Modeling of response to endocrine therapy in a panel of human luminal breast cancer xenografts.

Resistance to endocrine therapy is a major complication of luminal breast cancer and studies of the biological features of hormonal resistance are limited by the lack of adequate preclinical models. The aim of this study is to establish and characterize a panel of primary human luminal breast carcinoma xenografts, and to evaluate their response to endocrine therapies. Four hundred and twenty-three tumor fragments obtained directly from patients have been grafted in the interscapular fatpad of Swiss nude mice. After stable engraftment with estradiol supplementation, xenografted tumors have been validated by conventional pathology and immunohistochemistry examination, and additional molecular studies. In vivo tumor growth and response to different endocrine treatments were evaluated. We have engrafted 423 tumors including 314 ER+ tumors, and 8 new luminal breast cancer xenografts have been obtained (2.5%). Tumor take was much lower for luminal tumors than for non-luminal tumors (2.5 vs. 24.7%, P < 0.0001), and was associated with two independent criteria, i.e., ER status (P < 0.0001) and a high grade tumor (P = 0.05). Histological and immunohistochemical analyses performed on patient's tumors and xenografts showed striking similarities in the tumor morphology as well as in the expression level of ER, PR, and HER2. Response to hormone therapy, evaluated in 6 luminal models, showed different sensitivities, thus exhibiting heterogeneity similar to what is observed in the clinic. We have established a panel of primary human luminal breast cancer xenografts, recapitulating the biological and clinical behaviors of patient tumors, and therefore suitable for further preclinical experiments.

Establishment and characterisation of a new breast cancer xenograft obtained from a woman carrying a germline BRCA2 mutation.

BACKGROUND: The BRCA2 gene is responsible for a high number of hereditary breast and ovarian cancers, and studies of the BRCA2 biological functions are limited by the lack of models that resemble the patient's tumour features. The aim of this study was to establish and characterise a new human breast carcinoma xenograft obtained from a woman carrying a germline BRCA2 mutation. METHODS: A transplantable xenograft was obtained by grafting a breast cancer sample into nude mice. The biological and genetic profiles of the xenograft were compared with that of the patient's tumour using histology, immunohistochemistry (IHC), BRCA2 sequencing, comparative genomic hybridisation (CGH), and qRT-PCR. Tumour response to standard chemotherapies was evaluated. RESULTS: Histological profile identified the tumour as a basal-like triple-negative breast cancer. Targeted BRCA2 DNA sequencing of the xenograft showed the presence of the mutation previously identified in the carrier. Comparative genomic hybridisation array profiles of the primary tumour and the xenograft revealed a high number of similar genetic alterations. The therapeutic assessment of the xenograft showed sensitivity to anthracyclin-based chemotherapy and resistance to docetaxel. The xenograft was also highly sensitive to radiotherapy and cisplatin-based treatments. CONCLUSIONS: This study describes a new human breast cancer xenograft obtained from a BRCA2-mutated patient. This xenograft provides a new model for the pre-clinical drug development and for the exploration of the drug response biological basis.

Primary hypoaldosteronism in a dog with pituitary and adrenal T-cell lymphoma.

A 7-year-old mixed breed dog was presented with a 2-week history of vomiting, diarrhoea, weakness and loss of appetite. Initial laboratory tests revealed hyponatraemia and hyperkalaemia consistent with hypoadrenocorticism. Basal plasma cortisol and adrenocorticotropic hormone concentrations were not suggestive of primary hypoadrenocorticism but the aldosterone concentration was undetectable. Abdominal ultrasound scan showed a mass within the left kidney and a nodular enlargement of the left adrenal gland. Cytological analysis revealed a large granular lymphoma. The dog died 17 days later. Post mortem histological and immunohistochemical examinations revealed a diffuse large granular T-cell lymphoma involving the mediastinal lymph node, kidneys, pancreas, adrenal and pituitary glands.

A small rab GTPase is distributed in cytoplasmic vesicles in non polarized cells but colocalizes with the tight junction marker ZO-1 in polarized epithelial cells.

