n=10) and placebo (Group C; n=10). The variables analyzed were: from the first to the 10th day - body weight (before and after treatment); food and water daily consumption; on the 10th day - glycemia, insulinemia, HOMA-beta and HOMA-IR. The blood samples for laboratory analysis were obtained by intracardiac puncture. Also on the 10th day liver fragments were taken for analyzing glycogen and fattty. RESULTS: Group D animals compared to group C had: weight reduction (g), (D=226.5±24.7 vs C=295.0±25.4; p=0.001); increased glycemia (mmol/l) (D=19.5±2.1 vs C=14.2±3.1; p=0.0001); diminished insulinemia (mU/l) (D=0.2±0.1 vs C=2.0±0.4; p=0.0001); reduced HOMA-β (D=0.2±0.1 vs C=4.2±1.7; p=0.0002); diminished HOMA-IR (D=0.2±0.1 vs C=1.3±0.4; p=0.0002). Histological examination of the liver showed that 100% of group D and none of group C had moderate fatty. (p=0.2). CONCLUSION: Animals treated with glucocorticoid, in this experimental model, expressed hyperglycemia, hypoinsulinism and decreased peripheral insulin sensitivity.

Specific upregulation of RHOA and RAC1 in cancer-associated fibroblasts found at primary tumor and lymph node metastatic sites in breast cancer.

The importance of tumor-stromal cell interactions in breast tumor progression and invasion is well established. Here, an evaluation of differential genomic profiles of carcinoma-associated fibroblasts (CAFs) compared to fibroblasts derived from tissues adjacent to fibroadenomas (NAFs) revealed altered focal adhesion pathways. These data were validated through confocal assays. To verify the possible role of fibroblasts in lymph node invasion, we constructed a tissue microarray consisting of primary breast cancer samples and corresponding lymph node metastasis and compared the expression of adhesion markers RhoA and Rac1 in fibroblasts located at these different locations. Two distinct tissue microarrays were constructed from the stromal component of 43 primary tumors and matched lymph node samples, respectively. Fibroblasts were characterized for their expression of α-smooth muscle actin (α-SMA) and vimentin. Moreover, we verified the level of these proteins in the stromal compartment from normal adjacent tissue and in non-compromised lymph nodes. Our immunohistochemistry revealed that 59 % of fibroblasts associated with primary tumors and 41 % of the respective metastatic lymph nodes (p = 0.271) displayed positive staining for RhoA. In line with this, 57.1 % of fibroblasts associated with primary tumors presented Rac1-positive staining, and the frequency of co-positivity within the lymph nodes was 42.9 % (p = 0.16). Expression of RhoA and Rac1 was absent in fibroblasts of adjacent normal tissue and in compromised lymph nodes. Based on our findings that no significant changes were observed between primary and metastatic lymph nodes, we suggest that fibroblasts are active participants in the invasion of cancer cells to lymph nodes and support the hypothesis that metastatic tumor cells continue to depend on their microenvironment.

MYC is expressed in the stromal and epithelial cells of primary breast carcinoma and paired nodal metastases.

The MYC oncogene is directly involved in the proliferation, metabolism, progression and distant metastasis of breast cancer. Since metastatic spread to the lymph nodes is often the first indication of propensity for metastatic dissemination, the MYC status in nodal disease may represent a decision-making variable. However, the analysis of MYC expression in stromal cells, namely cancer-associated fibroblasts (CAFs), which are known to play a critical role in cancer progression, remains poorly reported. The aim of this study was to determine the expression of MYC and other markers, including estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), p53, Ki67, epidermal growth factor receptor (EGFR), phosphorylated AKT (p-AKT) and phospho-mammalian target of rapamycin (p-mTOR) by immunohistochemistry in representative samples from 80 patients with ductal infiltrative breast cancer and 43 paired compromised axillary lymph nodes allocated in tissue microarrays (TMAs). The epithelial and stromal components of primary tumors and respective lymph node metastases were separately analyzed. MYC expression (cytoplasmic and nuclear) was a frequent event in the epithelial and stromal components of the primary tumors. The epithelial cells in the nodal metastases exhibited a trend for decreased MYC expression compared to that in the primary tumors (P=0.08) but retained the original status of the primary tumors for all other markers. The stromal cells were uniformly negative for ER, PR, HER2, p53, Ki67 and EGFR. Comparison of the stromas of primary tumors and respective lymph node metastases revealed a reduced frequency of nuclear MYC in 15% of the cases (P=0.003), whereas p-mTOR followed a similar trend (P=0.09). Analyses of the possible correlations among markers revealed that epithelial nuclear MYC was associated with p53 (P=0.048). This is an original study demonstrating a significant proportion of MYC expression (nuclear or cytoplasmic), as well p-mTOR and p-AKT expression, in the epithelial and stromal components of either the primary tumor or the nodal metastases. CAFs expressing MYC may establish an angiogenic microenvironment supporting cancer survival and facilitating colonization at the nodal metastatic site.

