Development of an oral nanovaccine for dogs against Echinococcus granulosus.

Dogs are the main source of animal and human cystic echinococcosis caused by the Cestode parasite Echinococcus granulosus. Dog vaccination seems to be a good strategy to control this parasitic disease. Here we present the development of a polymeric nanoparticle-based oral vaccine for dogs against Echinococcus granulosus delivered in enteric-coated capsules. To achieve our target, we encapsulated two recombinant antigens into biodegradable polymeric nanoparticles in the presence of Monophosphoryl lipid A as an adjuvant to ensure efficient delivery and activation of a protective mucosal immune response. The formulated delivery system showed a nanoparticle size less than 200 nm with more than 80 % antigen encapsulation efficiency and conserved integrity and immunogenicity. The nanoparticle surface was coated with chitosan to enhance adhesion to the gut mucosa and a subsequent antigen delivery. Chitosan-coated nanoparticles showed a higher cell internalization in murine macrophages and dendritic cells as well as a higher penetration into Caco-2 cells in vitro. Antigen-loaded nanoparticles were freeze-dried and enteric-coated capsules were filled with the obtained powder. The obtained results show a promising nanoparticles delivery system for oral vaccination.

Implication of Netrin-1 Gain of Expression in Canine Nodal Lymphoma.

Netrin-1 is a member of the laminin superfamily, and is known to interact with specific receptors, called dependence receptors. While upon netrin-1 binding these receptors initiate positive signaling, in absence of netrin-1, these receptors trigger apoptosis. Tumor cells can avoid apoptosis by inactivating these receptors or by gaining ligand expression. The aim of the present study was to investigate the expression of netrin-1, the ligand of dependence receptors, in canine healthy lymph nodes (LN), and in lymphomas and to evaluate efficiency of a netrin-1 interfering compound in cell cultures from canine lymphoma. Thirty-two control LN and 169 lymphomas were analyzed through immunohistochemistry. Netrin-1 was expressed in the nucleoli of lymphoid and non-lymphoid cells in controls. Acquisition of a cytoplasmic expression was present in B-cell lymphomas (23.1 % in low-grade and 50.6% in high-grade) and T-cell lymphomas (50.0 % in low-grade and 78.8 % in high-grade), with a significant difference between the high- and low-grade in B-cell lymphomas. Through flow cytometry, we showed a significant increase in netrin-1 expression in either high-grade B-cell and T-cell lymphomas (19 and 5, respectively) compared with healthy LN (5), likewise an RT-qPCR analysis demonstrated a significant increase in netrin-1 expression level in 14 samples of lymphomas compared with eight samples of healthy LN. A T-cell aggressive canine lymphoma cell line and four primary canine nodal lymphomas cell cultures were treated with a netrin-1 interfering antibody. Apoptosis by measuring caspase 3 activity was significantly increased in the cell line and viability was decreased in three of the four primary cell cultures. Together, these data suggest that netrin-1 expression is increased in lymphoma, and more specifically in high-grade lymphomas, and that netrin-1 can act as a survival factor for the neoplastic cells, and so be a therapeutic target.

Toward A Regulatory Pathway for the Use of in Silico Trials in the CE Marking of Medical Devices.

In Silico Trials methodologies will play a growing and fundamental role in the development and de-risking of new medical devices in the future. While the regulatory pathway for Digital Patient and Personal Health Forecasting solutions is clear, it is more complex for In Silico Trials solutions, and therefore deserves a deeper analysis. In this position paper, we investigate the current state of the art towards the regulatory system for in silico trials applied to medical devices while exploring the European regulatory system toward this topic. We suggest that the European regulatory system should start a process of innovation: in principle to limit distorted quality by different internal processes within notified bodies, hence avoiding that the more innovative and competitive companies focus their attention on the needs of other large markets, like the USA, where the use of such radical innovations is already rapidly developing.

Anatomical features of the canine C2-C3 spinal cord vascular environment.

