Alexandra Rêma

CD117 immunoexpression in canine mast cell tumours: correlations with pathological variables and proliferation markers

BackgroundCutaneous mast cell tumours are one of the most common neoplasms in dogs and show a highly variable biologic behaviour. Several prognosis tools have been proposed for canine mast cell tumours, including histological grading and cell proliferation markers. CD117 is a receptor tyrosine kinase thought to play a key role in human and canine mast cell neoplasms. Normal (membrane-associated) and aberrant (cytoplasmic, focal or diffuse) CD117 immunoexpression patterns have been identified in canine mast cell tumours. Cytoplasmic CD117 expression has been found to correlate with higher histological grade and with a worsened post-surgical prognosis. This study addresses the role of CD117 in canine mast cell tumours by studying the correlations between CD117 immunoexpression patterns, two proliferation markers (Ki67 and AgNORs) histological grade, and several other pathological variables.ResultsHighly significant (p < 0,001) correlations were found between CD117 immunostaining patterns and histological grade, cell proliferation markers (Ki67, AgNORs) and tumoral necrosis. Highly significant (p < 0,001) correlations were also established between the two cellular proliferation markers and histological grade, tumour necrosis and epidermal ulceration. A significant correlation (p = 0.035) was observed between CD117 expression patterns and epidermal ulceration. No differences were observed between focal and diffuse cytoplasmic CD117 staining patterns concerning any of the variables studied.ConclusionThese findings highlight the key role of CD117 in the biopathology of canine MCTs and confirm the relationship between aberrant CD117 expression and increased cell proliferation and higher histological grade. Further studies are needed to unravel the cellular mechanisms underlying focal and diffuse cytoplasmic CD117 staining patterns, and their respective biopathologic relevance.

Effects of Human Mesenchymal Stem Cells Isolated from Wharton's Jelly of the Umbilical Cord and Conditioned Media on Skeletal Muscle Regeneration Using a Myectomy Model

Skeletal muscle has good regenerative capacity, but the extent of muscle injury and the developed fibrosis might prevent complete regeneration. The in vivo application of human mesenchymal stem cells (HMSCs) of the umbilical cord and the conditioned media (CM) where the HMSCs were cultured and expanded, associated with different vehicles to induce muscle regeneration, was evaluated in a rat myectomy model. Two commercially available vehicles and a spherical hydrogel developed by our research group were used. The treated groups obtained interesting results in terms of muscle regeneration, both in the histological and in the functional assessments. A less evident scar tissue, demonstrated by collagen type I quantification, was present in the muscles treated with HMSCs or their CM. In terms of the histological evaluation performed by ISO 10993-6 scoring, it was observed that HMSCs apparently have a long-term negative effect, since the groups treated with CM presented better scores. CM could be considered an alternative to the in vivo transplantation of these cells, as it can benefit from the local tissue response to secreted molecules with similar results in terms of muscular regeneration. Searching for an optimal vehicle might be the key point in the future of skeletal muscle tissue engineering.

Local iron homeostasis in the breast ductal carcinoma microenvironment.

BackgroundWhile the deregulation of iron homeostasis in breast epithelial cells is acknowledged, iron-related alterations in stromal inflammatory cells from the tumor microenvironment have not been explored.MethodsImmunohistochemistry for hepcidin, ferroportin 1 (FPN1), transferrin receptor 1 (TFR1) and ferritin (FT) was performed in primary breast tissues and axillary lymph nodes in order to dissect the iron-profiles of epithelial cells, lymphocytes and macrophages. Furthermore, breast carcinoma core biopsies frozen in optimum cutting temperature (OCT) compound were subjected to imaging flow cytometry to confirm FPN1 expression in the cell types previously evaluated and determine its cellular localization.ResultsWe confirm previous results by showing that breast cancer epithelial cells present an ‘iron-utilization phenotype’ with an increased expression of hepcidin and TFR1, and decreased expression of FT. On the other hand, lymphocytes and macrophages infiltrating primary tumors and from metastized lymph nodes display an ‘iron-donor’ phenotype, with increased expression of FPN1 and FT, concomitant with an activation profile reflected by a higher expression of TFR1 and hepcidin. A higher percentage of breast carcinomas, compared to control mastectomy samples, present iron accumulation in stromal inflammatory cells, suggesting that these cells may constitute an effective tissue iron reservoir. Additionally, not only the deregulated expression of iron-related proteins in epithelial cells, but also on lymphocytes and macrophages, are associated with clinicopathological markers of breast cancer poor prognosis, such as negative hormone receptor status and tumor size.ConclusionsThe present results reinforce the importance of analyzing the tumor microenvironment in breast cancer, extending the contribution of immune cells to local iron homeostasis in the tumor microenvironment context.

