Experimental Infections Reveal Acquired Zoonotic Capacity of Human Schistosomiasis Trough Hybridization.

We are currently witnessing the endemization of urogenital schistosomiasis in southern Europe. The incriminated parasite is a hybrid between a human parasite and a livestock parasite. Using an experimental evolutionary protocol, we created hybrid lines from pure strains of both parasite species. We showed that the host spectrum of the human parasite is enlarged to the livestock parasite after genomic introgression. We also evidenced that the tropism of the parasites within the host changes and that some hybrid lines are more virulent than the parental strains. These results engage a paradigm shift from human to zoonotic transmission of urogenital schistosomiasis.

Toxoplasma gondii and Alternaria sp.: An Original Association in an Immunosuppressed Dog with Persistent Skin Lesions.

Dogs and cats may suffer from a variety of diseases, mainly immune mediated, that require the administration of immunosuppressive drugs. Such therapies can cause adverse effects either by the toxicity of the drugs or as a consequence of immune suppression and associated opportunistic infections. Here we present an, yet unknown, association of Toxoplasma gondii and Alternaria fungus, within cutaneous lesions in a dog under long-term immunosuppressive therapy. The diagnosis of such infections is laborious and not obvious at first glance, since the clinical signs of cutaneous toxoplasmosis, neosporosis or alternariosis are not specific. A further laboratory confirmation is needed. Therefore, we currently recommend that dogs and cats should undergo serologic testing for toxoplasmosis or neosporosis prior to immunosuppressive therapy and a regular dermatological evaluation during the immunosuppressive therapy.

Potential applications of aptamers in veterinary science.

Aptamers are small nucleic acids that fold in a three-dimensional conformation allowing them to bind specifically to a target. This target can be an organic molecule, free or carried in cells or tissues, or inorganic components, such as metal ions. Analogous to monoclonal antibodies, aptamers however have certain advantages over the latter: e.g., high specificity for their target, no to low immunogenicity and easy in vitro selection. Since their discovery more than 30 years ago, aptamers have led to various applications, although mainly restricted to basic research. This work reviews the applications of aptamers in veterinary science to date. First, we present aptamers, how they are selected and their properties, then we give examples of applications in food and environmental safety, as well as in diagnosis and medical treatment in the field of veterinary medicine. Because examples of applications in veterinary medicine are scarce, we explore the potential avenues for future applications based on discoveries made in human medicine. Aptamers may offer new possibilities for veterinarians to diagnose certain diseases-particularly infectious diseases-more rapidly or "at the patient's bedside". All the examples highlight the growing interest in aptamers and the premises of a potential market. Aptamers may benefit animals as well as their owners, breeders and even public health in a "One Health" approach.

Activation of IFN/STAT1 signalling predicts response to chemotherapy in oestrogen receptor-negative breast cancer.

BACKGROUND: Oestrogen receptor-negative (ER-) breast cancer is intrinsically sensitive to chemotherapy. However, tumour response is often incomplete, and relapse occurs with high frequency. The aim of this work was to analyse the molecular characteristics of residual tumours and early response to chemotherapy in patient-derived xenografts (PDXs) of breast cancer. METHODS: Gene and protein expression profiles were analysed in a panel of ER- breast cancer PDXs before and after chemotherapy treatment. Tumour and stromal interferon-gamma expression was measured in xenografts lysates by human and mouse cytokine arrays, respectively. RESULTS: The analysis of residual tumour cells in chemo-responder PDX revealed a strong overexpression of IFN-inducible genes, induced early after AC treatment and associated with increased STAT1 phosphorylation, DNA-damage and apoptosis. No increase in IFN-inducible gene expression was observed in chemo-resistant PDXs upon chemotherapy. Overexpression of IFN-related genes was associated with human IFN-γ secretion by tumour cells. CONCLUSIONS: Treatment-induced activation of the IFN/STAT1 pathway in tumour cells is associated with chemotherapy response in ER- breast cancer. Further validations in prospective clinical trials will aim to evaluate the usefulness of this signature to assist therapeutic strategies in the clinical setting.