Preventive use of Bach flower Rescue Remedy in the control of risk factors for cardiovascular disease in rats.

OBJECTIVES: To evaluate the effect of Bach flower Rescue Remedy on the control of risk factors for cardiovascular disease in rats. DESIGN: A randomized longitudinal experimental study. METHODS: Eighteen Wistar rats were randomly divided into three groups of six animals each and orogastrically dosed with either 200 µl of water (group A, control), or 100 µl of water and 100 µl of Bach flower remedy (group B), or 200 µl of Bach flower remedy (group C) every 2 days, for 20 days. All animals were fed standard rat chow and water ad libitum. MAIN OUTCOME MEASURES: Urine volume, body weight, feces weight, and food intake were measured every 2 days. On day 20, tests of glycemia, hyperuricemia, triglycerides, high-density lipoprotein (HDL) cholesterol, and total cholesterol were performed, and the anatomy and histopathology of the heart, liver and kidneys were evaluated. Data were analyzed using Tukey's test at a significance level of 5%. RESULTS: No significant differences were found in food intake, feces weight, urine volume and uric acid levels between groups. Group C had a significantly lower body weight gain than group A and lower glycemia compared with groups A and B. Groups B and C had significantly higher HDL-cholesterol and lower triglycerides than controls. Animals had mild hepatic steatosis, but no cardiac or renal damage was observed in the three groups. CONCLUSION: Bach flower Rescue Remedy was effective in controlling glycemia, triglycerides, and HDL-cholesterol and may serve as a strategy for reducing risk factors for cardiovascular disease in rats. This study provides some preliminary "proof of concept" data that Bach Rescue Remedy may exert some biological effects.

Markers of breast cancer stromal fibroblasts in the primary tumour site associated with lymph node metastasis: a systematic review including our case series.

CAFs (cancer-associated fibroblasts), the most abundant cell type in breast cancer stroma, produce a plethora of chemokines, growth factors and ECM (extracellular matrix) proteins, that may contribute to dissemination and metastasis. Axillary nodes are the first metastatic site in breast cancer; however, to the present date, there is no consensus of which specific proteins, synthesized by CAFs, might be related with lymph node involvement. The purpose of this study was to perform a systematic review of CAF biomarkers associated with the presence of regional metastasis. PubMed was searched using the words: 'breast cancer' and 'lymph node' and fibroblast or stroma or microenvironment. After exclusions, eight studies evaluating biomarkers immunoexpression in CAFs and lymph node status were selected. Biomarkers evaluated in these studies may be divided in two groups, according to their ontology: extracellular matrix components [MMP13 (matrix metalloproteinase 13), TIMP2 (tissue inhibitor of metalloproteinases-2), THBS1 (thrombospondin 1), LGALS1 (lectin, galactoside-binding, soluble, 1)] and response to wounding [PDPN (podoplanin), PLAU (plasminogen activator, urokinase), PLAUR (plasminogen activator, urokinase receptor), CAV1 (caveolin 1), THBS1, LGALS1]. A positive expression of MMP13 and LGALS1 in CAFs was associated with enhanced OR (odds ratio) for regional metastasis. Contrariwise, CAV1 positive staining of fibroblasts was associated with decreased OR for nodal involvement. Expression of MMP13, PDPN and CAV1 was further tested in a new series of 65 samples of invasive ductal breast carcinomas by immunohistochemistry and no association between biomarkers expression in CAFs and nodal status was found. It was suggested that breast cancer subtypes may differentially affect CAFs behaviour. It would be interesting to evaluate the prognostic significance of these biomarkers in CAFs from different tumour types.

Contribuição da imuno-histoquímica no diagnóstico do câncer de próstata / Immunohistochemistry contribution to the diagnosis of prostate cancer

INTRODUCTION: Efforts for the identification of prostate cancer in the initial clinical and pathological stages led to an increase in the number of biopsies, sometimes making the histological diagnosis of adenocarcinoma difficult. This is due to the presence of minimal carcinoma or atypical glands suspicious for carcinoma, also known as atypical small acinar proliferation (ASAP). In these cases, the use of immunohistochemistry (IHC) has become a common practice in laboratories of pathology. OBJECTIVES: The aims of this study were to assess the incidence of diagnoses of ASAP and minimal adenocarcinoma in two laboratories of pathology and to evaluate the contribution of IHC and repeat biopsy to the diagnosis of prostate cancer. METHODS: We reviewed 641 sets of modified sextant needle biopsies of the prostate performed in two laboratories of pathology between January 2005 and December 2010. IHC using 34âE12 and p63 antibodies was performed on 35 of 73 (11.38%) cases diagnosed as ASAP and on 7 (1.1%) cases diagnosed as minimal adenocarcinoma. RESULTS: The incidence of ASAP diagnosis was 11.38% (n = 73). IHC was performed in 35 of the 73 ASAP cases and provided conclusive results in 31 cases (88.57%), resulting in a final diagnosis of adenocarcinoma in 19 patients (54.28%), benign lesions in 12 patients (34 28%); only 4 (11.42%) were inconclusive. CONCLUSION: The results suggest that IHC should be routinely used in evaluation of borderline biopsies and in ASAP cases. IHC strongly contributes to the diagnosis of prostate cancer.