OBJECTIVE: Interarcuate branch (IAB) is a vascular structure, particularly developed in C2-3 intervertebral space, forming a dorsal bridge that connects ventral venous plexi in the vertebral canal. While precisely described in the human, the precise anatomical features of IABs have not been reported in the veterinary literature. The purpose of this study is to describe the features and relations of IABs in the C2-3 vertebral canal. ANIMALS: 10 dogs were enrolled; 5 dogs for necropsy and 5 dogs for histology. PROCEDURES: The ventral venous plexi in the cervical spine of 5 dogs were injected with latex and underwent vertebral canal dissection for visual assessment of the IAB. Two out of 5 dogs were injected with the addition of barium sulfate and underwent a CT scan. The C2-3 regions of 5 small-breed dogs were harvested for histological examinations. RESULTS: IABs arose from the ventral venous plexus at the level of the intervertebral vein; they originated from 2 separate branches located caudally and cranially to the intervertebral foramen, forming a ventrodorsal triangle surrounding the spinal nerve root. No dorsal anastomosis was observed on the CT scan nor at dissection but were observed histologically. A cervical fibrous sheath was observed all around the vertebral canal. CLINICAL RELEVANCE: IABs are voluminous venous structures at the C2-3 intervertebral space in dogs and found within a split of the cervical fibrous sheath, which is adherent to the interarcuate ligament and the ligamentum flavum. This anatomical description is paramount when planning an approach to the C2-3 intervertebral space.

Microbial risk assessment of Nocardia cyriacigeorgica in polluted environments, case of urban rainfall water.

Urban Infiltration Basins (UIBs) are used to manage urban runoff transfers and feed aquifers. These UIBs can accumulate urban pollutants and favor the growth of potentially pathogenic biological agents as Nocardia. OBJECTIVES: To assess the spatio-temporal dynamics of pathogenic Nocardia in UIBs and to stablish phylogenetic relationships between clinical and UIB N. cyriacigeorgica strains. To assess pathogenicity associated with environmental N. cyriacigeorgica using an animal model, and to identify genetic elements that may be associated to its virulence. METHODS: A well-characterized UIB in terms of chemical pollutants from Lyon area was used in this study during a whole year. Cultural and Next-Generation-Sequencing methods were used for Nocardia detection and typing. Clinical and environmental isolates phylogenetic relationships and virulences were compared with Multilocus-Sequence-Analysis study together with a murine model. RESULTS: In autumn, N. cyriacigeorgica and N. nova were the pathogenic most prevalent species in the UIB. The complex N. abscessus/asiatica was also detected together with some other non-pathogenic species. The presence of pathogenic Nocardia was positively correlated to metallic trace elements. Up to 1.0 × 103 CFU/g sediment of N. cyriacigeorgica and 6 OTUs splited in two different phylogroups were retrieved and were close to clinical strains. The EML446 tested UIB isolate showed significant infectivity in mice with pulmonary damages similar to clinical clone (GUH-2). CONCLUSION: Hsp65 marker-based metabarcoding approach allowed detecting N. cyriacigeogica as the most abundant Nocardia pathogenic species in a UIB. Metal trace elements-polluted environments can be reservoirs of pathogenic Nocardia which may have a similar virulence to clinical strains.

FOXP3, CD208, and CD206 Expression in Canine Cutaneous Histiocytoma.

