Correlation between mastitis occurrence and the count of microorganisms in bulk raw milk of bovine dairy herds in four selective culture media

Milk is the normal secretion of the mammary gland, practically free of colostrum and obtained by the complete milking of oil(? or more healthy animals. Mastitis is all inflammatory process of the mammary gland and it may cause alterations in the milk. The present work aimed to verify whether it is possible, by means of the counts of microorganism in the bulk raw milk ill four selective culture media, to establish a correlation with the occurrence of mastitis and therefore, to monitor this disease in bovine dairy herds. The following selective culture media were Used: KF Streptococcus Agar, Edwards Agar, Baird-Parker Agar, Blood Agar plus potassium tellurite. Spearman`s correlation coefficient was calculated in order to compare the Occurrence of mastitis (percentage) in each herd with respective selective culture media counts of microorganisms in bulk raw milk. Thirty-six possibilities were analysed (Tamis and CMT-positive rates were compared with the log-transformed count in four selective Culture media) and there was a negative correlation between Tamis 3 and the Baird-Parker Agar plate count. The total results of microbiological tests showed that there were three correlations of the counts in selective culture media. Fifty-two possibilities were analysed and there was a negative correlation between no-bacteria I-growth mastitis rates and log(10) of KF Streptoccocus Agar plate Count and there were two positive correlations between coagulase-positive staphylococci and log(10) of Baird-Parker Agar plate count and Blood Agar plus potassium tellurite plate count.

Evaluation of somatic cell count thresholds to detect subclinical mastitis in Gyr cows

The objectives of this study were (1) to determine the sensitivity (Se) and specificity (Sp) of somatic cell count (SCC) thresholds to identify subclinical mastitis in Gyr cows caused by major and minor pathogens; (2) to study the effects of month of sampling, rear or front mammary quarters, herd, intramammary infection (IMI), and bacterial species on SCC at quarter level; and (3) to describe the prevalence of IMI in Gyr cows in commercial dairy herds. In total, 221 lactating Gyr cows from 3 commercial dairy farms were selected. Milk samples were collected from individual quarters once a month for 1 yr from all lactating cows for SCC and bacteriological analysis. Mammary quarters were considered the experimental units and the SCC results were log(10)-transformed. Four SCC thresholds (100, 200, 300 and 400 x 10(3) cells/mL) were used to determine Se and Sp to identify infected mammary quarters. The overall prevalence of IMI in quarter milk samples of Gyr cows was 49.8%, and the prevalence of minor pathogens was higher (31.9%) than that of major pathogens (17.8%). Quarter samples with microbial isolation presented higher SCC compared with negative samples. Sensitivity and Sp of selected SCC thresholds varied according to the group of pathogen (major and minor) involved in the IMI definition. Sensitivity increased and Sp decreased when mammary quarters with only major pathogens isolation were considered positive. The use of a single SCC analysis to classify quarters as uninfected or infected in Gyr cows may not be a useful test for this breed because Se and Sp of SCC at the studied thresholds were low. The occurrence of IMI and the bacterial species are the main factors responsible for SCC variation in mammary quarters of Gyr cows. Milk samples with major pathogens isolation elicited higher SCC than those with minor pathogens.

The severity of epithelial dysplasia is associated with loss of maspin expression in actinic cheilitis

Background Prolonged exposure of the lip to sunlight may cause actinic cheilitis (AC) and squamous cell carcinoma (SCC). Maspin is a serpin with tumor suppressor functions. This work analyzed the presence and distribution of maspin in AC and lip SCC. Methods Sections from 36 cases diagnosed as AC (18 cases with mild epithelial dysplasia, 11 with moderate and 7 with severe), 18 cases diagnosed as lip SCC and 7 specimens containing normal lip vermillion epithelium were submitted for immunohistochemical analysis to detect maspin. Results All AC cases with mild and two cases with moderate dysplasia were scored 3. The remaining nine cases with moderate dysplasia were identified as score 2, whereas all cases with severe dysplasia were scored 1. Positive staining for maspin decreased from the basal layer to the surface. Among the 18 lip SCCs studied, 15 cases showed abundant staining for maspin. Epithelium adjacent to the SCCs also showed intense positive staining in all cells. Conclusions Our results suggest that the loss of maspin expression occurs from the basal layer to the surface. Lip SCCs related to solar radiation show an intense presence of maspin protein in almost all tumor cells as well as the neighboring epithelium. Fontes A, Sousa SM, Santos E, Martins MT. The severity of epithelial dysplasia is associated with loss of maspin expression in actinic cheilitis.

