Immortalization of primary sheep embryo kidney cells.

Sheep primary epithelial cells are short-lived in cell culture systems. For long-term in vitro studies, primary cells need to be immortalized. This study aims to establish and characterize T immortalized sheep embryo kidney cells (TISEKC). In this study, we used fetal lamb kidneys to derive primary cultures of epithelial cells. We subsequently immortalized these cells using the large T SV40 antigen to generate crude TISEKC and isolate TISEKC clones. Among numerous clones of immortalized cells, the selected TISEKC-5 maintained active division and cell growth over 20 passages but lacked expression of the oncogenic large T SV40 antigen. Morphologically, TISEKC-5 maintained their epithelial aspect similar to the parental primary epithelial cells. However, their growth properties showed quite different patterns. Crude TISEKC, as well as the clones of TISEKC proliferated highly in culture compared to the parental primary cells. In the early passages, immortalized cells showed heterogeneous polyploidy but in the late passages the karyotype of immortalized cells became progressively stable, identical to that of the primary cells, because the TISEKC-5 cell line has lost the large SV40 T antigen expression, this cell line is a valuable tool for veterinary sciences and biotechnological productions.

Can indoor sports centers be allowed to re-open during the COVID-19 pandemic based on a certificate of equivalence?

Within a time span of only a few months, the SARS-CoV-2 virus has managed to spread across the world. This virus can spread by close contact, which includes large droplet spray and inhalation of microscopic droplets, and by indirect contact via contaminated objects. While in most countries, supermarkets have remained open, due to the COVID-19 pandemic, authorities have ordered many other shops, restaurants, bars, music theaters and indoor sports centers to be closed. As part of COVID-19 (semi)lock-down exit strategies, many government authorities are now (May-June 2020) allowing a gradual re-opening, where sometimes indoor sport centers are last in line to be permitted to re-open. This technical note discusses the challenges in safely re-opening these facilities and the measures already suggested by others to partly tackle these challenges. It also elaborates three potential additional measures and based on these additional measures, it suggests the concept of a certificate of equivalence that could allow indoor sports centers with such a certificate to re-open safely and more rapidly. It also attempts to stimulate increased preparedness of indoor sports centers that should allow them to remain open safely during potential next waves of SARS-CoV-2 as well as future pandemics. It is concluded that fighting situations such as the COVID-19 pandemic and limiting economic damage requires increased collaboration and research by virologists, epidemiologists, microbiologists, aerosol scientists, building physicists, building services engineers and sports scientists.

Estudio histológico sobre el efecto de la quimioembolización con partículas precargadas con doxorrubicina en el hepatocarcinoma / Histologic study of the effects of chemoembolization with preloaded doxorubicin beads in patients with hepatocellular carcinoma

Objetivo. Determinar el grado de necrosis tumoral en la pieza quirúrgica de hepatocarcinomas tratados con microesferas precargadas con doxorrubicina (QETA-DEB) y su relación con factores morfológicos y biomarcadores de imagen. Material y Métodos. 21 pacientes a quienes se realizó quimioembolización arterial selectiva con partículas DC-Beads (Biocompatibles, UK) fueron posteriormente trasplantados y analizados los hígados histológicamente. Resultados. Se detectaron por diferentes técnicas de imagen 43 nódulos con un tamaño medio de 25 mm. Angiográficamente eran 25 nódulos hipervascularizados, 12 discretamente vascularizados y 6 avasculares. En la pieza se encontraron 81 HCCs con un tamaño medio de 15 mm.; dos presentaron infiltración vascular y otros dos infiltración capsular. La necrosis media conseguida tras QETA-DEB fue del 39% siendo >60% en 28 HCCs y <60% en 52. Encontramos correlación estadística entre el índice de necrosis conseguida con el tiempo que transcurre desde la última QETA-DEB hasta el trasplante (siendo significativamente menor la necrosis conforme aumenta el tiempo), con el número de nódulos encontrados en la pieza y con la infiltración capsular. Asimismo, podemos predecir que cuando por técnicas de imagen detectamos 1 ó 2 nódulos tendremos más probabilidad de conseguir de manera significativa índices de necrosis > 90%. Sin embargo, ni el tamaño de los nódulos detectados por técnicas de imagen, ni el patrón de realce post-contraste ni el número de QETA-DEBs realizadas influye significativamente en la necrosis conseguida. Conclusión. La necrosis producida depende del tiempo en lista de espera, del número de nódulos en la pieza y de la infiltración capsular del tumor (AU)

