Somporn Techangamsuwan

Nectin4 Is an Epithelial Cell Receptor for Canine Distemper Virus and Involved in Neurovirulence

ABSTRACT Canine distemper virus (CDV) uses signaling lymphocyte activation molecule (SLAM), expressed on immune cells, as a receptor. However, epithelial and neural cells are also affected by CDV in vivo. Wild-type CDV strains showed efficient replication with syncytia in Vero cells expressing dog nectin4, and the infection was blocked by an anti-nectin4 antibody. In dogs with distemper, CDV antigen was preferentially detected in nectin4-positive neurons and epithelial cells, suggesting that nectin4 is an epithelial cell receptor for CDV and also involved in its neurovirulence.

Similar behaviour and primate-like properties of adult canine Schwann cells and olfactory ensheathing cells in long-term culture.

Adult canine Schwann cells and olfactory ensheathing cells (OECs) have been shown to promote neural regeneration in vivo. Since the majority of studies have been performed in rodents, it is not yet clear in how far OECs from large animals and humans share the reported properties. Moreover, due to the lack of comparative studies, it remains to be established whether Schwann cells and OECs display cell type-specific characteristics. In the present study, adult canine Schwann cells and OECs were comparatively analyzed regarding long-term growth, morphology, growth factor responsiveness, and antigenic expression. Adult canine Schwann cells and OECs displayed the same typical spindle-shaped morphology and expressed the cell type-specific marker p75(NTR). Moreover, the proliferation of both cell types was promoted by the same mitogens, including fibroblast growth factor-2 (FGF-2) and heregulin-1beta (HRG-1beta). Several observations indicate that canine OECs differ from the well characterized rodent OECs and display properties reminiscent on primate cells. Both cell types (i) proliferated through multiple passages in the absence of growth factors and did not enter a senescent state until 3 months in culture, (ii) were not responsive to the cAMP-elevating agent forskolin, and (iii) stably expressed p75(NTR) in long-term culture. Taken together, this is the first report demonstrating that adult canine Schwann cells and OECs in long-term culture share the same in vitro characteristics and display primate-like properties. This underscores the relevance of the dog as a translational species between rodents and humans.

Differential expression of HNK-1 and p75NTR in adult canine Schwann cells and olfactory ensheathing cells in situ but not in vitro

Olfactory ensheathing cells (OECs) are promising candidates for autologous cell transplantation therapies of nervous system injury and disease. Large animal models are relevant for transferring experimental data into clinical practice. In vivo studies have suggested that adult canine OECs may display similar regenerating capacities as their rodent counterpart. However, data on their molecular phenotype required for generating pure cell preparations are still scarce. In the present study, we comparatively analyzed expression of the carbohydrate HNK‐1 epitope and the neurotrophin receptor p75NTR in adult canine Schwann cells and olfactory ensheathing cells in situ and in vitro. Myelinating and nonmyelinating Schwann cells in situ exclusively expressed HNK‐1 and p75NTR, respectively, whereas OECs were negative for both markers. In vitro, OECs and Schwann cells shared cell surface expression of p75NTR but not of HNK‐1, which could be detected transiently in intracellular vesicles. This suggests that Schwann cells and OECs in vitro phagozytose HNK‐1+ cellular debris. The cultivation‐induced downregulation of HNK‐1 expression in Schwann cells and upregulation of p75NTR in OECs argues for the possibility that axonal signals control the expression of both markers in situ. Whereas HNK‐1 expression in Schwann cells is most likely controlled by signals inducing myelination, e.g., neuregulin, the mechanisms that may suppress p75NTR expression in OECs in situ remain to be elucidated. Interestingly, HNK‐1 expression in the adult dog was found in both sensory and motor nerve myelinating Schwann cells. This is reminiscent of humans and differs from rodents; it also underscores the importance of large animal models for translational research. J. Comp. Neurol. 505:572–585, 2007.

Transfection of adult canine Schwann cells and olfactory ensheathing cells at early and late passage with human TERT differentially affects growth factor responsiveness and in vitro growth

Adult canine Schwann cells and olfactory ensheathing cells (OECs) are closely related cell types that are considered attractive candidates for translational studies of neural repair. To establish a reliable cell source by comparing the in vitro properties of immortalized Schwann cells and OECs for transplantation purposes, we transfected both cell types with human telomerase reverse transcriptase (hTERT). Ectopic hTERT expression has been shown to induce immortalization of various cell types without substantial alterations of their phenotypes. Schwann cells and OECs were isolated from adult dogs, transfected with hTERT at early (P4) and late passage (P26), characterized regarding in vitro proliferation, antigenic expression and senescence-associated genes in the presence and absence of fibroblast growth factor-2 (FGF-2). Ectopic hTERT expression in late passage glia treated with but not without FGF-2 prevented the decline in proliferation observed in non-transfected cells. Immortalization did not alter p75(NTR) and GFAP but O4 and A2B5 expression. Contrary to this, early passage hTERT transfection significantly reduced proliferation independent of FGF-2 and lowered expression of O4 and GFAP in both cell types. Transfection did not alter mRNA expression of senescence-associated genes such as p53 and p16. No substantial differences were found between Schwann cells and OECs underscoring the close relationship of both cell types. Taken together, we established a stable source of adult canine Schwann cells and OECs and demonstrated that the effects of hTERT expression on in vitro growth and growth factor responsiveness depend on the replicative age.

New aspects of the pathogenesis of canine distemper leukoencephalitis.

