Lorenzo Ressel

Characterization and potential applications of progenitor-like cells isolated from horse amniotic membrane.

The aim of this work was to isolate, for the first time, progenitor‐like cells from the epithelial (AECs) and mesenchymal (AMCs) portions of the horse amniotic membrane, and to define the biological properties of these cells. AECs displayed polygonal epithelial morphology, while AMCs were fibroblast‐like. Usually, six to eight passages were reached before proliferation decreased, with 13.08 and 26.5 cell population doublings attained after 31 days for AECs and AMCs, respectively. Immunocytochemical studies performed at passage 3 (P3) showed that both cell populations were positive for the expression of specific embryonic markers (TRA‐1‐60, SSEA‐3, SSEA‐4 and Oct‐4). Meanwhile, RT–PCR performed at P1 and P5 showed expression of mesenchymal stem/stromal cell markers (CD29, CD105, CD44 and CD166) with negativity for CD34 at P1, although this marker began to be expressed by P5. The cells also expressed MHC‐I at both P1 and P5, but lacked MHC‐II expression at P1. Both AECs and AMCs demonstrated high plasticity, differentiating in vitro toward the osteogenic, adipogenic, chondrogenic and neurogenic lineages. Equine amnion‐\derived cells could also be frozen and recovered without loss of their functional integrity in terms of morphology, presence of specific stemness markers and differentiation ability, although the renewal capacity was lower than that observed for freshly isolated cells. To investigate potential therapeutic effects and cell tolerance in vivo, horse amnion‐derived cells were allogeneically injected into three horses with tendon injuries, resulting in a quick reduction in tendon size and ultrasonographic cross‐sectional area measurements. These results suggest that horse amnion‐derived cells may be useful for cell therapy applications. Copyright

Amniotic Membrane Application Reduces Liver Fibrosis in a Bile Duct Ligation Rat Model

Biliary fibrosis and resultant cirrhosis are among the most common outcomes of chronic liver diseases. Currently, liver transplantation remains the only effective treatment. In seeking alternative therapeutic approaches, we focused on the potential use of the human amniotic membrane (AM). Indeed, AM has gained increasing importance for its antiscarring, anti-inflammatory, and wound-healing properties, as well as for the multipotent differentiation ability and immunomodulatory features of AM-derived cells. Intriguingly, we have recently demonstrated that placenta-derived cells reduce lung fibrosis in bleomycin-treated mice, and that AM patches reduce postischemic cardiac injury in rats. Hence, we have now investigated the effects of human AM on biliary fibrosis induced in rats through the bile duct ligation (BDL) procedure. A fragment of human AM was applied onto the liver surface after BDL and the effects on fibrosis establishment and progression were evaluated at different time points in comparison with fibrosis progression in control BDL rats. The degree of liver fibrosis was first assessed by the semiquantitative Knodell scoring system and, thereafter, by digital image morphometric analysis to quantify the area occupied by ductular reaction, activated myofibroblasts, and collagen deposition. We demonstrated a significant reduction in the severity of BDL-induced fibrosis in AM-treated rats. Indeed, while fibrosis progressed rapidly in control BDL rats, leading to cirrhosis within 6 weeks, AM-treated rats showed confined fibrosis at the portal/periportal area with no signs of cirrhosis, and a reduction in collagen deposition to about 50% of levels observed in control BDL rats. In addition, the AM was able to significantly slow the gradual progression of the ductular reaction and reduce, at all time points, the area occupied by activated myofibroblasts. These findings suggest that human AM, when applied as a patch onto the liver surface, might inhibit fibrosis progression in BDL-injured livers, and could protect against hepatic damage associated with fibrotic degeneration.

Conditioned medium from amniotic mesenchymal tissue cells reduces progression of bleomycin-induced lung fibrosis

Background and aims We have demonstrated recently that transplantation of placental membrane-derived cells reduces bleomycin-induced lung fibrosis in mice, despite a limited presence of transplanted cells in host lungs. Because placenta-derived cells are known to release factors with potential immunomodulatory and trophic activities, we hypothesized that transplanted cells may promote lung tissue repair via paracrine-acting molecules. To test this hypothesis, we examined whether administration of conditioned medium (CM) generated from human amniotic mesenchymal tissue cells (AMTC) was able to reduce lung fibrosis in this same animal model. Methods Bleomycin-challenged mice were either treated with AMTC-CM or control medium, or were left untreated (Bleo group). After 9 and 14 days, the distribution and severity of lung fibrosis were assessed histologically with a scoring system. Collagen deposition was also evaluated by quantitative image analysis. Results At day 14, lung fibrosis scores in AMTC-CM-treated mice were significantly lower (P<0.05) compared with mice of the Bleo group, in terms of fibrosis distribution [1.0 (interquartile range, IQR 0.9) versus 3.0 (IQR 1.8)], fibroblast proliferation [0.8 (IQR 0.4) versus 1.6 (IQR 1.0)], collagen deposition [1.4 (IQR 0.5) versus 2.0 (IQR 1.2)] and alveolar obliteration [2.3 (IQR 0.8) versus 3.2 (IQR 0.5)]. No differences were observed between mice of the Bleo group and mice treated with control medium. Quantitative analysis of collagen deposition confirmed these findings. Importantly, AMTC-CM treatment significantly reduced the fibrosis progression between the two observation time-points. Conclusions This pilot study supports the notion that AMTC exert anti-fibrotic effects through release of yet unknown soluble factors.