Canine cutaneous histiocytoma (CCH) is a noninfectious tumor that spontaneously regresses. It is suggested that this regression is due to tumor cell maturation, which is responsible for CD8 lymphocyte activation and tumor cell destruction. Nevertheless, the possible role of the immune microenvironment in tumor regression has not been investigated to date. The aim of this study was to investigate the expression of CD208 and FoxP3 as markers of dendritic cells and regulatory T lymphocytes, respectively, and tumor cell expression of CD206 as a marker of Langerhans cell activation, and relate these parameters to the different phases of CCH and to intratumoral T cell infiltration. Formalin-fixed, paraffin-embedded samples from 31 CCH were evaluated. In each case, the mitotic count and regression phase were recorded. Within the tumor, a quantitative evaluation of immunolabeled CD208+ cells, FoxP3+ cells, and CD3+ lymphocytes was performed, as well as the CD206+ tumor cell location. Intratumoral CD208+ cells correlated with CD3+ lymphocytic infiltration. The possible role of dendritic cells in tumor regression was not confirmed since CD208 seemed to be a nonspecific marker for canine dendritic cells. FoxP3+ lymphocyte density was not correlated with any parameter. Neoplastic Langerhans cells presented progressive CD206 expression, from the bottom of the tumor to the epidermis, which correlated with the tumor regression phase and with intratumoral T lymphocyte infiltration. In conclusion, we confirmed a CD206 phenotype change in tumor cells in a spatial group-related pattern, supporting the hypothesis that tumoral Langerhans cells acquire a mature phenotype with tumor regression.

Influence of wind and relative humidity on the social distancing effectiveness to prevent COVID-19 airborne transmission: A numerical study.

It has been confirmed that the coronavirus disease 2019 (COVID-19) can transmit through droplets created when an infected human coughs or sneezes. Accordingly, 1.83-m (6-feet) social distancing is advised to reduce the spread of the disease among humans. This is based on the assumption that no air circulation exists around people. However, it is not well investigated whether the ambient wind and relative humidity (RH) will cause SARS-CoV-2 laden droplets to transport farther in the air, and make the current social distancing policy insufficient. To provide evidence and insight into the "social distancing" guidelines, a validated computational fluid-particle dynamics (CFPD) model was employed to simulate the transient transport, condensation/evaporation, and deposition of SARS-CoV-2 laden droplets emitted by coughs, with different environmental wind velocities and RHs. Initial droplet diameters range from 2 to 2000 µm, and the wind velocities range from 0 to 16 km/h, representing different wind forces from calm air to moderate breeze. The comparison between a steady-state wind and a gust with a constant frequency has also been performed. Ambient RHs are 40% and 99.5%. The distances between the two virtual humans are 1.83 m and 3.05 m (6 feet and 10 feet). The facial covering effect on reducing the airborne transmission of the cough droplets has also been evaluated. Numerical results indicate that the ambient wind will enhance the complexity of the secondary flows with recirculation between the two virtual humans. Microdroplets follow the airflow streamlines well and deposit on both human bodies and head regions, even with the 3.05-m (10-feet) separation distance. The rest of the microdroplets can transport in the air farther than 3.05 m (10 feet) due to wind convection, causing a potential health risk to nearby people. High RH will increase the droplet sizes due to the hygroscopic growth effect, which increases the deposition fractions on both humans and the ground. With the complex environmental wind and RH conditions, the 6-feet social distancing policy may not be sufficient to protect the inter-person aerosol transmission, since the suspending micro-droplets were influenced by convection effects and can transport from the human coughs/sneezes to the other human in less than 5 seconds. Due to the complex real-world environmental ventilation conditions, a social distance longer than 1.83 m (6 feet) needs to be considered. Wearing masks should also be recommended for both infected and healthy humans to reduce the airborne cough droplet numbers.

Interplay between enterohaemorrhagic Escherichia coli and nitric oxide during the infectious process.

Enterohaemorrhagic Escherichia coli (EHEC) are bacterial pathogens responsible for life-threatening diseases in humans such as bloody diarrhoea and the hemolytic and uremic syndrome. To date, no specific therapy is available and treatments remain essentially symptomatic. In recent years, we demonstrated in vitro that nitric oxide (NO), a major mediator of the intestinal immune response, strongly represses the synthesis of the two cardinal virulence factors in EHEC, namely Shiga toxins (Stx) and the type III secretion system, suggesting NO has a great potential to protect against EHEC infection. In this study, we investigated the interplay between NO and EHEC in vivo using mouse models of infection. Using a NO-sensing reporter strain, we determined that EHEC sense NO in the gut of infected mice. Treatment of infected mice with a specific NOS inhibitor increased EHEC adhesion to the colonic mucosa but unexpectedly decreased Stx activity in the gastrointestinal tract, protecting mice from renal failure. Taken together, our data indicate that NO can have both beneficial and detrimental consequences on the outcome of an EHEC infection, and underline the importance of in vivo studies to increase our knowledge in host-pathogen interactions.