Oral and Dental Abnormalities in Barber-Say Syndrome

A previously unreported case of Barber Say syndrome is described with special attention to dental manifestations. A 7-year-old female with multiple congenital anomalies such mammary gland hypoplasia, hypertrichosis, ectropion, and redundant skin was seen at the School of Dentistry of the University of Sao Paulo. Oral examination revealed macrostomia, broad alveolar ridges, gingival fibromatosis, taurodontism, delayed tooth eruption, and malocclusion. Dental treatment included gingivoplasty and orthodontic treatment. (C) 2010 Wiley-Liss, Inc.

Tissue-specific induction of SOCS gene expression by PRL

The mechanisms whereby tissue sensitivity to PRL is controlled are not well understood. Here we report that expression of mRNA and protein for members of the SOCS/CIS/JAB family of cytokine signaling inhibitors is increased by PRL administration in ovary and adrenal gland of the lactating rat deprived of circulating PRL and pups for 24 h but not in mammary gland. Moreover, suckling increases SOCS mRNA in the ovary but not in the mammary gland of pup-deprived rats. Deprivation of PRL and pups for 48 h allows the mammary gland to induce SOCS genes in response to PRL administration, and this is associated with a decrease in basal SOCS-3 mRNA and protein expression to the level seen in other tissues, suggesting that SOCS-3 induced refractoriness related to filling of the gland. In reporter assays, SOCS-1, SOCS-3, and CIS, but not SOCS-2, are able to inhibit transactivation of the STAT 5-responsive beta -lactoglobulin promoter in transient transfection assays. Moreover, suckling results in loss of ovarian and adrenal responsiveness to PRL administered 2 h after commencement of suckling, as determined by STAT 5 gel shift assay. Immunohistochemistry was used to localize the cellular sites of SOCS-3 and CIS protein expression in the ovary and adrenal gland. We propose that induced SOCS-1, SOCS-3, and CIS are actively involved in the cellular inhibitory feedback response to physiological PRL surges in the corpus luteum and adrenal cortex during lactation, but after pup withdrawal, the mammary gland is rendered unresponsive to PRL by increased levels of SOCS-3.

Characterization of baboon (Papio hamadryas) milk proteins

The major proteins of baboon milk were identified as beta -lactoglobulin (beta LG), alpha -lactalbumin (alpha LA), lysozyme, lactoferrin, casein, and albumin by immobiline isoelectric focusing, SDS-PAGE, immunoblotting of gels with rabbit antisera to human alpha LA, lysozyme, and albumin and bovine beta LG and casein, and N-terminal sequencing of proteins blotted from gels. The first 30 N-terminal residues of baboon polymorphism at residue 2. The complete cDNA sequence and derived amino acid composition of beta LG were elucidated using RT-PCR amplification of poly(A)(+) mRNA purified from lactating mammary gland. Baboon beta LG identified to date. beta LG and alpha LA polymorphisms with three (A, B, and C) and two (A and B) variants, respectively, were detected by immobiline IEF, pH 4-6, of individual baboon milk samples at varying stages of lactation.

Proteomics of breast cancer for marker discovery and signal pathway profiling

Breast cancer is the most common form of cancer among women and the identification of markers to discriminate tumorigenic from normal cells, as well as the different stages of this pathology, is of critical importance. Two-dimensional electrophoresis has been used before for studying breast cancer, but the progressive completion of human genomic sequencing and the introduction of mass spectrometry, combined with advanced bioinformatics for protein identification, have considerably increased the possibilities for characterizing new markers and therapeutic targets. Breast cancer proteomics has already identified markers of potential clinical interest (such as the molecular chaperone 14-3-3 sigma) and technological innovations such as large scale and high throughput analysis are now driving the field. Methods in functional proteomics have also been developed to study the intracellular signaling pathways that underlie the development of breast cancer. As illustrated with fibroblast growth factor-2, a mitogen and motogen factor for breast cancer cells, proteomics is a powerful approach to identify signaling proteins and to decipher the complex signaling circuitry involved in tumor growth. Together with genomics, proteomics is well on the way to molecularly characterizing the different types of breast tumor, and thus defining new therapeutic targets for future treatment.