Small rab/Ypt1/Sec4 GTPase family have been involved in the regulation of membrane traffic along the biosynthetic and endocytic pathways in eucaryotic cells. Polarized epithelial cells have morphologically and functionally distinct apical and basolateral surfaces separated by tight junctions. The establishment and maintenance of these structures require delivery of membrane proteins and lipids to these domains. In this work, we have isolated a cDNA clone from a human intestinal cDNA library encoding a small GTPase, rab13, closely related to the yeast Sec4 protein. Confocal microscopy analysis on polarized Caco-2 cells shows that rab13 protein colocalized with the tight junction marker ZO-1. Cryostat sections of tissues confirm that rab13 localized to the junctional complex region of a variety of epithelia, including intestine, kidney, liver, and of endothelial cells. This localization requires assembly and integrity of the tight junctions. Disruption of tight junctions by incubation in low Ca2+ media induces the redistribution of rab13. In cells devoid of tight junctions, rab13 was found associated with vesicles dispersed throughout the cytoplasm. Cell-cell contacts initiated by E-cadherin in transfected L cells do not recruit rab13 to the resulting adherens-like junction complexes. The participation of rab13 in polarized transport, in the assembly and/or the activity of tight junctions is discussed.

In vivo, villin is required for Ca(2+)-dependent F-actin disruption in intestinal brush borders.

Villin is an actin-binding protein localized in intestinal and kidney brush borders. In vitro, villin has been demonstrated to bundle and sever F-actin in a Ca(2+)-dependent manner. We generated knockout mice to study the role of villin in vivo. In villin-null mice, no noticeable changes were observed in the ultrastructure of the microvilli or in the localization and expression of the actin-binding and membrane proteins of the intestine. Interestingly, the response to elevated intracellular Ca(2+) differed significantly between mutant and normal mice. In wild-type animals, isolated brush borders were disrupted by the addition of Ca(2+), whereas Ca(2+) had no effect in villin-null isolates. Moreover, increase in intracellular Ca(2+) by serosal carbachol or mucosal Ca(2+) ionophore A23187 application abolished the F-actin labeling only in the brush border of wild-type animals. This F-actin disruption was also observed in physiological fasting/refeeding experiments. Oral administration of dextran sulfate sodium, an agent that causes colonic epithelial injury, induced large mucosal lesions resulting in a higher death probability in mice lacking villin, 36 +/- 9.6%, compared with wild-type mice, 70 +/- 8.8%, at day 13. These results suggest that in vivo, villin is not necessary for the bundling of F-actin microfilaments, whereas it is necessary for the reorganization elicited by various signals. We postulate that this property might be involved in cellular plasticity related to cell injury.

Parental care and clutch sizes in North and South American birds.

The evolutionary causes of small clutch sizes in tropical and Southern Hemisphere regions are poorly understood. Alexander Skutch proposed 50 years ago that higher nest predation in the south constrains the rate at which parent birds can deliver food to young and thereby constrains clutch size by limiting the number of young that parents can feed. This hypothesis for explaining differences in clutch size and parental behaviors between latitudes has remained untested. Here, a detailed study of bird species in Arizona and Argentina shows that Skutch's hypothesis explains clutch size variation within North and South America. However, neither Skutch's hypothesis nor two major alternatives explain differences between latitudes.

Proteomic and phosphoproteomic analysis of cellular responses in medaka fish (Oryzias latipes) following oral gavage with microcystin-LR.