In vitro evaluations on canine monocyte-derived dendritic cells of a nanoparticles delivery system for vaccine antigen against Echinococcus granulosus.

Since dogs play a central role in the contamination of humans and livestock with Echinococcus granulosus, the development of an effective vaccine for dogs is essential to control the disease caused by this parasite. For this purpose, a formulation based on biodegradable polymeric nanoparticles (NPs) as delivery system of recombinant Echinococcus granulosus antigen (tropomyosin EgTrp) adjuved with monophosphoryl lipid A (MPLA) has been developed. The obtained nanoparticles had a size of approximately 200 nm in diameter into which the antigen was correctly preserved and encapsulated. The efficiency of this system to deliver the antigen was evaluated in vitro on canine monocyte-derived dendritic cells (cMoDCs) generated from peripheral blood monocytes. After 48 h of contact between the formulations and cMoDCs, we observed no toxic effect on the cells but a strong internalization of the NPs, probably through different pathways depending on the presence or not of MPLA. An evaluation of cMoDCs activation by flow cytometry showed a stronger expression of CD80, CD86, CD40 and MHCII by cells treated with any of the tested formulations or with LPS (positive control) in comparison to cells treated with PBS (negative control). A higher activation was observed for cells challenged with EgTrp-NPs-MPLA compared to EgTrp alone. Formulations with MPLA, even at low ratio of MPLA, give better results than formulations without MPLA, proving the importance of the adjuvant in the nanoparticles structure. Moreover, autologous T CD4+ cell proliferation observed in presence of cMoDCs challenged with EgTrp-NPs-MPLA was higher than those observed after challenged with EgTrp alone (p<0.05). These first results suggest that our formulation could be used as an antigen delivery system to targeting canine dendritic cells in the course of Echinococcus granulosus vaccine development.

Milk Fat Globules Hamper Adhesion of Enterohemorrhagic Escherichia coli to Enterocytes: In Vitro and in Vivo Evidence.

Enterohemorrhagic Escherichia coli (EHEC; E. coli) are food-borne agents associated with gastroenteritis, enterocolitis, bloody diarrhea and the hemolytic-uremic syndrome (HUS). Bovine milk glycans have been shown to contain oligosaccharides which are similar to host epithelial cell receptors and can therefore prevent bacterial adhesion. This study aimed to describe interactions between EHEC O157:H7 EDL933 and O26:H11 21765 and milk fat globules (MFGs) in raw milk and raw milk cheese, and the impact of MFGs on EHEC strains adhesion to the intestinal tract in vitro and in vivo. Both EHEC serotypes clearly associated with native bovine MFGs and significantly limited their adhesion to a co-culture of intestinal cells. The presence of MFGs in raw milk cheese had two effects on the adhesion of both EHEC serotypes to the intestinal tracts of streptomycin-treated mice. First, it delayed and reduced EHEC excretion in mouse feces for both strains. Second, the prime implantation site for both EHEC strains was 6 cm more proximal in the intestinal tracts of mice fed with contaminated cheese containing less than 5% of fat than in those fed with contaminated cheese containing 40% of fat. Feeding mice with 40% fat cheese reduced the intestinal surface contaminated with EHEC and may therefore decrease severity of illness.

Characterization of a novel canine T-cell line established from a spontaneously occurring aggressive T-cell lymphoma with large granular cell morphology.