INTRODUÇÃO: Os esforços de detecção precoce do câncer de próstata, identificados em fases clínicas e patológicas iniciais, levou ao aumento no número de biópsias e, por vezes, indefinição do diagnóstico histológico de adenocarcinoma devido à presença de carcinomas mínimos ou alterações pseudoneoplásicas, como proliferação atípica de pequenos ácinos (PAPA). Nesses casos, o uso da imuno-histoquímica (IMH) para evidenciar a presença de células basais tornou-se uma prática comum em laboratórios de patologia. OBJETIVOS: O presente estudo visa a avaliar a incidência de PAPA e de adenocarcinoma mínimo em dois laboratórios de patologia do interior, bem como avaliar a contribuição da IMH e da rebiópsia no diagnóstico do câncer de próstata. MÉTODOS: Foram revistas 641 biópsias de próstata por agulha realizadas entre janeiro de 2005 e dezembro de 2010. Dos 73 casos diagnosticados como PAPA (11,38%), 35 foram submetidos ao exame imuno-histoquímico, usando anticorpos anti-34ßE12 e p63, assim como sete casos diagnosticados como adenocarcinoma mínimo (1,1%). Resultados: Os resultados mostraram que a técnica foi conclusiva em 31 casos (88,57%), com diagnóstico final de adenocarcinoma em 19 pacientes (54,28%); 12 (34,28%) com lesões benignas; e apenas quatro (11,42%) inconclusivos (PAPA). CONCLUSÃO: Os resultados sugerem que IMH deve ser rotineiramente usada em biópsias limítrofes e casos de PAPA, pois contribui significativamente para o diagnóstico de câncer de próstata.

Carcinoma mamário secretório em criança de sete anosde idade: relato de caso e revisão da literatura / Carcinoma mamário secretório em criança de sete anos de idade: relato de caso e revisão da literatura

O carcinoma secretório da mama é uma entidade extremamente rara, inicialmente descrita em crianças e adolescentes, mas um número relativamente frequente de casos foi relatado em adultos. É usualmente associado ao bom prognóstico. O presente caso refere-se a uma menina de sete anos de idade com crescimento tumoral progressivo na mama esquerda. O exame clínico identificou um nódulos ubareolar duro, móvel, indolor e margens regulares; macroscopicamente, o espécime cirúrgico mostrou nódulo com 1,3 cm de diâmetro e superfície lisa. O diagnóstico final de neoplasia maligna levou à mastectomia, e a avaliação intraoperatória do linfonodo sentinela foi negativa. O perfil imunoistoquímico incluiu coloração fortemente positiva para citoqueratinas (AE1/AE3) e TP53, expressão negativa para receptores de estrógeno e progesterona e proteína Her-2/neu; o antígeno de proliferação celualar Ki-67 (MIB-1) mostrou índice de marcação de 10%. Exames adicionais de acompanhamento não mostraram nenhuma evidência de metástases à distância, com desenvolvimento normal das características sexuais secundárias (pubarca, telarca direita e menarca, que ocorreu em julho de 2009). Sete anos mais tarde, a adolescente ainda está livre da doença e aguarda cirurgia plástica reconstrutiva da mama esquerda.

Secretory breast carcinoma is an extremely rare entity initially described in children and in adolescents, but a relatively frequent number of cases has been reported in adults. It is often associated with good prognosis. The present case refers to a seven-year-old girl with a progressively growing tumor in the left breast. Clinical examination identified a hard, mobile, non-painful,subareolar nodule with regular margins; further macroscopy of surgical specimen showed a smooth surface and 1.3 cm in diameter. The final diagnosis of malignancy led to mastectomy and intra-operatively negative sentinel lymph node assessment. Immunohistochemical profile included a strong positive stain for cytokeratins (AE1/AE3) and TP53, and estrogen-receptor and progesterone-receptor and Her-2/neu protein expression negative; and MIB-1 (Ki-67) labeling index was 10%. Further follow-up examinations have shown no evidence of distant metastases, with normal development of secondary sexual characteristics (pubarche, right thelarche, and menarche occurring in July, 2009). Seven years later, the teenager still does not have the disease and waits reconstructive plastic surgery of the left breast.