Ultrahigh dose-rate FLASH irradiation increases the differential response between normal and tumor tissue in mice.

In vitro studies suggested that sub-millisecond pulses of radiation elicit less genomic instability than continuous, protracted irradiation at the same total dose. To determine the potential of ultrahigh dose-rate irradiation in radiotherapy, we investigated lung fibrogenesis in C57BL/6J mice exposed either to short pulses (≤ 500 ms) of radiation delivered at ultrahigh dose rate (≥ 40 Gy/s, FLASH) or to conventional dose-rate irradiation (≤ 0.03 Gy/s, CONV) in single doses. The growth of human HBCx-12A and HEp-2 tumor xenografts in nude mice and syngeneic TC-1 Luc(+) orthotopic lung tumors in C57BL/6J mice was monitored under similar radiation conditions. CONV (15 Gy) triggered lung fibrosis associated with activation of the TGF-ß (transforming growth factor-ß) cascade, whereas no complications developed after doses of FLASH below 20 Gy for more than 36 weeks after irradiation. FLASH irradiation also spared normal smooth muscle and epithelial cells from acute radiation-induced apoptosis, which could be reinduced by administration of systemic TNF-α (tumor necrosis factor-α) before irradiation. In contrast, FLASH was as efficient as CONV in the repression of tumor growth. Together, these results suggest that FLASH radiotherapy might allow complete eradication of lung tumors and reduce the occurrence and severity of early and late complications affecting normal tissue.

Acquired resistance to endocrine treatments is associated with tumor-specific molecular changes in patient-derived luminal breast cancer xenografts.

PURPOSE: Patients with luminal breast cancer (LBC) often become endocrine resistant over time. We investigated the molecular changes associated with acquired hormonoresistances in patient-derived xenografts of LBC. EXPERIMENTAL DESIGN: Two LBC xenografts (HBCx22 and HBCx34) were treated with different endocrine treatments (ET) to obtain xenografts with acquired resistances to tamoxifen (TamR) and ovariectomy (OvaR). PI3K pathway activation was analyzed by Western blot analysis and IHC and responses to ET combined to everolimus were investigated in vivo. Gene expression analyses were performed by RT-PCR and Affymetrix arrays. RESULTS: HBCx22 TamR xenograft was cross-resistant to several hormonotherapies, whereas HBCx22 OvaR and HBCx34 TamR exhibited a treatment-specific resistance profile. PI3K pathway was similarly activated in parental and resistant xenografts but the addition of everolimus did not restore the response to tamoxifen in TamR xenografts. In contrast, the combination of fulvestrant and everolimus induced tumor regression in vivo in HBCx34 TamR, where we found a cross-talk between the estrogen receptor (ER) and PI3K pathways. Expression of several ER-controlled genes and ER coregulators was significantly changed in both TamR and OvaR tumors, indicating impaired ER transcriptional activity. Expression changes associated with hormonoresistance were both tumor and treatment specific and were enriched for genes involved in cell growth, cell death, and cell survival. CONCLUSIONS: PDX models of LBC with acquired resistance to endocrine therapies show a great diversity of resistance phenotype, associated with specific deregulations of ER-mediated gene transcription. These models offer a tool for developing anticancer therapies and to investigate the dynamics of resistance emerging during pharmacologic interventions. Clin Cancer Res; 20(16); 4314-25. ©2014 AACR.

Optimization of tumor xenograft dissociation for the profiling of cell surface markers and nutrient transporters.

Metabolic adaptations and changes in the expression of nutrient transporters are known to accompany tumorigenic processes. Nevertheless, in the context of solid tumors, studies of metabolism are hindered by a paucity of tools allowing the identification of cell surface transporters on individual cells. Here, we developed a method for the dissociation of human breast cancer tumor xenografts combined with quantification of cell surface markers, including metabolite transporters. The expression profiles of four relevant nutrient transporters for cancer cells' metabolism, Glut1, ASCT2, PiT1 and PiT2 (participating to glucose, glutamine and inorganic phosphate, respectively), as detected by new retroviral envelope glycoprotein-derived ligands, were distinctive of each tumor, unveiling underlying differences in metabolic pathways. Our tumor dissociation procedure and nutrient transporter profiling technology provides opportunities for future basic research, clinical diagnosis, prognosis and evaluation of therapeutic responses, as well as for drug discovery and development.