Objective. To determine the degree of tumor necrosis in surgical specimens of hepatocellular carcinomas treated with microspheres preloaded with doxorubicin and to analyze the relationship between the degree of necrosis and a) morphologic factors and b) imaging biomarkers. Material and methods. We studied the livers of 21 patients who had undergone selective arterial chemoembolization with DC beads (Biocompatibles, UK) before receiving liver transplants. Results. Imaging techniques detected 43 nodules (mean size, 25 mm). Angiography showed 25 hypervascularized nodules, 12 slightly vascularized nodules, and 6 avascular nodules. A total of 81 hepatocellular carcinomas (mean size, 15 mm) were detected in the specimens: two were capsular and two had vascular infiltration. The mean degree of necrosis after chemoembolization was 39%; necrosis was greater than 60% in 28 hepatocellular carcinomas and less than 60% in 52. The degree of necrosis correlated significantly with the time elapsed between the last chemoembolization treatment and liver transplantation (the degree of necrosis decreased as time increased), with the number of nodules in the specimen, and with capsular infiltration. When imaging techniques detected 1 or 2 nodules, there was a greater probability of achieving greater than 90% necrosis. No relation with the degree of necrosis achieved was found for the size of the nodules detected at imaging, the enhancement pattern, or the number of chemoembolization treatments. Conclusion. The degree of necrosis achieved depends on the time spent on the waiting list, on the number of nodules in the specimen, and on whether capsular infiltration is present (AU)

Histologic study of the effects of chemoembolization with preloaded doxorubicin beads in patients with hepatocellular carcinoma.

OBJECTIVE: To determine the degree of tumor necrosis in surgical specimens of hepatocellular carcinomas treated with microspheres preloaded with doxorubicin and to analyze the relationship between the degree of necrosis and a) morphologic factors and b) imaging biomarkers. MATERIAL AND METHODS: We studied the livers of 21 patients who had undergone selective arterial chemoembolization with DC beads (Biocompatibles, UK) before receiving liver transplants. RESULTS: Imaging techniques detected 43 nodules (mean size, 25 mm). Angiography showed 25 hypervascularized nodules, 12 slightly vascularized nodules, and 6 avascular nodules. A total of 81 hepatocellular carcinomas (mean size, 15 mm) were detected in the specimens: two were capsular and two had vascular infiltration. The mean degree of necrosis after chemoembolization was 39%; necrosis was greater than 60% in 28 hepatocellular carcinomas and less than 60% in 52. The degree of necrosis correlated significantly with the time elapsed between the last chemoembolization treatment and liver transplantation (the degree of necrosis decreased as time increased), with the number of nodules in the specimen, and with capsular infiltration. When imaging techniques detected 1 or 2 nodules, there was a greater probability of achieving greater than 90% necrosis. No relation with the degree of necrosis achieved was found for the size of the nodules detected at imaging, the enhancement pattern, or the number of chemoembolization treatments. CONCLUSION: The degree of necrosis achieved depends on the time spent on the waiting list, on the number of nodules in the specimen, and on whether capsular infiltration is present.

Digital squamous cell carcinoma in dogs: epidemiological, histological, and immunohistochemical study.