Cytokine regulation of syndecan-1 and-2 gene expression in human periodontal fibroblasts and osteoblasts

Cell-surface proteoglycans participate in several biological functions including interactions with a variety of growth factors and cytokines. Regulation of syndecan-1 and -2 gene expression was investigated in human periodontal ligament fibroblasts (PDLF), osteoblasts (OB) and gingival fibroblasts (GF), in response to platelet-derived growth factor (PDGF-BB), transforming growth factor (TGF-beta(1)), and interleukin (IL-1beta) by Northern blot analyses. We also compared the effect of PDGF-BB and TGF-beta(1), separately and in combination, in the prolonged presence of IL-1beta on the expression of both syndecan genes. The results demonstrated that the three cell lines regulated the expression of syndecan-1 and -2 in response to growth factors and cytokines in different manners. These cell lines increased syndecan-1 mRNA levels in response to either PDGF-BB or TGF-beta(1) and decreased levels in response to IL-1beta. The effect of IL-1beta on syndecan-1 mRNA synthesis was partially reversed after adding PDGF-BB and TGF-beta(1), separately or in combination, in the presence of IL-1beta. In contrast, syndecan-2 mRNA level was markedly upregulated in response to either TGF-beta(1) or IL-1beta in OB when compared with the other two cell lines. However, the stimulatory effect of TGF-beta(1) on syndecan-2 mRNA production in OB was abolished in the prolonged presence of IL-1beta. These findings lend support to the notion that syndecan-1 and syndecan-2 have distinct functions which correlate with their source and functions within the periodontium.

Differential expression and distribution of syndecan-1 and-2 in periodontal wound healing of the rat

Cell-surface proteoglycans participate in several biological functions including interactions with adhesion molecules, growth factors and a variety of other effector molecules. Accordingly, these molecules play a central role in various aspects of cell-cell and cell-matrix interactions. To investigate the expression and distribution of the cell surface proteoglycans, syndecan-1 and -2, during periodontal wound healing, immunohistochemical analyses were carried out using monoclonal antibodies against syndecan-1, or -2 core proteins. Both syndecan-1 and -2 were expressed and distributed differentially at various stages of early inflammatory cell infiltration, granulation tissue formation, and tissue remodeling in periodontal wound healing. Expression of syndecan-1 was noted in inflammatory cells within and around the fibrin clots during the earliest stages of inflammatory cell infiltration. During granulation tissue formation it was noted in fibroblast-like cells and newly formed blood vessels. Syndecan-1 was not seen in newly formed bone or cementum matrix at any of the time periods studied. Syndecan-1 expression was generally less during the late stages of wound healing but was markedly expressed in cells that were close to the repairing junctional epithelium. In contrast, syndecan-2 expression and distribution was not evident at the early stages of inflammatory cell infiltration. During the formation of granulation tissue and subsequent tissue remodeling, syndecan-2 was expressed extracellularly in the newly formed fibrils which were oriented toward the root surface. Syndecan-2 was found to be significantly expressed on cells that were close to the root surface and within the matrix of repaired cementum covering root dentin as well as at the alveolar bone edge. These findings indicate that syndecan-1 and -2 may have distinctive functions during wound healing of the periodontium. The appearance of syndecan-1 may involve both cell-cell and cell-matrix interactions, while syndecan-2 showed a predilection to associate with cell-matrix interactions during hard tissue formation.

Arsenic inhibits the repair of DNA damage induced by benzo(a)pyrene

In order to study the effect of arsenic on DNA damage, Sprague-Dawley rats were dosed with sodium arsenite (10 mg/kg) with or without 800 mug of benzo(a)pyrene (BP) by intramammilary injection. The animals were sacrificed on day 1, 3, 5, 10 and 27 and the mammary gland tissues were collected for DNA adduct measurement using a P-32 post-labeling assay. Animals dosed with arsenic alone did not show any DNA adducts. DNA adduct levels in rats dosed with BP alone reached a maximum level by day 5, reducing to 13% of this level by day 27. Adduct levels in rats dosed with arsenic and BP also reached a maximum by day 5 but only 80% of the level observed in the BP group. However, 84% of this amount still remained by day 27. The First Nucleotide Change (FNC) technique was used for the screening of 115 samples of various tissues from mice that had been chronically exposed to sodium arsenate for over 2 years revealed that inorganic arsenic did not attack the two putative hotspots (codons 131 and 154) of the hOGG1 gene. These results support the hypothesis that arsenic exerts its biological activity through DNA repair inhibition. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Peroxisome proliferator-activated receptor beta expression in human breast epithelial cell lines of tumorigenic and non-tumorigenic origin