Inhibiting aurora kinases reduces tumor growth and suppresses tumor recurrence after chemotherapy in patient-derived triple-negative breast cancer xenografts.

Triple-negative breast cancers (TNBC) have an aggressive phenotype with a relatively high rate of recurrence and poor overall survival. To date, there is no approved targeted therapy for TNBCs. Aurora kinases act as regulators of mammalian cell division. They are important for cell-cycle progression and are frequently overexpressed or mutated in human tumors, including breast cancer. In this study, we investigated the therapeutic potential of targeting Aurora kinases in preclinical models of human breast cancers using a pan-inhibitor of Aurora kinases, AS703569. In vitro, AS703569 was tested in 15 human breast cancer cell lines. TNBC cell lines were more sensitive to AS703569 than were other types of breast cancer cells. Inhibition of proliferation was associated with cell-cycle arrest, aneuploidy, and apoptosis. In vivo, AS703569 administered alone significantly inhibited tumor growth in seven of 11 patient-derived breast cancer xenografts. Treatment with AS703569 was associated with a decrease of phospho-histone H3 expression. Finally, AS703569 combined to doxorubicin-cyclophosphamide significantly inhibited in vivo tumor recurrence, suggesting that Aurora kinase inhibitors could be used both in monotherapy and in combination settings. In conclusion, these data indicate that targeting Aurora kinases could represent a new effective approach for TNBC treatment.

Molecular profiling of patient-derived breast cancer xenografts.

INTRODUCTION: Identification of new therapeutic agents for breast cancer (BC) requires preclinical models that reproduce the molecular characteristics of their respective clinical tumors. In this work, we analyzed the genomic and gene expression profiles of human BC xenografts and the corresponding patient tumors. METHODS: Eighteen BC xenografts were obtained by grafting tumor fragments from patients into Swiss nude mice. Molecular characterization of patient tumors and xenografts was performed by DNA copy number analysis and gene expression analysis using Affymetrix Microarrays. RESULTS: Comparison analysis showed that 14/18 pairs of tumors shared more than 56% of copy number alterations (CNA). Unsupervised hierarchical clustering analysis showed that 16/18 pairs segregated together, confirming the similarity between tumor pairs. Analysis of recurrent CNA changes between patient tumors and xenografts showed losses in 176 chromosomal regions and gains in 202 chromosomal regions. Gene expression profile analysis showed that less than 5% of genes had recurrent variations between patient tumors and their respective xenografts; these genes largely corresponded to human stromal compartment genes. Finally, analysis of different passages of the same tumor showed that sequential mouse-to-mouse tumor grafts did not affect genomic rearrangements or gene expression profiles, suggesting genetic stability of these models over time. CONCLUSIONS: This panel of human BC xenografts maintains the overall genomic and gene expression profile of the corresponding patient tumors and remains stable throughout sequential in vivo generations. The observed genomic profile and gene expression differences appear to be due to the loss of human stromal genes. These xenografts, therefore, represent a validated model for preclinical investigation of new therapeutic agents.

A preclinical therapeutic schedule optimizing docetaxel plus estramustine administration in prostate cancer.

Androgen-dependent and castration-resistant prostate cancer (PC) is usually sensitive to docetaxel chemotherapy. Nevertheless, docetaxel resistance frequently appears after several cycles of treatment, raising the problem of salvage treatment for docetaxel-resistant PC patients. Although the combination of docetaxel and estramustine prolongs metastasis-free and overall survival of patients with androgen-independent PC, the use of this modality remains limited in elderly patients or patients with several comorbidities, especially vascular disease or gastrointestinal toxicity, because of unacceptable toxicity including venous thrombosis. The aims of this study were therefore (i) to evaluate the in-vivo efficacy of estramustine combined with docetaxel since initial tumor growth and following the appearance of docetaxel resistance in the androgen-dependent human PC xenograft PAC120, and (ii) to evaluate the efficacy of estramustine in six human androgen-independent PC models derived from PAC120. In docetaxel-resistant tumor-bearing mice, estramustine alone induced a TGD2 of 18 days, whereas the combination of docetaxel and estramustine induced a TGD2 of 50 days (P<0.05) with no significantly different overall survival of mice treated by docetaxel and estramustine since day 1 or since the onset of resistance to docetaxel. Among the six human androgen-independent tumors treated with estramustine alone, two highly sensitive models, two intermediate responding tumors, and two resistant models were observed. Altogether, these results suggest that estramustine should be combined with docetaxel in PC patients, but the use of this treatment could be limited, particularly in elderly patients, to docetaxel-resistant cases.