Squamous cell carcinoma represents 47.4% of all malignant canine digital lesions, but despite its frequency, there are few published studies available. Pathology submission records of 154 cases and follow-up of 49 animals were analyzed. On the 49 cases, histological evaluation was performed of the differentiation degree, mitotic index, presence of emboli, and immunohistochemical expression of vimentin and E-cadherin. The mean (SD) age of affected animals was 10.2 (2.3) years; no sex predisposition was recorded. Beauceron and Briard were 2 new overrepresented breeds. Dark-haired animals comprised 97 of 105 (92%); 94 dogs of 125 (75.2%) belonged to large and giant breeds. The forelimb was affected twice more than the hind limb. Probable metastases were observed in 4 dogs; new tumor development was recorded in 11 of 49 (22.4%). Epidemiologic factors, histological grade, mitotic index, and expression of immunohistochemical markers seemed not to be related to the clinical outcome.

A morphological study of 608 cases of canine malignant lymphoma in France with a focus on comparative similarities between canine and human lymphoma morphology.

This study reports cytomorphological, histomorphological, and immunological characterization of 608 biopsy cases of canine malignant lymphoma, with epidemiological and clinical data, collected from 7 French veterinary pathology laboratories. It compares morphological characteristics of malignant lymphoma in canines, per the updated Kiel classification system, with those reported in humans, per the World Health Organization (WHO) classification system. Of tumors described, 24.5% and 75.5% were classified as low- and high-grade malignant lymphomas, respectively. Presenting clinical signs included generalized or localized lymphadenopathy (82.4%) and extranodal diseases (17.6%) involving the skin (12.34%) and other sites (5.26%). Immunohistochemistry confirmed 63.8% B-cell (CD3-, CD79a+), 35.4% T-cell (CD3+, CD79a-), and 0.8% null-cell (CD3-, CD79a-) lymphomas. Most B-cell cases (38.49%) were of high-grade centroblastic polymorphic subtype; most T-cell cases (8.55%), high-grade pleomorphic mixed and large T-cell lymphoma subtypes. Some B-cell tumors showed morphologic characteristics consistent with follicular lymphomas and marginal zone lymphomas per the Revised European American Classification of Lymphoid Neoplasms and WHO canine classification systems and the WHO human classification system. Unusual high-grade B-cell subtypes included an atypical high-grade small B-cell lymphoma (0.66%), Burkitt-type B-cell lymphoma (1.64%), plasmacytoid lymphoma (0.99%), and mediastinal anaplastic large B-cell lymphoma (0.16%). Unusual T-cell subtypes included a previously undescribed high-grade canine immunoblastic T-cell type (1.15%), a rare low-grade prolymphocytic T-cell lymphoma (0.16%), and a recently described high-grade canine T-cell entity--aggressive granulocytic large-cell lymphoma (0.16%). Marginal zone lymphomas were common (10.86%); follicular lymphomas were rare (0.49%). Canine primary cutaneous malignant lymphoma subtypes were present (11.84%). There was no significant difference between B- and T-cell malignant lymphoma in regard to canine age and sex. A significant overrepresentation of Boxers (24.19%) was found for T-cell lymphomas.

Immunohistochemical detection of survivin in canine lymphoma.

Survivin is a member of the family of proteins known as 'inhibitors of apoptosis proteins'. Survivin has a role in cellular decisions concerning division and survival and is frequently expressed in neoplastic cells. The aim of the present study was to investigate immunohistochemically the expression of survivin in normal canine tissues and in canine lymphoma. A representative range of fetal and adult normal tissues as well as biopsy samples from dogs with lymphoma were assembled in tissue arrays. The lymphomas were classified according to the revised Kiel and to the Revised European American Lymphoma-World Health Organization (REAL-WHO) schemes. Polyclonal and monoclonal antisera cross-reactive with canine survivin identified cytoplasmic expression of the molecule in a broad range of normal canine cells. The same reagents demonstrated cytoplasmic labelling of more than 5% of cells in all 83 lymphoma samples tested with polyclonal antiserum and in 67 of 82 (82%) of samples tested with monoclonal antiserum. Survivin was expressed by a wide range of canine lymphoma subtypes, but the expression of this molecule in normal canine tissues must be considered if novel therapies targeting survivin are applied to the management of canine lymphoma.