Chronic and subchronic toxicity resulting from exposure to microcystins (MCs) receives increasing attention due to the risk of bioaccumulation of these toxins by aquatic animals, including fish. The mechanisms of action of MCs that target the liver, involve modifications of protein phosphorylation resulting from phosphatases 1 and 2A inhibition. Therefore, studying phosphoprotein modifications by using a specific phosphoprotein stain Pro-Q Diamond in fish liver contaminated with MC-leucine-arginine (MC-LR), the most toxic MC, should help dissecting disturbed signaling and metabolic networks. We have recently used this technology to identify several proteins that are modulated either in expression or phosphorylation in the liver of medaka following short-term exposure to MC-LR by balneation. In the present study, we have decided to use an alternative way of introducing the toxin into fish; that is by gavage (force-feeding). This was first achieved using tritiated MC-LR and allowed us to quantify the quantity of toxin incorporated into fish and to demonstrate that the toxin is mainly accumulated in liver. Afterwards a proteomics study limited to liver cytosolic proteins of contaminated animals showed that several proteins were up or down regulated either in quantity or in phosphorylation or both. Some of them had been previously detected as modified in balneation experiments but new molecules were identified as involved in signal transduction pathways activated by the toxin. In addition, in the conditions used (5 microg toxin/g body weight) anatomopathological studies supported a process of apoptonecrosis established after 24h, which was suggested to proceed by the evolution of some of the proteins after 2h contamination.

Parent birds assess nest predation risk and adjust their reproductive strategies.

Avian life history theory has long assumed that nest predation plays a minor role in shaping reproductive strategies. Yet, this assumption remains conspicuously untested by broad experiments that alter environmental risk of nest predation, despite the fact that nest predation is a major source of reproductive failure. Here, we examined whether parents can assess experimentally reduced nest predation risk and alter their reproductive strategies. We experimentally reduced nest predation risk and show that in safer environments parents increased investment in young through increased egg size, clutch mass, and the rate they fed nestlings. Parents also increased investment in female condition by increasing the rates that males fed incubating females at the nest, and decreasing the time that females spent incubating. These results demonstrate that birds can assess nest predation risk at large and that nest predation plays a key role in the expression of avian reproductive strategies.

Life-history and ecological correlates of geographic variation in egg and clutch mass among passerine species.

Broad geographic patterns in egg and clutch mass are poorly described, and potential causes of variation remain largely unexamined. We describe interspecific variation in avian egg and clutch mass within and among diverse geographic regions and explore hypotheses related to allometry, clutch size, nest predation, adult mortality, and parental care as correlates and possible explanations of variation. We studied 74 species of Passeriformes at four latitudes on three continents: the north temperate United States, tropical Venezuela, subtropical Argentina, and south temperate South Africa. Egg and clutch mass increased with adult body mass in all locations, but differed among locations for the same body mass, demonstrating that egg and clutch mass have evolved to some extent independent of body mass among regions. A major portion of egg mass variation was explained by an inverse relationship with clutch size within and among regions, as predicted by life-history theory. However, clutch size did not explain all geographic differences in egg mass; eggs were smallest in South Africa despite small clutch sizes. These small eggs might be explained by high nest predation rates in South Africa; life-history theory predicts reduced reproductive effort under high risk of offspring mortality. This prediction was supported for clutch mass, which was inversely related to nest predation but not for egg mass. Nevertheless, clutch mass variation was not fully explained by nest predation, possibly reflecting interacting effects of adult mortality. Tests of the possible effects of nest predation on egg mass were compromised by limited power and by counterposing direct and indirect effects. Finally, components of parental investment, defined as effort per offspring, might be expected to positively coevolve. Indeed, egg mass, but not clutch mass, was greater in species that shared incubation by males and females compared with species in which only females incubate eggs. However, egg and clutch mass were not related to effort of parental care as measured by incubation attentiveness. Ecological and life-history correlates of egg and clutch mass variation found here follow from theory, but possible evolutionary causes deserve further study.

Habitat selection responses of parents to offspring predation risk: an experimental test.

The ability of nest predation to influence habitat settlement decisions in birds is widely debated, despite its importance in limiting fitness. Here, we experimentally manipulated nest predation risk across a landscape and asked the question, do migratory birds assess and respond to variation in nest predation risk when choosing breeding habitats? We examined habitat preference by quantifying the density and settlement date of eight species of migratory passerines breeding in areas with and without intact nest predator communities. We found consistently more individuals nesting in areas with reduced nest predation than in areas with intact predator assemblages, although predation risk had no influence on settlement or breeding phenology. Additionally, those individuals occupying safer nesting habitats exhibited increased singing activity. These findings support a causal relationship between habitat choice and nest predation risk and suggest the importance of nest predation risk in shaping avian community structure and breeding activity.