Viral protein expression and phenotyping of inflammatory responses in the central nervous system of phocine distemper virus-infected harbor seals (Phoca vitulina).

The central nervous system (CNS) represents an important target organ of the phocine distemper virus (PDV). The aim of the present study was to characterize pathological changes in the CNS of harbor seals suffering from natural PDV-infection. The distribution of virus protein and mRNA was investigated by immunohistochemistry (IHC) and in situ hybridization, respectively. In addition, inflammatory and glial cells were characterized by IHC. Polioencephalitis with glial activation, neuronal death and perivascular mononuclear infiltrations in the cerebral cortex was the main histopathological finding. Inflammatory responses, dominated by CD3(+) T-cells and activated microglia/macrophages were associated with a prominent MHC-II upregulation within the CNS. Viral protein was found predominantly in neurofilament-expressing neurons within inflamed areas as demonstrated by immunohistochemical double-labeling. Morbillivirus nucleo-, phospho-, matrix-, fusion- and hemagglutinin-proteins were found in CNS-lesions. The expressions of viral matrix- and fusion-proteins were reduced in severely inflamed plaques. Comparison of viral protein and mRNA expression revealed a diminished amount of viral phosphoprotein preferentially associated with perivascular inflammation. In summary, CNS-lesions in PDV-infected seals are similar to canine distemper virus-induced acute polioencephalitis in dogs and measles virus inclusion body polioencephalitis in men, respectively.

An atypical peripheral nerve sheath tumour with pseudoglandular architecture in a dog.

This case describes a subcutaneous soft tissue tumour in a German Shepherd dog. Histologically, the lesion was characterized by proliferating ovoid cells, loosely arranged in a collagenous to myxoid stroma, and by numerous pseudoglandular structures lined by neoplastic cells. Immunohistochemically, neoplastic cells were labelled with vimentin, glial fibrillary acidic protein and S100 antibodies, but not with cytokeratin, desmin and smooth muscle actin antibodies. Ultrastructurally, neoplastic cells were characterized by numerous mitochondria surrounded by endoplasmic reticulum and contained few secondary lysosomes. This tumour was diagnosed as a subcutaneous peripheral nerve sheath tumour (PNST) with pseudoglandular architecture. This case illustrates the morphological diversity of PNST and provides new insight into the differential diagnosis of cutaneous tumours of similar morphology in the dog.

Preclinical assessment of cisplatin-based therapy versus docetaxel-based therapy on a panel of human non-small-cell lung cancer xenografts.

The success of treatment of advanced non-small-cell lung cancer (NSCLC) remains very poor. The aim of this study was, on a series of NSCLC xenografts, to compare the efficacy of standard cisplatin-based or docetaxel-based chemotherapy. Seven human xenografts were obtained from six patients (two xenografts were derived from primary or metastatic tumors of the same patient). Three xenografts were adenocarcinomas and four were squamous cell carcinomas. All xenografts reproduced the same histology as that of the patient's original tumor. Docetaxel, administered as single-agent chemotherapy, induced a significant response in five of the seven NSCLC xenografts (71%), without significant increase after combination with cisplatin, vinorelbine, or gemcitabine. Relative expression of genes putatively involved in drug response was also studied in all xenografts and did not explain the variability of drug sensitivity. In conclusion, this panel of human NSCLC xenografts reliably reproduces the data obtained in patient tumors and the relative sensitivity to docetaxel reported in NSCLC patients.