Genetic and environmental risk indicators in canine non-Hodgkin's lymphomas: breed associations and geographic distribution of 608 cases diagnosed throughout France over 1 year.

BACKGROUND: The etiology of non-Hodgkin's lymphomas (NHL) is multifactorial. Environmental and genetic factors are frequently incriminated both in humans and dogs. OBJECTIVES: Our purpose was to study the geographic distribution of canine NHL (CNHL) in France and to evaluate genetic and environmental influences. ANIMALS: Six hundred and eight cases of CNHL, diagnosed throughout France over 1 year, were collected from 7 Veterinary Histopathologic Laboratories. METHODS: Retrospective study. Breeds affected by lymphomas were compared with the national population and associations between breed and immunophenotype were studied. The distribution of CNHL and canine T-cell NHL per 100,000 dogs per department was compared with the distribution of waste incinerators, polluted sites, and radioactive waste. RESULTS: The breeds significantly overrepresented among lymphoma cases were Boxer, Setter, and Cocker Spaniel (P < .001). There was a significant association between Boxer and T-cell NHL (P < .001), and between German Shepherd and Rottweiler and B-cell NHL (P < .01). The geographic distribution of CNHL and canine T-cell NHL indicated significant heterogeneity. Significant association between distributions of CNHL and waste incinerators (rho= 0.25, P < .05), polluted sites (rho= 0.36, P < .001), and radioactive waste (rho= 0.51, P < .001) was found. CONCLUSIONS AND CLINICAL IMPORTANCE: Influence of genetics in the development of CNHL was supported by the existence of an association between breed and immunophenotype. Waste incinerators, polluted sites, and radioactive waste could just be considered as risk indicators of CNHL, but not as risk factors. Case-control studies around critical sites are necessary to confirm the implication of those environmental factors in the development of CNHL.

[Histological study of the effect of pre-transplant chemoembolization of hepatocellular carcinoma]. / Estudio histopatológico del efecto de la quimioembolización pretrasplante en el hepatocarcinoma.

OBJECTIVE: To retrospectively evaluate the degree of necrosis brought about by chemoembolization of hepatocellular carcinoma by correlating the histological study of livers explanted in liver transplantations with morphological (number, size, histological type, encapsulation, and vascular invasion) and technical (angiographic patterns, number of sessions, and level of chemoembolization) factors of the tumors. MATERIAL AND METHODS: Seventeen cirrhotic patients that underwent chemoembolization of hepatocellular carcinoma prior to liver transplantation were studied. Chemoembolization consisted of introducing adriamycin, lipiodol, and particles of polyvinyl alcohol into the hepatic artery. The explanted livers were studied macroscopically and microscopically, evaluating the degree of necrosis achieved in each of the nodules found. RESULTS: A total of 32 nodules (26 hepatocellular carcinomas and 7 type II dysplastic nodules) were detected in 16 patients; the remaining patient had multiple hepatocellular carcinomas. The mean size of the hepatocellular carcinomas was 3.2 cms (range 0.4-7.5) and the mean size of the dysplastic nodules was 1.2 cm (range 1-1.7). Greater than 90% necrosis was achieved in 17 (68%) of the 25 hepatocellular carcinomas; 6 of these lesions were encapsulated and none had vascular infiltration. In the patient with multiple hepatocellular carcinomas (all were hypervascularized and had no capsule), the degree of necrosis achieved was < 30% in each lesion. In three patients with nodular vascular infiltration, multiple nodules were present; the necrosis achieved in these lesions ranged from 0%-70%, and extrahepatic metastases after transplantation. Eighteen of the hepatocellular carcinomas had a hypervascularized angiographic pattern and seven were hypovascular or avascular. In the seven type II dysplastic nodules (none of which was encapsulated), no necrosis was achieved. These nodules were found in three patients; all but one were avascular and coincided with simultaneous hepatocellular carcinomas in two patients. CONCLUSION: The greatest degree of necrosis was achieved in single, encapsulated, hypervascularized hepatocellular carcinomas without vascular infiltration. Chemoembolization fails to achieve necrosis in dysplastic nodules.