Subcellular localization of tetanus neurotoxin-insensitive vesicle-associated membrane protein (VAMP)/VAMP7 in neuronal cells: evidence for a novel membrane compartment.

The clostridial neurotoxin-insensitive soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors, tetanus neurotoxin-insensitive (TI)-vesicle-associated membrane protein (VAMP)/VAMP7, SNAP23, and syntaxin 3 have recently been implicated in transport of exocytotic vesicles to the apical plasma membrane of epithelial cells. This pathway had been shown previously to be insensitive to tetanus neurotoxin and botulinum neurotoxin F. TI-VAMP/VAMP7 is also a good candidate to be implicated in an exocytotic pathway involved in neurite outgrowth because tetanus neurotoxin does not inhibit this process in conditions in which it abolishes neurotransmitter release. We have now found that TI-VAMP/VAMP7 has a widespread distribution in the adult rat brain in which its localization strikingly differs from that of nerve terminal markers. TI-VAMP/VAMP7 does not enrich in synaptic vesicles nor in large dense-core granules but is associated with light membranes. In hippocampal neurons developing in vitro, TI-VAMP/VAMP7 localizes to vesicles in the axonal and dendritic outgrowths and concentrates into the leading edge of the growth cone, a region devoid of synaptobrevin 2, before synaptogenesis. After the onset of synaptogenesis, TI-VAMP/VAMP7 is found predominantly in the somatodendritic domain. In PC12 cells, TI-VAMP/VAMP7 does not colocalize with synaptobrevin 2, chromogranin B, or several markers of endocytic compartments. At the electron microscopic level, TI-VAMP/VAMP7 is mainly associated with tubules and vesicles. Altogether, these results suggest that TI-VAMP/VAMP7 defines a novel membrane compartment in neurite outgrowths and in the somatodendritic domain.

Differentiation of the epithelial apical junctional complex during mouse preimplantation development: a role for rab13 in the early maturation of the tight junction.

We have investigated the mechanisms by which the epithelial apicolateral junctional complex (AJC) is generated during trophectoderm differentiation in the mouse blastocyst using molecular, structural and functional analyses. The mature AJC comprises an apical tight junction (TJ), responsible for intercellular sealing and blastocoel formation, and subjacent zonula adherens E-cadherin/catenin adhesion complex which also extends along lateral membrane contact sites. Dual labelling confocal microscopy revealed that the AJC derived from a single 'intermediate' complex formed following embryo compaction at the 8-cell stage in which the TJ-associated peripheral membrane protein, ZO-1alpha- isoform, was co-localized with both alpha- and beta-catenin. However, following assembly of the TJ transmembrane protein, occludin, from the early 32-cell stage when blastocoel formation begins, ZO-1alpha- and other TJ proteins (ZO-1alpha+ isoform, occludin, cingulin) co-localized in an apical TJ which was separate from a subjacent E-cadherin/catenin zonula adherens complex. Thin-section electron microscopy confirmed that a single zonula adherens-like junctional complex present at the AJC site following compaction matured into a dual TJ and zonula adherens complex at the blastocyst stage. Embryo incubation in the tracer FITC-dextran 4 kDa showed that a functional TJ seal was established coincident with blastocoel formation. We also found that rab13, a small GTPase previously localized to the TJ, is expressed at all stages of preimplantation development and relocates from the cytoplasm to the site of AJC biogenesis from compaction onwards with rab13 and ZO-1alpha- co-localizing precisely. Our data indicate that the segregation of the two elements of the AJC occurs late in trophectoderm differentiation and likely has functional importance in blastocyst formation. Moreover, we propose a role for rab13 in the specification of the AJC site and the formation and segregation of the TJ.

Ovine mononuclear phagocytes in situ: identification by monoclonal antibodies and involvement in experimental pyogranulomas.