Atypical vimentin expression in a feline salivary gland adenocarcinoma with widespread metastases.

We report herein a feline salivary gland adenocarcinoma with widespread metastases to draining lymph nodes, liver and lung, as well as an unusual metastasis to the spleen. Histologically, the primary salivary gland tumor consisted of low columnar to polygonal epithelial cells forming tubules and trabeculae. The spleen was infiltrated with sheets of poorly differentiated large round cells. Interestingly, morphologic change in epithelial cells was accompanied with the acquisition of vimentin intermediate filaments, a feature particularly evident in the splenic metastasis. This study highlights the role of epithelial cell plasticity during carcinogenesis and metastasis.

A mammary gland adenomyoepithelioma in a C57BL/6 mouse.

Mammary gland adenomyoepitheliomas are benign complex mammary gland tumors composed of neoplastic cells of epithelial and myoepithelial origins, described in many species (humans, dogs, cats, rats) and rarely in mice. We report here an adenomyoepithelioma in a C57BL/6 female mouse. Histologically, tubes and cords formed by neoplastic epithelial cells were separated by bundles of neoplastic myoepithelial cells in a clear and partially mucinous matrix. The tumor displayed characteristics of a benign neoplastic proliferation with a compressive growth pattern, and moderate cellular pleomorphism and mitotic index. At immunohistochemistry, the epithelial cells were strongly cytokeratin positive; the myoepithelial cells were weakly cytokeratin positive and strongly smooth muscle actin positive. This is to our knowledge, the first report of a mammary gland adenomyoepithelioma in a C57BL/6 mouse.

An atypical case of histiocytic sarcoma in a Wistar rat (Rattus norvegicus).

Histiocytic sarcoma is the most frequent hematopoietic tumor in rats. We report here a histiocytic sarcoma infiltrating the liver, the spleen and the pancreas from a Wistar rat. In the liver, the tumor was associated with oval cell and bile duct hyperplasia. The cells looked like neoplastic histocytic cells described in this species but with some particularities (e.g. lack of multinucleated giant cells). At immunohistochemistry, neoplastic cells in the liver were vimentine positive but lysozyme and CD68 negative. In the kidney, lysozyme-positive cytoplasmic droplets were observed. We describe here an atypical case of histiocytic sarcoma in the rat and we compare the nature of these neoplastic cells to other species.

Immunohistochemical characterization of a hepatic neuroendocrine carcinoma in a cat.

A hepatic mass was identified in a 5-year-old, female mixed-breed cat that died spontaneously after a clinical history of progressive emaciation, ptyalism, and persistent coryza. At necropsy, a 7-cm-diameter, yellow-brown, firm, multilobulated tumor was identified in the liver. Microscopically, the mass consisted of neoplastic cells arranged in small, closely packed nests within a thin fibrovascular stroma. These cells were of medium sized and polygonal, with fine argyrophilic cytoplasmic granules. Nuclei were predominantly round with finely stippled chromatin and indistinct nucleoli. Mitotic figures were numerous. Immunohistochemically, most of the neoplastic cells were immunoreactive for chromogranin A, neuron-specific enolase (NSE), and cytokeratin AE1/AE3 and weakly labeled for synaptophysin. The tumor was negative for glial fibrillary acidic protein (GFAP), vimentin, and cytokeratins 5, 6, 8, and 17. Vascular emboli and intrahepatic micrometastasis were also identified with chromogranin A. All these features were consistent with a hepatic neuroendocrine carcinoma and emphasized the importance of using a panel of antibodies to diagnose such rare tumors.

Clinical, mycological and pathological findings in turkeys experimentally infected by Aspergillus fumigatus.