Thyroid disruption in the lizard Podarcis bocagei exposed to a mixture of herbicides: a field study

Pesticide exposure has been related with thyroid disrupting effects in different vertebrate species. However, very little is known about the effects of these compounds in reptiles. In the Mediterranean area, lacertid lizards are the most abundant vertebrate group in agroecosystems, and have been identified as potential model species for reptile ecotoxicology. The aim of this study was to understand if the herbicides applied in corn fields have thyroid disruptive effects in the lizard Podarcis bocagei. Adult male lizards were captured in north-western Portugal in corn fields treated with herbicides (exposed sites), and in organic agricultural fields (reference sites). Thyroid and male gonad morphology and functionality, and testosterone levels were investigated through histological, immunohistochemical and biochemical techniques. Lizards from exposed locations displayed thyroid follicular lumens with more reabsorption vacuoles and significantly larger follicular area than those from reference fields. Furthermore, testes of lizards from exposed locations had significantly larger seminiferous tubule diameters, significantly higher number of spermatogenic layers and displayed an up-regulation of thyroid hormone receptors when compared with lizards from reference areas. These findings strongly suggest that the complex mixture of herbicides that lizards are exposed to in agricultural areas have thyroid disrupting effects which ultimately affect the male reproductive system. Alachlor, which has demonstrated thyroid effects in mammals, may be largely responsible for the observed effects.

Endocrine disruption effects of p,p'-DDE on juvenile zebrafish.

The persistent organic pollutant p,p′‐DDE, the major metabolite of the insecticide DDT, has displayed evidence of endocrine disruption through the inhibition of androgen binding to androgen receptors in different species. Although p,p′‐DDE was continuously detected in wild fish with abnormal gonad development such as intersex, little is known about its mode of action during gonad development in fish. To elucidate the potential endocrine effects of this pollutant in zebrafish (Danio rerio), juveniles (30 days post hatch) were exposed to p,p′‐DDE during the critical window of sexual differentiation. Fish were exposed to sublethal concentrations ranging from 0.01 to 20 µg l–1 over 14 days and were maintained in control water for an additional 4 months. As core endpoints, the vitellogenin (vtg) concentration was measured at the end of exposure, and sex ratio and the gonadosomatic index were assessed 4 months after the end of exposure. An increase in vtg production in whole body homogenate was observed in fish exposed to 0.2 and 2.0 µg l–1 p,p′‐DDE. No significant differences were displayed in morphological parameters such as the gonadosomatic index of males and females or sex ratio. However, exposed females presented histopathological changes that include the reduction of the number of mature oocytes, which might impair their successful reproduction. These results demonstrate the ability of p,p′‐DDE to cause endocrine disruption in zebrafish exposed during gonad differentiation of juvenile specimens. Furthermore, vtg induction by p,p′‐DDE in juvenile zebrafish arises as a predictive marker for adverse effects of this DDT metabolite on the ovarian function of female zebrafish. Copyright

A new methodology for the improvement of diagnostic immunohistochemistry in canine veterinary pathology: automated system using human monoclonal and polyclonal antibodies

The authors describe their experience with an automated immunohistochemical system applied to canine tissue samples. Twenty human cellular markers specific monoclonal and polyclonal antibodies and two different antigen retrieval methods were used in normal and neoplastic breast tissue, as well as skin samples obtained from female dogs of pure and mixed breeds. The antibodies tested were the most frequently used in human and veterinary medicine studies, employed with diagnostic purposes in breast pathology, as well as in cancer research. Most of them may be used to study other normal and abnormal tissues and included cytokeratins, progesterone receptor, c-erbB2, p53, MIB-1, PCNA, EMA, vimentin, desmin, a-actin, S-100, pan-cadherin, and E-cadherin. The results demonstrated that using an automated staining system it is possible to use different human markers in veterinary pathology. The advantages of automated immunohistochemistry are improved quality, reproducibility, speed, and standardisation.

Reduced expression of claudin-2 is associated with high histological grade and metastasis of feline mammary carcinomas.

Claudins (CLDNs) are a family of tight junction (TJ) proteins that play an important role in maintaining cell polarity, in controlling paracellular ion flux and in regulating cell proliferation and differentiation. There is a growing body of evidence that associates changes in CLDN expression with the development of human breast cancer. In the present study CLDN-2 expression was examined immunohistochemically in samples of normal feline mammary tissue (n = 5) and mammary carcinomas (n = 52), including metastatic lesions (n = 29). Seventy-seven percent of carcinomas showed reduced CLDN-2 expression compared with that observed in normal mammary gland. Reduced expression of CLDN-2 was significantly associated with a high histological grade of carcinoma (P = 0.011), with 88.6% of grade II/III carcinomas showing decreased expression. Furthermore, CLDN-2 down-regulation was significantly associated with metastatic disease (P = 0.0027), with 93.1% of cases with signs of metastasis showing decreased expression of this protein. CLDN-2 may constitute a molecular marker for identification of a subgroup of feline mammary carcinomas characterized by high histological grade and the development of metastasis.