Expression of Vascular Endothelial Growth Factor in Canine Inflammatory and Non-inflammatory Mammary Carcinoma

Inflammatory mammary carcinoma (IMC) is the most aggressive type of mammary tumour in the dog and has been proposed as a model for human inflammatory breast cancer. The aim of this study was to investigate angiogenesis in canine IMC by immunohistochemical assessment of the expression of vascular endothelial growth factor (VEGF). Tissues from 19 cases of IMC were compared with tissues from 27 cases of invasive mammary carcinoma without inflammation (non-IMC). Immunohistochemical expression of oestrogen receptor (ER), progesterone receptor (PR) and HER-2 receptor was also assessed. VEGF was strongly expressed in all IMCs and the percentage of VEGF-immunoreactive tumour cells was significantly higher in IMC than in non-IMC (P=0.02). There was no difference in HER-2 receptor expression between IMC and non-IMC, and no IMC expressed ER or PR. These results suggest that VEGF may contribute to the high angiogenic phenotype of canine IMC and that this expression may underlie the tendency towards local and systemic metastasis of these tumours.

Reduced PTEN Protein Expression and Its Prognostic Implications in Canine and Feline Mammary Tumors

Phosphatase and tensin homolog (PTEN) belongs to the group of gatekeeper tumor suppressor genes and is involved in multiple mechanisms leading to cellular defense against neoplastic transformation and progression. Twenty-four dogs and 17 cats were submitted to a 2-year follow-up study, and clinicopathologic features were recorded and compared with immunohistochemical PTEN staining. PTEN-negative status occurred in 33% of canine and 76% of feline mammary carcinomas. In canine mammary carcinomas, there was a significant (P < .05) correlation between loss of PTEN protein expression and simple carcinoma histotype, lymphatic vessel invasion, lymph node metastases, distant organ metastases, tumor dedifferentiation, tumor recurrence, and shorter overall survival. In feline mammary tumors, a significant correlation between loss of PTEN protein expression and lymphatic vessel invasion was found. Loss of PTEN expression could be a useful prognostic marker in canine mammary carcinomas.

Integrated transcriptomic and proteomic analyses uncover regulatory roles of Nrf2 in the kidney

The transcription factor Nrf2 exerts protective effects in numerous experimental models of acute kidney injury, and is a promising therapeutic target in chronic kidney disease. To provide a detailed insight into the regulatory roles of Nrf2 in the kidney, we performed integrated transcriptomic and proteomic analyses of kidney tissue from wild-type and Nrf2 knockout mice treated with the Nrf2 inducer methyl-2-cyano-3,12-dioxooleano-1,9-dien-28-oate (CDDO-Me, also known as bardoxolone methyl). After 24 hours, analyses identified 2561 transcripts and 240 proteins that were differentially expressed in the kidneys of Nrf2 knockout mice, compared to wild-type counterparts, and 3122 transcripts and 68 proteins that were differentially expressed in wild-type mice treated with CDDO-Me, compared to vehicle control. In light of their sensitivity to genetic and pharmacological modulation of renal Nrf2 activity, genes/proteins that regulate xenobiotic disposition, redox balance, the intra/extracellular transport of small molecules, and the supply of NADPH and other cellular fuels were found to be positively regulated by Nrf2 in the kidney. This was verified by qPCR, immunoblotting, pathway analysis and immunohistochemistry. In addition, the levels of NADPH and glutathione were found to be significantly decreased in the kidneys of Nrf2 knockout mice. Thus, Nrf2 regulates genes that coordinate homeostatic processes in the kidney, highlighting its potential as a novel therapeutic target.

HER-2 expression in canine morphologically normal, hyperplastic and neoplastic mammary tissues and its correlation with the clinical outcome