Experimental aspergillosis was induced in 1-day-old turkeys by intra-air-sac inoculation of a spore suspension of a 3-day-old Aspergillus fumigatus culture (CBS 144.89) containing 10(7) spores. Ten additional poults were used as controls. Infected and non-infected animals were closely observed at least twice a day for the appearance of clinical signs and were sequentially sacrificed at days 1, 2, 3, 5 and 7 post-inoculation. In the infected group, most lung tissues and air sac swabs were culture positive from day 1 to day 5. At 1 day post-inoculation, air sac membranes were multifocally and moderately to severely thickened by an oedema and covered by an exudate. A small number of germinating conidia were present in the superficial exudate, already giving rise to small radiating hyphae. Lung lesions were mild, dominated by a diffuse congestion and a mild heterophilic infiltration. From 2 to 3 days post-inoculation, air sac membranes were more severely affected and several granulomas were observed. Both granulomas and exudates were rich in germinated conidia and hyphae. Pulmonary lesions consisted in a diffuse pneumonia. Five days post-inoculation, air sac membrane lesions progressed to a severe, multifocal, heterophilic and granulomatous inflammation. Seven days post-inoculation, a reduction of the severity of the diffuse pneumonia was detected. Concomitantly, the fungal elements were mainly observed as fragmented tubules in the cytoplasm of multinucleate giant cells. The present study demonstrated that healthy turkey poults might be able to withstand exposure to 10(7) A. fumigatus spores.

Rumenic acid significantly reduces plasma levels of LDL and small dense LDL cholesterol in hamsters fed a cholesterol- and lipid-enriched semi-purified diet.

Conjugated linoleic acids (CLAs) consist of a series of positional and geometrical isomers of linoleic acid. CLA have been reported to beneficially affect cardiovascular risk factors in animal models. In order to assess the role of individual CLA isomers on lipoprotein cholesterol concentration, 30 hamsters were fed for 12 weeks an hyperlipidic diet containing pure cis-9,trans-11 CLA (c9,t11) or pure trans-10, cis-12 CLA (t10,c12) isomers given alone or as a mixture. Plasma total cholesterol, LDL and HDL cholesterol concentrations were significantly lower in the c9,t11 CLA isomer fed hamsters relative to the Control group, with the most substantially effect on LDL cholesterol (-56%; P < 0.05). Plasma triacylglycerol concentrations did not differ significantly regarding those two groups. Plasma cholesterol parameters showed a tendency to decrease in the t10,c12 CLA isomer and CLA mixture fed hamsters compared with the Control group, but differences were not significant. For the first time, the atherogenic fraction of small dense LDL was investigated. Plasma small dense LDL cholesterol concentration was lower in the c9,t11 CLA relative to Control, while the t10,c12 and CLA mixture groups showed only a non significant tendency to decrease. Taken together, these data indicate that feeding rumenic acid (c9,t11 CLA) may beneficially affect lipoprotein profile in hamster fed a cholesterol- and lipid-enriched semi-purified diet, when t10,c12 CLA isomer or CLA mixture would be less active.

Microcystin-LR and embryo-larval development of medaka fish, Oryzias latipes. I. Effects on the digestive tract and associated systems.

The cyanobacterial hepatotoxin microcystin-LR (MC-LR) is a specific potent PP1 and PP2A protein phosphatase inhibitor. In view to obtain an integrated whole-body, understanding of the key target organs of MC-LR subsequent to embryonic exposure on the anatomy of medaka fish hatchlings, embryos at stage 19 were microinjected with a sublethal dose of MC-LR corresponding to 0.2 pg/vitellus. MC-LR-induced histo-pathological modifications of the alimentary system (i.e. digestive tract, pancreas, liver) were analysed in newly hatched embryos. Our data are indicative of an MC-LR-induced inhibition of both yolk sac resorption and gas concentrating swimbladder expansion. In contrast to control hatchlings, (i) no mucus-secreting cells in the oesophagus, (ii) a decreased folding of the stomach and intestine, (iii) a clear reduction in size of the exocrine pancreas associated with a destructuration of acinar units, and (iv) a strong decrease in the mass and size of the liver were observed in MC-LR treated embryos. Furthermore, as an indication of MC-LR-induced hepatic glycogen store depletion, unstained cytoplasmic areas present in control hatchling hepatocytes, were fully absent in all liver examined in treated embryos. Finally, as a general observation in MC-LR-treated embryos, our data clearly indicated terminal differentiation disorders in all organs associated with the digestive tract.