Estudio histopatológico del efecto de la quimioembolización pretrasplante en el hepatocarcinoma / Histological study of the effect of pre-transplant chemoembolization of hepatocellular carcinoma

Objetivo. Valorar retrospectivamente el grado de necrosis en el hepatocarcinoma producido por la quimioembolización hepática correlacionando el estudio histológico de los hígados explantados en el trasplante hepático con factores morfológicos del tumor (número, tamaño, tipo histológico, encapsulación e infiltración vascular) y técnicos (patrón angiográfico, número de sesiones y nivel de la quimioembolización). Material y métodos. Se estudian 17 pacientes cirróticos trasplantados hepáticos por hepatocarcinoma y a los que previamente se les realizó quimioembolización. Este procedimiento se realizó introduciendo en ramas de la arteria hepática adriamicina, lipiodol y partículas de alcohol de polivinilo. Los hígados explantados se estudiaron macro y microscópicamente, valorando el grado de necrosis alcanzado en cada uno de los nódulos encontrados. Resultados. En el hígado explantado se detectaron 32 nódulos en 16 pacientes: 25 hepatocarcinomas y 7 nódulos displásicos tipo II; en el paciente restante fueron múltiples hepatocarcinomas. El tamaño medio de los hepatocarcinomas fue de 3,2 cm (0,4-7,5) y el de los nódulos displásicos de 1,2 cm (1-1,7). En relación con los 25 hepatocarcinomas se consiguió una necrosis mayor del 90% en 17 de ellos (68%), 6 de los cuales estaban encapsulados y ninguno presentó infiltración vascular. En los pacientes con múltiples hepatocarcinomas la necrosis fue menor del 30% en cada uno de ellos (todos sin cápsula e hipervascularizados). En tres pacientes donde apareció infiltración vascular por alguno de los nódulos coincidió con multiplicidad de los mismos, necrosis del 0-70% de éstos y aparición de metástasis extrahepáticas en el período postrasplante. Dieciocho de los hepatocarcinomas tenían un patrón angiográfico hipervascularizado y 7 eran hipo/avasculares. En relación con los 7 nódulos displásicos tipo II (todos no encapsulados) la necrosis fue nula en todos ellos; aparecieron en tres pacientes, todos (excepto uno) eran avasculares y coincidieron con hepatocarcinomas simultáneos en dos pacientes. Conclusión. Las mayores necrosis parecen obtenerse en hepatocarcinonas únicos, encapsulados, hipervascularizados y sin infiltración vascular. La necrosis por el procedimiento es nula en los nódulos displásicos (AU)

Objective. To retrospectively evaluate the degree of necrosis brought about by chemoembolization of hepatocellular carcinoma by correlating the histological study of livers explanted in liver transplantations with morphological (number, size, histological type, encapsulation, and vascular invasion) and technical (angiographic patterns, number of sessions, and level of chemoembolization) factors of the tumors. Material and methods. Seventeen cirrhotic patients that underwent chemoembolization of hepatocellular carcinoma prior to liver transplantation were studied. Chemoembolization consisted of introducing adriamycin, lipiodol, and particles of polyvinyl alcohol into the hepatic artery. The explanted livers were studied macroscopically and microscopically, evaluating the degree of necrosis achieved in each of the nodules found. Results. A total of 32 nodules (26 hepatocellular carcinomas and 7 type II dysplastic nodules) were detected in 16 patients; the remaining patient had multiple hepatocellular carcinomas. The mean size of the hepatocellular carcinomas was 3.2 cms (range 0.4-7.5) and the mean size of the dysplastic nodules was 1.2 cm (range 1-1.7). Greater than 90% necrosis was achieved in 17 (68%) of the 25 hepatocellular carcinomas; 6 of these lesions were encapsulated and none had vascular infiltration. In the patient with multiple hepatocellular carcinomas (all were hypervascularized and had no capsule), the degree of necrosis achieved was < 30% in each lesion. In three patients with nodular vascular infiltration, multiple noduleswere present; the necrosis achieved in these lesions ranged from 0%-70%, and extrahepatic metastases after transplantation. Eighteen of the hepatocellular carcinomas had a hypervascularized angiographic pattern and seven were hypovascular or avascular. In the seven type II dysplastic nodules (none of which was encapsulated), no necrosis was achieved. These nodules were found in three patients; all but one were avascular and coincided with simultaneous hepatocellular carcinomas in two patients. Conclusion. The greatest degree of necrosis was achieved in single, encapsulated, hypervascularized hepatocellular carcinomas without vascular infiltration. Chemoembolization fails to achieve necrosis in dysplastic nodules (AU)