Peroxisome proliferator-activated receptor beta (PPARbeta) is a member of the nuclear hormone receptor superfamily and is a ligand activated transcription factor. although the precise genes that it regulates and its physiological and pathophysiological role remain unclear. In view of the association of PPARbeta with colon cancer and increased mRNA levels of PPARbeta in colon tumours we sought in this study to examine the expression of PPARbeta in human breast epithelial cells of tumorigenic (MCF-7 and MDA-MB-231) and non-tumorigenic origin (MCF-10A). Using quantitative RT-PCR we measured PPARbeta mRNA levels in MCF-7. MDA-MB-231 and MCF-10A cells at various stages in culture. After serum-deprivation, MDA-MB-231 and MCF-10A cells had a 4.2- and 3.8-fold statistically greater expression of PPARbeta compared with MCF-7 cells. The tumorigenic cell lines also exhibited a significantly greater level of PPARbeta mRNA after serum deprivation compared with subconfluence whereas such an effect was not observed in non-tumorigenic MCF-10A cells. The expression of PPARbeta was inducible upon exposure to the PPARbeta ligand bezafibrate. Our results suggest that unlike colon cancer. PPARbeta overexpression is not an inherent property of breast cancer cell lines. However, the dynamic changes in PPARbeta mRNA expression and the ability of PPARbeta in the MCF-7 cells to respond to ligand indicates that PPARbeta may play a role in mammary gland carcinogenesis through activation of downstream genes via endogenous fatty acid ligands or exogenous agonists. (C) 2002 Elsevier Science Ltd. All rights reserved.

Peroxisome proliferator-activated receptor alpha in the human breast cancer cell lines MCF-7 and MDA-MB-231

Peroxisome proliferator-activated receptor (PPAR) alpha is a ligand-activated transcription factor that has been linked with rodent hepatocarcinogenesis. It has been suggested that PPARalpha mRNA expression levels are an important determinant of rodent hepatic tumorigenicity. Previous work in rat mammary gland epithelial cells showed significantly increased PPARalpha mRNA expression in carcinomas, suggesting the possible role of this isoform in rodent mammary gland carcinogenesis. In this study we sought to determine whether PPARalpha is expressed and dynamically regulated in human breast cancer MCF-7 and MDA-MB-231 cells. Having established the presence of PPARalpha in both cell types, we then examined the consequence of PPARa activation, by its ligands Wy-14,643 and clofibrate, on proliferation. With real-time reverse transcriptase-polymerase chain reaction, we showed that PPARalpha mRNA was dynamically regulated in MDA-MB-231 cells and that PPARalpha activation significantly increased proliferation of the cell line. In contrast, PPARalpha expression in MCF-7 cells did not change with proliferation during culture and was present at significantly lower levels than in MDA-MB-231 cells. However, PPARalpha ligand activation still significantly increased the proliferation of MCF-7 cells. The promotion of proliferation in breast cancer cell lines following PPARalpha activation was in stark contrast to the effects of PPARgamma-activating ligands that decrease proliferation in human breast cancer cells. our results established the presence of PPARalpha in human breast cancer cell lines and showed for the first time that activation of PPARalpha in human breast cancer cells promoted proliferation. Hence, this pathway may be significant in mammary gland tumorigenesis. (C) 2002 Wiley-Liss, Inc.

Quantificação da difusão na ressonância magnética da mama: ADC e Kurtosis

Mestrado em Radiações Aplicadas às Tecnologias da Saúde. Área de especialização: Ressonância Magnética