The proto-oncogene HER-2/neu (c-erbB-2) encodes a transmembrane receptor protein with tyrosine-kinase activity. Previous studies have shown that HER-2 protein over-expression is present in canine mammary tumours, however, possible prognostic and predictive analogies between protein over-expression patterns in canine and human species are still controversial. Thirty-five canine mammary carcinomas, 11 mammary adenomas, and normal, hyperplastic or dysplastic tissues taken at the marginal area of the tumours were evaluated by immunohistochemistry (IHC) for HER-2 expression, using the Hercept Test® system scoring guidelines. HER-2 over-expression was detected in 3/11 adenomas and 10/35 carcinomas. Normal, hyperplastic and dysplastic mammary tissues were also found to be positive. The correlations between HER-2 expression and tumour histological grading, mitotic index, the presence of lymphatic invasion, and overall survival (OS) were evaluated. In carcinomas, HER-2 positive status only correlated with the mitotic index. A positive correlation was also found between HER-2 positive status and the presence of HER-2 over-expression in normal, hyperplastic or/and dysplastic mammary tissues surrounding the tumours. The percentage of HER-2 over-expressing tumours was similar to the percentage previously observed in canine benign and malignant mammary tumours. However an investigation regarding morphologically normal and hyperplastic or dysplastic tissues surrounding neoplastic lesions also showed HER-2 over-expression. In contrast with human mammary tumours, this study confirmed that in canine species, HER-2 over-expression does not identify a subgroup of tumours with a poor prognosis. In fact, we found HER-2 over-expression in morphologically non-neoplastic mammary tissues, surrounding hyperplastic and neoplastic lesions.

Blunt Force Trauma in Veterinary Forensic Pathology

Veterinary pathologists commonly encounter lesions of blunt trauma. The development of lesions is affected by the object’s mass, velocity, size, shape, and angle of impact and by the plasticity and mobility of the impacted organ. Scrape, impact, and pattern abrasions cause localized epidermal loss and sometimes broken hairs and implanted foreign material. Contusions are best identified after reflecting the skin, and must be differentiated from coagulopathies and livor mortis. Lacerations—traumatic tissue tears—may have irregular margins, bridging by more resilient tissue, deviation of the wound tail, crushed hairs, and unilateral abrasion. Hanging or choking can cause circumferential cervical abrasions, contusions and rupture of hairs, hyoid bone fractures, and congestion of the head. Other special forms of blunt trauma include fractured nails, pressure sores, and dog bites. Ocular blunt trauma causes extraocular and intraocular hemorrhages, proptosis, or retinal detachment. The thoracic viscera are relatively protected from blunt trauma but may develop hemorrhages in intercostal muscles, rib fractures, pulmonary or cardiac contusions or lacerations with subsequent hemothorax, pneumothorax, or cardiac arrhythmia. The abdominal wall is resilient and moveable, yet the liver and spleen are susceptible to traumatic laceration or rupture. Whereas extravasation of blood can occur after death, evidence of vital injury includes leukocyte infiltration, erythrophagocytosis, hemosiderin, reparative lesions of fibroblast proliferation, myocyte regeneration in muscle, and callus formation in bone. Understanding these processes aids in the diagnosis of blunt force trauma including estimation of the age of resulting injuries.

Equine Cutaneous Mast Cell Tumours Exhibit Variable Differentiation, Proliferation Activity and KIT Expression.

Equine cutaneous mast cell tumours (CMCTs) are generally considered to be benign skin lesions, although recurrent and multicentric tumours have been described. For canine CMCTs, grading and prognostic approaches are well established and aberrant KIT expression as well as high proliferation indices are associated with poor outcome. However, in the case of equine CMCTs, morphological features, proliferative activity and KIT expression pattern have not been assessed or related to biological behaviour, and there is discussion as to whether CMCTs are true neoplastic processes. The present study describes 45 equine CMCTs in terms of their morphology and KIT and PCNA expression by immunohistochemistry. KIT expression was classified as membranous (I), cytoplasmic and focally stippled (II) or diffuse cytoplasmic (III). A large proportion of the tumours were multinodular or diffuse dermal infiltrates of mast cells with mild anisokaryosis, a low proliferative rate and a dominance of KIT pattern I, representing well-differentiated CMCTs. In approximately one third of the cases, the mast cells exhibited more infiltrative growth, moderate to marked anisokaryosis and a higher degree of proliferation. These were classified as poorly differentiated CMCTs and exhibited only KIT patterns II and III. These findings indicate that there is a subgroup of poorly differentiated equine CMCTs, in which there is an association between aberrant KIT expression, high proliferative rate and potential aggressive behaviour, all features that confirm at least the poorly differentiated CMCT as a true neoplastic processes.

Preliminary report on the expression of leptin and leptin receptor (ObR) in normal, hyperplastic and neoplastic canine mammary tissues.

Leptin and its receptor (ObR) expression were investigated by immunohistochemistry in normal, hyperplastic and neoplastic canine mammary tissues and related to clinical-pathological features. Leptin expression was detected in healthy mammary tissues, adenosis and in benign mammary tumours and was lower in ductal hyperplasias and malignant tumours. A high percentage of ObR-positive cells were present in adenosis, benign tumours and in complex carcinomas, while ObR expression was lower in healthy mammary tissues, in ductal hyperplasias and in a large part of invasive mammary carcinomas. Our data demonstrated that cancer cells expressed at low level leptin and ObR in canine mammary tumours with a more aggressive behaviour, as well as in lymph node metastases. Consequently, leptin and ObR expressions in this species resulted to be not associated with a reduced overall survival.