Severe necrotizing encephalitis in a Yorkshire terrier: topographic and immunohistochemical study.

Necrotizing encephalitis of the Yorkshire terrier is a chronic non-suppurative encephalitis that was reported in approximately 15 cases worldwide. We report the case of a 10-year-old female Yorkshire terrier with gross evidence of severe cortical degeneration and necrosis. Microscopically, affected areas were mainly located in the cortical white matter and in the mesencephalon without implication of the cerebellum. Cavitation necrosis, demyelination, gemistocytic astrocytosis, marked perivascular lymphocytic cuffing with a diffuse lymphocytic/histiocytic/gitter cell infiltration characterized the lesions. Immunohistochemical analysis identified the major infiltration of T lymphocytes and macrophages with implication of some cytotoxic lymphocytes and IgG-producing plasma cells; depositions of IgG in the affected white matter were also observed. Specific stains did not reveal fungal, protozoal or bacterial organisms and reverse transcriptase-polymerase chain reaction analysis for distemper virus was also negative. The lympho-histiocytic inflammation suggests a T-cell-mediated and a delayed-type immune reaction as a possible pathogenic mechanism for this brain disorder.

Adjuvant chemotherapy for prevention of recurrence of invasive hepatocellular carcinoma after orthotopic liver transplantation.

UNLABELLED: Orthotopic liver transplantation (OLT) is the best treatment for nonresectable hepatocellular carcinoma (HCC), but tumor recurrence reduces long-term and medium-term survival. The effectiveness of adjuvant chemotherapy to prevent tumor recurrence has not been fully established. METHODS: Three hundred eighty-seven consecutive patients, including 43 with HCC superimposed on liver cirrhosis, underwent OLT. Twelve patients with one or more prognostic criteria for HCC recurrence were entered into a prospective prophylaxis protocol with monthly cycles of cisplatin (60 mg/m(2)) and adriamycin (30 mg/m(2)), beginning the fourth week post-OLT for a maximum of seven sessions. RESULTS: The 5-year survival of the non-HCC patients was 65.7% and that of the HCC patients was 60.46% (P = NS). Chemotherapy was reasonably well tolerated, but the 9 patients with hepatitis C- or B-associated cirrhosis showed viral and histological recurrence of the primary disease. A high proportion of patients (7 of 12) developed tumor recurrence during the first year after OLT. Six of these patients died, all but one due to HCC relapse. Five patients remain healthy and tumor free at 58 to 130 months. Post-OLT adjuvant chemotherapy does not avoid tumor recurrence and its fatal consequences but may contribute to prolonged tumor-free survival among a significant proportion of patients with high-risk HCC. However, the uncertain implications on viral recurrence and the lack of control groups do not allow post-OLT chemotherapy to be recommended outside controlled clinical trials, which are clearly warranted.

Mycobacterium tuberculosis infection in a dog from Africa.