Dogs with lymphoma are established as good model for human non-Hodgkin lymphoma studies. Canine cell lines derived from lymphomas may be valuable tools for testing new therapeutic drugs. In this context, we established a canine T-cell line, PER-VAS, from a primary aggressive T-cell lymphoma with large granular morphology. Flow cytometric analysis revealed a stable immunophenotype: PER-VAS cells were positively labelled for CD5, CD45, MHC II and TLR3, and were negative for CD3, CD4 and CD8 expression. Although unstable along the culture process, IL-17 and MMP12 proteins were detectable as late as at passages 280 and 325i.e. respectively 24 and 29 months post isolation. At passage 325, PER-VAS cells maintained the expression of IL-17, CD3, CD56, IFNγ and TNFα mRNAs as shown by RT-PCR analysis. Stable rearrangement of the TCRγ gene has been evidenced by PCR. PER-VAS cells have a high proliferation index with a doubling time of 16.5h and were tumorigenic in Nude mice. Compared to the canine cell lines already reported, PER-VAS cells display an original expression pattern, close to NKT cells, which makes them valuable tools for in vitro comparative research on lymphomas.

Phase I Clinical Pharmacology Study of F14512, a New Polyamine-Vectorized Anticancer Drug, in Naturally Occurring Canine Lymphoma.

PURPOSE: F14512 is a new topoisomerase II inhibitor containing a spermine moiety that facilitates selective uptake by tumor cells and increases topoisomerase II poisoning. F14512 is currently in a phase I/II clinical trial in patients with acute myeloid leukemia. The aim of this study was to investigate F14512 potential in a new clinical indication. Because of the many similarities between human and dog lymphomas, we sought to determine the tolerance, efficacy, pharmacokinetic/pharmacodynamic (PK/PD) relationship of F14512 in this indication, and potential biomarkers that could be translated into human trials. EXPERIMENTAL DESIGN: Twenty-three dogs with stage III-IV naturally occurring lymphomas were enrolled in the phase I dose-escalation trial, which consisted of three cycles of F14512 i.v. injections. Endpoints included safety and therapeutic efficacy. Serial blood samples and tumor biopsies were obtained for PK/PD and biomarker studies. RESULTS: Five dose levels were evaluated to determine the recommended dose. F14512 was well tolerated, with the expected dose-dependent hematologic toxicity. F14512 induced an early decrease of tumoral lymph node cells, and a high response rate of 91% (21/23) with 10 complete responses, 11 partial responses, 1 stable disease, and 1 progressive disease. Phosphorylation of histone H2AX was studied as a potential PD biomarker of F14512. CONCLUSIONS: This trial demonstrated that F14512 can be safely administered to dogs with lymphoma resulting in strong therapeutic efficacy. Additional evaluation of F14512 is needed to compare its efficacy with standards of care in dogs, and to translate biomarker and efficacy findings into clinical trials in humans.

Spinal neurenteric cyst in a dog.

A 2-year-old female crossbreed dog was presented with progressive ataxia and paraparesis. A T3-L3 spinal lesion was determined by neurological examination. Magnetic resonance imaging (MRI) revealed an ovoid-shaped, well-circumscribed mass affecting the spinal cord at the level of the T9 vertebra. A left hemilaminectomy and a durotomy at the level of T9 allowed discovery of an ovoid deformation of the meninges with a cystic appearance. En bloc removal was performed and appeared to be complete. Pathological analysis showed a voluminous cystic lesion lined by a heterogeneous epithelium. Three types of epithelium were present: a pseudostratified columnar epithelium, a stratified squamous epithelium and a transitional epithelium. Mucus production, the morphology of some cells with microvilli at the apical pole and immunohistochemical assays were highly in favor of an endodermal origin of the cyst. The age of the dog, anamnesis, MRI study and histological findings were consistent with an intradural neurenteric cyst as described in humans. Total surgical removal led to a progressive clinical improvement with no recurrence at 18 months. We report an unusual intradural extramedullary cyst, called a neurenteric cyst, in a 2-year-old female crossbreed dog. This type of cyst is well-known in humans but has never been described in dogs. We propose that neurenteric cysts should be included in the differential diagnoses for tumor-like or cystic intradural lesions in the young dog. Prognosis for this type of cyst seems to be good, as total surgical removal led to a progressive clinical improvement with no recurrence at 18 months.