In order to characterize in situ the macrophages present in experimental pyogranulomas induced in lambs with Corynebacterium pseudotuberculosis, a set of monoclonal antibodies (MAbs) was produced following immunization of BALB/c mice with alveolar macrophages from healthy sheep. Three MAbs were retained after two steps of screening using alveolar macrophages, peripheral blood lymphocytes, and polymorphonuclear leukocytes as target cells. Their reactivity was tested not only on macrophages in pyogranulomas but also on sections of various organs in steady-state conditions. One MAb, termed OM1, recognized the monocytes and the majority of cells of the mononuclear phagocyte system in lymphoid and nonlymphoid organs. The two other MAbs, OM2 and OM3, reacted with a subpopulation of alveolar macrophages and with other cell types in tissues, in particular with endothelial cells for the MAb OM2. On sections of experimental pyogranulomas that developed in lymph nodes draining the C. pseudotuberculosis-injected sites, MAb OM1 reacted with all the macrophages distributed in a palisade surrounding the necrotic center of the lesion from day 6 to day 28 postinoculation. The two other MAbs, OM2 and OM3, enabled two types of granulomas to be distinguished: one type was characterized by a large number of epithelioid cells stained by OM2; and the other was characterized by a few OM2-positive macrophages, whereas the OM3-positive cells were more numerous. These results show that macrophages are predominant cells in pyogranulomas and suggest two different histological patterns in the evolution of pyogranulomas induced by C. pseudotuberculosis, according to the immunological status of the host.

Histopathology of the early phase during experimental Corynebacterium pseudotuberculosis infection in lambs.

Histological responses during experimental Corynebacterium pseudotuberculosis infection in lambs were investigated in parotid lymph nodes for ten days following inoculation. Lambs were infected by the subcutaneous route into the right eyelid with a virulent strain of C. pseudotuberculosis. Multiple microscopic acute abscesses, predominantly infiltrated with polymorphonuclear (PMN) leucocytes, were seen in the right parotid lymph node on the 1st day post-inoculation (PI). This massive PMN infiltration coincided with a peripheral blood granulocytosis. On day 3 PI, an influx of histiocytes was observed, while the microabscesses became confluent. From day 3 to day 10 PI, these lesions became enlarged and transformed into typical pyogranulomas with a central necrosis and a peripheral mantle of mononuclear cells composed of macrophages, epithelioid cells and lymphocytes; these histological changes were associated with a bacterial dissemination limited to the superficial lymph nodes. A lymphoid hyperplasia with prominent germinal centers was observed in the draining lymph nodes from day 3 PI. These results illustrate the dual role of granulomatous lesions in chronic bacterial infections: although they limit bacterial dissemination, the granulomas do not impair the persistence of infectious organisms in the host, leading to focal tissue damage.

Comparative histopathology of draining lymph node after infection with virulent or attenuated strains of Salmonella abortusovis in lambs.

Histological responses to the early phase of infection were compared in parotid lymph nodes of lambs infected by the subcutaneous route into the right eyelid with either a virulent or an attenuated strain of Salmonella abortusovis. The right parotid lymph nodes showed a massive PMN infiltration for the first days of infection for both strains. From day 6, the infected lymph nodes developed a lymphoid hyperplasia with prominent germinal centers independent of strain type. The virulent strain of S. abortusovis induced focal lesions in 2 out of 6 lambs necropsied on days 6 and 10, and provoked a systemic infection evidenced by the regular colonization of spleen on day 6. In contrast, no focal lesion and a restricted bacterial dissemination were observed in lambs infected with the vaccinal strain.

Non specific serological reactions in the diagnosis of bovine brucellosis: experimental oral infection of cattle with repeated doses of Yersinia enterocolitica O:9.

Eight heifers were orally infected with 4 x 10(9) colony forming units of a field cattle strain of Yersinia enterocolitica O:9 in a capsule, 5 days a week, for about 9 weeks (day 0-day 64 (D0-D64). The faecal shedding of Y. enterocolitica O:9 began on D5 for seven out of the eight challenged cattle with a high level of excretion during the first month, followed by a decrease till the day of slaughter (D76). Y. enterocolitica O:9 was not isolated from organs collected at slaughter. No clinical symptoms were observed. Hyperplasia of intestinal lymph formations was the sole microscopic lesions observed. Five animals showed a serological reaction against Brucella antigens in at least one of the following tests: Rose-Bengal test, complement fixation test, tube agglutination test or indirect ELISA (iELISA) tests. Only one animal showed a high level of serological response and a positive reaction in the dithiothreitol-microagglutination test. The observed variability in terms of individual sensitivity to the Y. enterocolitica O:9 infection is in agreement with the low individual prevalence rate and the transient serological reaction and faecal Y. entercolitica O:9 shedding observed in herds showing false positive serological reactions in brucellosis.