Development of an embryotoxicity test for Enchytraeus crypticus – The effect of Cd

The existing standard enchytraeid reproduction test (ERT) concerns the assessment of effects on survival and reproduction. In the present study we optimized and propose an embryotoxicity test using Enchytraeus crypticus. Cadmium (Cd) was used as a test substance. Endpoints evaluated were embryo development, number of embryonic structures, Calcium (Ca) channels quantification and hatching success with macroscopic monitoring, histological and immunohistochemistry analysis. Results showed that Cd is embryotoxic for this species, causing a decrease in the hatching success (EC50=3.1mg/kg), a delay or disruption in formation of embryonic structures depending on concentrations (<5mg Cd/kg or ⩾16mg Cd/kg). Results from immunohistochemistry suggest a competitive binding between Cd and Ca for Ca channels, resulting in changes in Ca homeostasis. The use of the E. crypticus embryotoxicity test with the combination of histological and immunohistological tools provided a good option towards mechanistic information enhancing the importance of these tests to evaluate the hazard of chemicals and possible use in risk assessment.

Immunohistochemical Expression of Cyclooxygenase‐2 (COX‐2) in Feline Endometrial Adenocarcinoma and in Normal and Hyperplastic Endometria

Cyclooxygenase-2 (COX-2) is overexpressed in several human and animal neoplasms, including the human endometrial carcinoma. It has been suggested as a prognostic marker and a potential therapeutic target. This study aimed to (i) clarify histological aspects of feline endometrial adenocarcinomas (FEA) of the papillary serous type and (ii) characterize COX-2 immunohistochemical expression in normal, hyperplastic and neoplastic endometrium in this species. Archived paraffin-embedded tissue samples of 33 FEA, eight cystic endometrial hyperplasias (CEHs) and 21 samples of normal, healthy endometrium in the follicular (FS; n = 10) and luteal (LS; n = 11) stages were evaluated. Histological evaluation of haematoxylin and eosin-stained sections of the FEA revealed a papillary proliferation of neoplastic cells of serous type, accompanied by clear and multinucleated cells. Other architectural arrangements mainly included solid and tubular growth. Randomly distributed areas of necrosis within the tumours were commonly observed. Invasion of the myometrium, of the serosa and of the vascular and/or lymphatic vessels was not constant features. The mean number of mitoses was higher in FEA compared to non-neoplastic endometrium. COX-2 scores were lower in FEA (p = 0.003) and CEH (p = 0.05) when compared to normal epithelium (NE). The loss of the membrane apical reinforcement in epithelial cells was observed in FEA samples, which was accompanied by the dislocation of COX-2 labelling into the cytoplasm and the perinuclear area; in contrast, in epithelial cells in the healthy and hyperplastic endometria, the immunoreaction showed the characteristic pattern of apical membrane reinforcement, suggestive of the membrane polarization. COX-2 epithelial scores were higher in the FS than in the LS. No differences were found in stromal COX-2 expression between normal, CEH and FEA groups, but it was higher in the LS than in the FS. In summary, loss of COX-2 compartmentalization in neoplastic epithelial cells might be one of the molecular events underlying endometrial carcinogenesis.

An immunohistochemical study on the expression of sex steroid receptors, Ki-67 and cytokeratins 7 and 20 in feline endometrial adenocarcinomas

BackgroundEndometrial adenocarcinomas are a rare type of tumour in cats. Though different morphologies have been reported, the most frequent histological type of feline endometrial adenocarcinoma (FEA) is the papillary serous. Characterization of molecular markers expression in FEA may contribute to clarify the pathogenesis of these tumours and to assess the differences between normal endometrium and FEA regarding the expression pattern of several proteins. Therefore, this study aimed to evaluate the immunohistochemical profile of a wide panel of antibodies (specific for ER-α, PR, Ki-67, CK7 and CK20) in twenty-four cases of FEA. Comparisons were made between FEA and feline normal cyclic endometrium in follicular (n = 13) and luteal (n = 10) stages. Except for Ki-67, all other molecular markers were assessed independently for the intensity of immunolabeling and for the percentage of cells expressing the protein.ResultsThis study showed that in FEA a loss of expression occurs for ER-α (P ≤ 0.0001) and less markedly also for PR. The lost in sex steroid receptors concerns a decrease in both the proportion of labelled cells and the intensity of immunolabelling (P = 0.002 and P = 0.024, respectively). Proliferative activity, estimated via Ki-67 immunoreaction, significantly increased in FEA as compared to normal endometrium (P ≤ 0.0001). Feline endometrial adenocarcinomas maintained the CK7+/CK20+ status of normal endometrium. However, FEA showed decreased CK7 intensity of labelling compared to normal endometria (P ≤ 0.0001) and loss of CK20 expression, both in intensity (P ≤ 0.0001) and in percentage of positive cells (P = 0.01), compared to normal tissues.ConclusionsData gathered in this study suggest that proliferation in FEA accompanies ER-α down-regulation, possibly following activation of pathways mediated by local growth factors. Moreover, FEA retains combined expression of CK7 and CK20, as evidenced in normal endometrial epithelia, although a decrease in CK7 expression was observed.