Presumed primary muscular lymphoma in a dog.

A case of presumed primary muscular lymphoma in an 8-year-old, intact, male Newfoundland dog is reported. The dog was presented for evaluation of an infiltrating ventral cervical mass, respiratory distress, and anorexia of 1-month duration. Fine-needle aspiration of the mass revealed anaplastic large cell lymphoma. Despite chemotherapy, health status declined and the animal was euthanized a few weeks later. At necropsy, the mass infiltrated the cervical muscles and extended ventrally to the left forelimb and cranially to the tongue and laryngeal musculature. Other muscles were infiltrated by the same neoplasm (diaphragm and intercostal, abdominal, and gluteal muscles) indicating a probable multicentric origin. Histological examination confirmed the diagnosis of anaplastic large cell lymphoma, which showed a strong muscular tropism. Immunohistochemical staining revealed neoplastic cell reactivity for cluster of differentiation 3 (CD3) and Ki-67 antigens (70% and 90%, respectively). The neoplastic cells were negative for CD79a. The presumed histological diagnosis in this dog was primary muscular anaplastic large T-cell lymphoma.

An oral recombinant vaccine in dogs against Echinococcus granulosus, the causative agent of human hydatid disease: a pilot study.

Dogs are the main source of human cystic echinococcosis. An oral vaccine would be an important contribution to control programs in endemic countries. We conducted two parallel experimental trials in Morocco and Tunisia of a new oral vaccine candidate against Echinococcus granulosus in 28 dogs. The vaccine was prepared using two recombinant proteins from adult worms, a tropomyosin (EgTrp) and a fibrillar protein similar to paramyosin (EgA31), cloned and expressed in a live attenuated strain of Salmonella enterica serovar typhimurium.In each country, five dogs were vaccinated with the associated EgA31 and EgTrp; three dogs received only the vector Salmonella; and six dogs were used as different controls. The vaccinated dogs received two oral doses of the vaccine 21 d apart, and were challenged 20 d later with 75,000 living protoscoleces. The controls were challenged under the same conditions. All dogs were sacrificed 26-29 d postchallenge, before the appearance of eggs, for safety reasons.We studied the histological responses to both the vaccine and control at the level of the duodenum, the natural localization of the cestode. Here we show a significant decrease of parasite burden in vaccinated dogs (70% to 80%) and a slower development rate in all remaining worms. The Salmonella vaccine EgA31-EgTrp demonstrated a high efficacy against E. granulosus promoting its potential role in reducing transmission to humans and animals.

Toll-like receptor 3 (TLR3): a new marker of canine monocytes-derived dendritic cells (cMo-DC).

Toll-like receptors (TLRs) are a family of functionally important receptors for recognition of pathogen-associated molecular pattern (PAMP) since they trigger the pro-inflammatory response and upregulation of costimulatory molecules, linking the rapid innate response to adaptative immunity. In human leukocytes, TLR3 has been found to be specifically expressed in dendritic cells (DC). This study examined the expression of TLR3 in canine monocytes-derived DC (cMo-DC) and PBMC using three new anti-TLR3 mAbs (619F7, 722E2 and 713E4 clones). The non-adherent cMo-DC generated after culture in canine IL-4 plus canine GM-CSF were labelled with the three anti-TLR3 clones by flow cytometry, with a strong expression shown for 619F7 and 722E2 clones. By contrast, TLR3 expression was low to moderate in canine monocytes and lymphocytes. These results were confirmed by Western blot using 619F7 and 722E2 clones and several polypeptide bands were observed, suggesting a possible cleavage of TLR3 molecule or different glycosylation states. In addition, TLR3 was detectable in immunocytochemistry by using 722E2 clone. In conclusion, this first approach to study canine TLR3 protein expression shows that three anti-TLR3 clones detect canine TLR3 and can be used to better characterize canine DC and the immune system of dogs.