A 4-year-old male Boxer dog with a history of vomiting, diarrhea, and weight loss moved from West Africa to Lyon, France, where it was further evaluated. Radiographs revealed pleural effusion and enlargement of tracheobronchial lymph nodes and liver. Cytologic examination of the pleural effusion and a fine needle aspirate specimen of the liver showed mixed mononuclear inflammation with nonstaining rod structures within epithelioid histiocytes. At necropsy, the main gross pathologic findings were exudative pleuritis, nodular hepatitis, and infarcts and caseous nodules in the kidneys. The main histologic lesions were granulomatous hepatitis, granulomatous pneumonia, fibrinous leukocytic pleuritis, necrotic and fibro-calcified granulomatous lymphadenitis, and granulomatous nephritis. A Ziehl-Neelsen stain applied to both cytologic and histologic samples was positive for acid-fast bacilli. Bacterial culture of the pleural fluid was positive for Mycobacterium tuberculosis. Cytology is a valuable tool in the diagnosis of this important zoonotic disease.

High-grade canine T-cell lymphoma/leukemia with plasmacytoid morphology: a clinical pathological study of nine cases.

The aim of this study is to determine the clinical, morphological, and immunophenotypical presentation of 9 cases of a particular type of canine T-cell lymphoma/leukemia. The morphological presentation was a diffuse infiltration of small, medium-sized, or large blast cells with eccentric nuclei, hyperbasophilic cytoplasm, and a juxtanuclear, pale cytoplasmic area, giving a plasmacytoid appearance and suggesting a B-cell morphology. Surprisingly, all 9 cases were of T-cell phenotype (CD3+). Among the 7 immunophenotyped cases, 4 were CD4-/CD8+, 2 CD8+/CD4+, and 1 CD4+/CD8-. The median Ki-67 index was 65.7%, which placed this lymphoma in the high-grade group. This type of lymphoma/leukemia was found in dogs between 1 and 11 years of age, with a median age of 5.8. The male-female ratio was 0.8 for a reference population of 1.04. The most significant clinical findings were lymphadenopathy either generalized or localized in all cases, a mediastinal mass in 4 cases, bone marrow involvement in 7 cases, hypercalcemia in 4 cases, along with an aggressive clinical course and a poor response to chemotherapy in all cases, with a median disease-free survival time of 3 months.

Probing complex RNA structures by mechanical force.

RNA secondary structures of increasing complexity are probed combining single molecule stretching experiments and stochastic unfolding/refolding simulations. We find that force-induced unfolding pathways cannot usually be interpreted by solely invoking successive openings of native helices. Indeed, typical force-extension responses of complex RNA molecules are largely shaped by stretching-induced, long-lived intermediates including non-native helices. This is first shown for a set of generic structural motifs found in larger RNA structures, and then for Escherichia coli's 1540-base long 16S ribosomal RNA, which exhibits a surprisingly well-structured and reproducible unfolding pathway under mechanical stretching. Using out-of-equilibrium stochastic simulations, we demonstrate that these experimental results reflect the slow relaxation of RNA structural rearrangements. Hence, micromanipulations of single RNA molecules probe both their native structures and long-lived intermediates, so-called "kinetic traps", thereby capturing -at the single molecular level- the hallmark of RNA folding/unfolding dynamics.

Canine T-cell lymphomas: a morphological, immunological, and clinical study of 46 new cases.