Quantificação por imagem ponderada em difusão (DWI) das lesões tumorais da mama

Introdução – A técnica de Difusão por Ressonância Magnética (RM), ao avaliar o movimento das moléculas de água nos tecidos, permite inferir sobre a arquitetura dos mesmos, em particular relativamente à celularidade, volume celular e permeabilidade das membranas. O Coeficiente de Difusão Aparente (ADC) é um parâmetro quantificável da imagem ponderada em difusão (DWI). A sua análise poderá fornecer informação clínica adicional sobre estas lesões, sobretudo em relação à sua caracterização histológica. Objetivos – Caracterizar e diferenciar tipos e alguns subtipos de lesões mamárias através da análise do ADC. Metodologia – 20 Mulheres com 23 lesões mamárias foram submetidas a RM mamária: 3 lesões benignas (3 Fibroadenomas-FA) e 20 malignas (16 Carcinomas Ductais Invasivos-CDI, 2 Carcinomas Ductais In Situ-CDIS e 2 Carcinomas Invasivos Lobulares-CLI). Num equipamento 1.5T aplicou-se uma sequência de Difusão (b=0,50,250,500,750,1000 s/mm2). Obteve-se o ADC através do ajuste exponencial da intensidade de sinal das lesões em função do valor de b, fazendo-se corresponder os valores de ADC à respetiva caracterização histológica e posterior comparação com a literatura. Resultados e Discussão – As lesões malignas apresentaram ADCs significativamente (p=0,014) inferiores [(0,94±0,22)x10-3 mm2/s] aos das benignas [(1,43±0,25)x10-3 mm2/s]. A justificação pode residir no aumento da celularidade e consequente restrição da Difusão que se observa nas lesões malignas. Os CDI apresentaram ADCs baixos [(0,88±0,17)x10-3 mm2/s], enquanto que os CDIS apresentaram ADCs mais elevados [(1,33±0,29)x10-3 mm2/s]. Estes resultados estão de acordo com o facto dos CDIS estarem limitados aos ductos mamários, mantendo-se menos alterada a estrutura do tecido adjacente e resultando numa menor restrição à difusão que nos CDI. Verificaram-se diferenças significativas entre FA e CDI (p=0,010) e entre CDI e CDIS (p=0,049). Conclusões – O ADC possibilita a diferenciação entre lesões mamárias benignas e malignas, bem como entre alguns tipos histológicos. O desenvolvimento deste conceito pode representar um avanço no papel da RM na avaliação destas neoplasias. ABSTRACT - Introduction – The Magnetic Resonance (MR) diffusion technique measures the movement of water molecules in tissues. Therefore, it provides useful information about tissue architecture, specially regarding tissue cellularity, cell volume and membrane permeability. The quantification of diffusion weighted imaging (DWI) data is done by measuring the so-called. Apparent Diffusion Coefficient (ADC). This parameter provides additional clinical information about breast lesions, and potentially allows for in-vivo histological characterization. Purpose – To characterize and differentiate breast lesions through ADC analysis. Methodology – The study comprised 20 women, with 23 breast lesions: 3 benign lesions - 3 Fibroadenomas (FA); and 20 malignant - 16 Invasive Ductal Carcinomas (CDI), 2 Ductal Carcinomas In Situ (CDIS), 2 Invasive Lobular Carcinoma (CLI). On a 1.5T equipment a diffusion-weighted sequence with 6 b-values (b=0,50,250,500,750,1000 s/mm2) was used to examine the patients. ADC was obtained by fitting an exponential to data of lesion signal intensity vs. b values. A correspondence of ADC values to histological lesion characterization was done and finally, the results were comparison with the literature. Results and Discussion – Malignant lesions showed inferior ADCs significantly (p=0.014) lower ((0.94±0.22)x10-3 mm2/s) than the benign lesions ((1.43±0.25)x10-3 mm2/s). This may be associated to increasead cellularity in malignant lesions, which result in higher tissue restriction to diffusion. CDI showed low ADC values ((0.88±0.17)x10-3 mm2/s), while the CDIS showed higher ADCs ((1.33±0.29)x10-3 mm2/s). These results agree with the fact that CDIS are limited to mammary ducts, maintaining a less altered neighboring tissue structure, which results in a lower restriction to diffusion than observed in CDI. Significant differences between FA and CDI (p=0.010) and between CDI and CDIS (p=0.049) were observed. Conclusion – The ADC parameter is able to differentiate between malignant and benign breast lesions, as well as between some histological types.

Análise quantitativa da saturação de gordura em ressonância magnética mamária: comparação das técnicas SPAIR e DIXON

Introdução – A ressonância magnética (RM) mamária com injeção de contraste é uma ferramenta indispensável para a caracterização de lesões mamárias. Para tal, é fulcral uma boa capacidade de saturação de gordura de forma a delimitar as lesões observadas. Este trabalho tem como objetivo analisar quantitativa e qualitativamente as técnicas de saturação de gordura em RM mamária, nomeadamente as técnicas SPAIR e Dixon. Metodologia – Utilizou-se um equipamento de RM de 1,5T com bobina específica para a mama. Aplicou-se, durante o exame, uma sequência adicional em que a técnica de saturação de gordura utilizada foi a Dixon. Posteriormente foram analisadas as imagens obtidas com as técnicas SPAIR e Dixon e colocadas regiões de interesse (ROI) na lesão, na glândula mamária, no tecido adiposo e no ar. Estes valores de sinal obtidos foram registados e calcularam-se as relações sinal-ruído (SNR), contraste-ruído (CNR) e uniformidade de saturação de gordura para as mesmas estruturas a analisar e nas distintas sequências. Resultados – Verificou-se que a técnica de supressão de gordura Dixon apresenta resultados quantitativos e qualitativos superiores à técnica SPAIR. Conclusões – Recomenda-se a utilização da técnica Dixon para saturação de gordura nas sequências de estudo dinâmico com agente de contraste, especificamente nos estudos de RM mamária.