CD86 molecule is a specific marker for canine monocyte-derived dendritic cells.

In this study, canine monocyte-derived dendritic cells (cMo-DC) were produced in presence of canine GM-CSF (cGM-CSF) and canine IL-4 (cIL-4), and they were characterized by their dendritic morphology, MLR functionality and phenotype. We noticed that cMo-DC were labelled with three anti-human CD86 (FUN-1, BU63 and IT2.2 clones), whereas resting and activated lymphocytes or monocytes were not stained. CD86 expression was induced by cIL-4 and was up-regulated during the differentiation of the cMo-DC, with a maximum at day 7. Furthermore, cMo-DC were very potent even in low numbers as stimulator cells in allogeneic MLR, and BU63 mAb was able to completely block the cMo-DC-induced proliferation in MLR. We also observed that cMo-DC highly expressed MHC Class II and CD32, but we failed to determine their maturation state since the lack of commercially available canine markers. Moreover, cMo-DC contained cytoplasmic periodic microstructures, potentially new ultrastructural markers of canine DC recently described. In conclusion, this work demonstrates that the CD86 costimulatory marker is now usable for a better characterization of in vitro canine DC.

Isolation, culture and characteristics of epididymal epithelial cells from adult cats.

A tissue-culture system in which cells retain defined ultrastructural and functional characteristics was established to provide a basis for functional investigations of the epididymal duct in the cat. A widely used culture protocol for rat epididymal epithelium was used as a starting point and subsequently modified. The cellular population of the cat's epididymal epithelium was isolated by successive collagenase and trypsin digestion. A high yield of isolated cells obtained with good viability, were cultured in DMEM/F12 medium supplemented with foetal bovine serum, in absence or in presence of additional dihydrotestosterone (1 nM). The plated primary cultures reached confluence within 5-8 days, producing a monolayer of cohesive cells. Samples taken after 6 days in culture were processed for transmission and scanning electron microscopies. Immunocytochemical staining was used to estimate the purity of the epithelial cell population in the monolayers. The cell cultures displayed several functional traits of in vivo epithelia, including [35S] hypotaurine and [35S] taurine production. These results demonstrate that primary cultures of epididymal epithelial cells isolated from sexually mature cats maintain several differentiated characteristics of the intact organ and therefore provide a valuable system for the study of epididymal epithelial cell functions, metabolic activities and their regulation in cats.

Acute megakaryoblastic leukemia with erythrophagocytosis and thrombosis in a dog.

A 7-year-old, intact male Dachshund was presented to the Lyon veterinary school for lethargy and anorexia of several weeks duration. The main clinical signs were pale and icteric mucous membranes, hepatomegaly, splenomegaly, and lymphadenopathy. Results of a CBC and plasma biochemistry tests revealed severe nonregenerative anemia, thrombocytopenia, and increased alanine aminotransferase and alkaline phosphatase activities. Blood smear evaluation and cytologic examination of lymph node and bone marrow aspirate specimens revealed a large population of poorly differentiated blast cells with morphologic features suggesting megakaryocytic lineage. A low number of well-differentiated but dysplastic megakaryocytes also were observed in lymph node and bone marrow smears. A few blast cells were erythrophagocytic. Blast cells were positive for glycoprotein IIIa, factor VIII-related antigen, and factor XIII using immunocytochemistry. The dog was euthanized and necropsied. Histologic findings consisted of diffuse, massive infiltration of lymph nodes, liver, and spleen by megakaryoblasts and atypical megakaryocytes, with widespread thrombosis. This case confirms the usefulness of immunochemistry, including for factor XIII, in the diagnosis of megakaryoblastic leukemia, and demonstrates the unique features of tumor cell erythrophagocytosis and marked fibrinous thrombosis, which have not been reported previously in dogs.