[Caprine arthritis-encephalitis: trial of an adjuvant vaccine preparation. I. Clinical and virological study]. / Arthrite-encéphalite caprine: essai d'une preparation vaccinale adjuvée--I. Etude clinique et virologique.

In purpose to protect goats against caprine arthritis encephalitis virus (CAEV), the first group of kids (I) was inoculated with purified, inactivated and adjuvant-treated virions, the second group (II) with adjuvant and the third one (III) with culture medium. 2-4 months later, the three groups were challenged with virulent CAEV by intraarticular route. On the clinical level, vaccinated and challenged kids show more early and severe arthritis than other groups. On the virological level, isolation of lentivirus from white blood cells and different organs is more important in group I than groups II and III. Therefore, vaccinations with inactivated and adjuvant-treated virions do not protect against a virulent challenge; there is an enhancement of lesions. We note that the adjuvant elicits a mild non-specific protection against virulent challenge.

Histopathological changes in lymph nodes of cats experimentally infected with the feline immunodeficiency virus (FIV).

Twelve specific-pathogen-free (SPF) kittens aged 8-12 weeks were serially infected in pairs every 6 weeks, by the intraperitoneal route, with the feline immunodeficiency virus (FIV). Three additional SPF kittens were kept as controls. The infected animals were killed 10 weeks after inoculation, during the primary phase of the FIV infection. Generalized lymphadenopathy (GL) was observed in the first three pairs of cats. All lymph nodes examined from the 12 infected cats showed histological changes. These included severe follicular hyperplasia with hyperactive follicular centres (FCs) which were either (1) naked, (2) infiltrated by lymphocytes, (3) seen to contain islets of lymphocytic mantle cells, or (4) disrupted by lymphocytes. The presence of both CD4+ and CD8+ T lymphocytes was demonstrated in the FCs immunocytochemically. The distribution of CD4 lymphocytes resembled that in control lymph nodes, but the CD8 cells were increased in number and either scattered or clustered in the follicles. In addition, varying degrees of interfollicular proliferation and medullary plasmacytosis were observed in the lymph nodes. These findings, which were common to all infected animals, represented distinct prodromal manifestations of FIV infection. The changes in lymphocyte subpopulation distribution observed in early FIV infection were reminiscent of findings encountered in human immunodeficiency virus (HIV) infection and reinforce the suggestion that FIV infection is an appropriate model for the study of HIV pathogenesis.

Preliminary report of familial thymic lymphosarcoma in Holstein calves.

Seventy-three cases of the thymic form of leukosis were found in Holstein calves in five departments of France over a period of five months. Most of the calves had been sired by the same bull. The calves were negative for specific antibodies to bovine leukaemia virus. Morphological studies including light and electron-microscopic cytology, and serological and virological studies of 14 of the cases suggest that the disease was transmitted genetically.

[Transmissible subacute spongiform encephalopathies in animals]. / Encéphalopathies spongiformes subaiguës transmissibles animales.

Transmissible subacute spongiform encephalopathies are a group of neurodegenerative diseases of man and of several species of domestic mammals. Scrapie has been recognised in sheep and goats for more than 250 years. Descriptions in 1986 of the first cases of bovine spongiform encephalopathy (BSE) in United Kingdom, its expansion with an enzootic pattern, and in 1996 of a new variant of Creutzfeldt-Jakob disease, probably related to the same agent, focused the attention of scientists, medias and public on what seems to be a new zoonosis. The biopathological study of BSE and scrapie is useful for evaluating what risk these animal diseases represent for human health.