The aim of this study is to report 46 new cases of canine T-cell lymphomas among a series of 140 lymphomas studied by immunophenotyping (incidence 32.8%). According to the updated Kiel classification adapted to the canine species, 13 were classified as low-grade and 33 as high-grade lymphomas. Among the low-grade lymphomas, five were small clear-cell lymphomas, three were pleomorphic small-cell lymphomas, and five mycosis fungoides. Among the high-grade cases, there were 11 pleomorphic mixed-, small-, and large-cell lymphomas, 6 pleomorphic large-cell lymphomas, 11 lymphoblastic lymphomas, and 5 unclassifiable high-grade plasmacytoid lymphomas. The cytohistologic features were highly suggestive of a T-cell phenotype on the basis of cell morphology (irregular nuclei and clear cytoplasms) (30/46 cases), a T-cell zone pattern, and the presence of hyperplastic postcapillary venules (22/46 cases). All 46 cases were CD3+ CD79a-, and among 34 cases investigated for CD4 and CD8 expression, 13 were CD4+CD8-, 13 were CD8+CD4-, and 8 were CD4CD8 double positive or double negative. The pleomorphic mixed lymphomas were mainly CD4+CD8- (6/7) and the lymphoblastic lymphomas were double positive or double negative (6/8). The main clinical, hematologic, and biochemical features were generalized (28/46) or regional lymphadenopathy (16/46), hepatosplenomegaly (15/46), extranodal involvement (11/46), mediastinal mass (9/46), and leukemia (8/46), which were mainly present in cases of lymphoblastic lymphomas and hypercalcemia (16/46).

Cellular immune response of lymph nodes from dogs following the intradermal injection of a recombinant antigen corresponding to a 66 kDa protein of Echinococcus granulosus.

A recombinant polypeptide (referred to as EgA31), which represents a 66kDa protein, was prepared from an Echinococcus granulosus cDNA library. In order to assess its potential to induce cellular immune responses, dog popliteal and prescapular lymph nodes were sensitized with this recombinant polypeptide. Subpopulations of lymphocytes were then analyzed by flow cytometry and immunohistochemistry on lymph node sections. Five days after the sensitization, the paracortical areas of the lymph nodes appeared hypertrophic, the number of CD3+, CD4+, CD8+ and CD5+ cells increased, the number of B-cells began to augment and some secondary follicles occurred, and a number of CD4+ cells appeared in germinal centers. Many large secondary follicles and a significantly augmented number of CD5+ cells in cords of medullae were observed 10 days after the sensitization. These active cellular responses strengthen the interest for further studies on the development of a vaccine with this recombinant polypeptide.

Effect of PGE1 on TNF-alpha status and hepatic D-galactosamine-induced apoptosis in rats.

Prostaglandin E1 has hepatoprotective properties in several clinical and experimental models of liver dysfunction. Hepatotoxicity induced by D-galactosamine (D-GalN) is a suitable animal model of human acute hepatic failure. The aim of the study was to investigate if prostaglandin E1 (PGE1) protection against hepatic D-GalN-induced apoptosis was related to tumour necrosis factor-alpha (TNF-alpha) content in serum. This cytokine is associated with in vitro apoptosis and general inflammatory disorders. In this study, PGE1 was administered 30 min before D-GalN to rats. In other experiments, several doses of TNF-alpha were administered 15min after PGE1 to D-Ga1N-treated rats. Several parameters related to apoptosis and necrosis were measured by flow cytometry, gel electrophoresis, biochemical analysis, and optical and electron microscopy. Tumour necrosis factor-alpha was quantified by competitive enzyme-linked immunosorbent assay (ELISA). PGE1 by itself did not modify the cell cycle of hepatocytes and liver toxicity, but increased TNF-alpha in serum in comparison with the control group. D-Galactosamine increased the percentage of hepatocytes in apoptosis and in the S phase of the cell cycle, and decreased those in G0/G1. Such an increase of hepatocytes in apoptosis was correlated with a higher number of apoptotic bodies and DNA fragmentation in liver than control samples. Also, D-GalN increased alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase and TNF-alpha in serum compared with the control group. Pre-administration of PGE1 to D-GalN-treated rats reduced all the parameters of apoptosis and necrosis in liver, and increased additionallyTNF-alpha content in serum. In those experiments where low doses of TNF-alpha were administered to PGE1 and D-GalN-treated rats an inverse relationship appeared between TNF-alpha and ALT content in serum. In conclusion, the protective effects of PGE1 on D-GalN-induced apoptosis may be linked to its capacity to modulate cell division and/or its immunomodulatory activity. In this sense, our experimental results suggest that TNF-alpha could be involved in protection or exacerbation of liver damage in relation to the pathophysiological status of the liver.