Canine distemper virus (CDV) is a member of the genus morbillivirus, which is known to cause a variety of disorders in dogs including demyelinating leukoencephalitis (CDV-DL). In recent years, substantial progress in understanding the pathogenetic mechanisms of CDV-DL has been made. In vivo and in vitro investigations provided new insights into its pathogenesis with special emphasis on axon-myelin-glia interaction, potential endogenous mechanisms of regeneration, and astroglial plasticity. CDV-DL is characterized by lesions with a variable degree of demyelination and mononuclear inflammation accompanied by a dysregulated orchestration of cytokines as well as matrix metalloproteinases and their inhibitors. Despite decades of research, several new aspects of the neuropathogenesis of CDV-DL have been described only recently. Early axonal damage seems to represent an initial and progressive lesion in CDV-DL, which interestingly precedes demyelination. Axonopathy may, thus, function as a potential trigger for subsequent disturbed axon-myelin-glia interactions. In particular, the detection of early axonal damage suggests that demyelination is at least in part a secondary event in CDV-DL, thus challenging the dogma of CDV as a purely primary demyelinating disease. Another unexpected finding refers to the appearance of p75 neurotrophin (NTR)-positive bipolar cells during CDV-DL. As p75NTR is a prototype marker for immature Schwann cells, this finding suggests that Schwann cell remyelination might represent a so far underestimated endogenous mechanism of regeneration, though this hypothesis still remains to be proven. Although it is well known that astrocytes represent the major target of CDV infection in CDV-DL, the detection of infected vimentin-positive astrocytes in chronic lesions indicates a crucial role of this cell population in nervous distemper. While glial fibrillary acidic protein represents the characteristic intermediate filament of mature astrocytes, expression of vimentin is generally restricted to immature or reactive astrocytes. Thus, vimentin-positive astrocytes might constitute an important cell population for CDV persistence and spread, as well as lesion progression. In vitro models, such as dissociated glial cell cultures, as well as organotypic brain slice cultures have contributed to a better insight into mechanisms of infection and certain morphological and molecular aspects of CDV-DL. Summarized, recent in vivo and in vitro studies revealed remarkable new aspects of nervous distemper. These new perceptions substantially improved our understanding of the pathogenesis of CDV-DL and might represent new starting points to develop novel treatment strategies.

Genetic characterization of a betanodavirus isolated from a clinical disease outbreak in farm‐raised tilapia Oreochromis niloticus (L.) in Thailand

Betanodavirus infection was diagnosed in larvae of farm-raised tilapia Oreochromis niloticus (L.), in central Thailand. Extensive vacuolar degeneration and neuronal necrosis were observed in histological sections with positive immunohistochemical staining for betanodavirus. Molecular phylogenetic analysis was performed based on the nucleotide sequences (1333 bases) of the capsid protein gene. The virus strain was highly homologous (93.07-93.88%) and closely related to red-spotted grouper nervous necrosis virus (RGNNV).

A retrospective study of clinical hematology and biochemistry of canine hepatozoonosis on hospital populations in Bangkok, Thailand

Canine hepatozoonosis is an endemic tick-borne disease of Thailand, but the clinical data for the disease are rarely reported. The objective of this study was intended to characterize the clinical hematology and biochemistry of admitted canine hepatozoonosis cases of the Chulalongkorn Small Animal Teaching Hospital, Bangkok from January 2001 to December 2003. A total of 342 dogs were examined in this study. Most of the dogs presented with hypocytic hypochromic anemia. Leukocyte and platelet counts were variable and nonspecific in these cases. Additionally, serum biochemistry including blood urea nitrogen, creatinine, liver enzymes, total protein, albumin, and glucose of infected dogs were within normal ranges. This study indicated that hypocytic hypochromic anemia was a consistent hematological value feature of canine hepatozoonosis, but clinical biochemical findings were not consistent in this endemic-hospital population of canine hepatozoonosis.

A retrospective study of the clinical hematology and biochemistry of canine ehrlichiosis in an animal hospital population in Bangkok, Thailand

A retrospective study on 687 canine ehrlichiosis cases was carried out during January 2001–December 2003 at the Chulalongkorn Small Animal Teaching Hospital, Faculty of Veterinary Science, Chulalongkorn University. Data on rectal temperature, clinical hematology, and biochemistry were analyzed on the first day of admission. The highest incidences each year were observed in December 2001 (24 cases), June and July 2002 (31 cases each), and October 2003 (38 cases). The clinical findings varied, although most cases showed a high fever (33%), severe anemia (18%), leukocytosis (18%) or leukopenia (19%), thrombocytopenia (93%), and hepatic enzyme elevation. It followed that the therapeutic plan should comprise both specific and clinically supportive treatments, including hematotonics and hepatotonics.

Distinct cell tropism of canine distemper virus strains to adult olfactory ensheathing cells and Schwann cells in vitro.

Canine distemper virus (CDV) can enter the brain via infection of olfactory neurons. Whether olfactory ensheathing cells (OECs) are also infected by CDV, and if yes, how they respond to the virus has remained enigmatic. Here, we exposed adult canine OECs in vitro to several attenuated (CDV-2544, CDV-R252, CDV-Ond, CDV-OndeGFP) and one virulent CDV strain (CDV-5804PeGFP) and studied their susceptibility compared to Schwann cells, a closely related cell type sharing the phagocytizing activity. We show that OECs and Schwann cells were infected by CDV strains albeit to different levels. Ten days post-infection (dpi), a mild to severe cytopathic effect ranging from single cell necrosis to layer detachment was noted. The percentage of infection increased during 10 dpi and viral progenies were detected in each culture using virus titration. Interestingly, CDV-2544, CDV-OndeGFP, and CDV-5804PeGFP predominantly infected OECs, while CDV-Ond targeted Schwann cells. No significant differences were found between the virulent and attenuated CDV strains. The observation of a CDV strain-specific cell tropism is evidence for significant molecular differences between OECs and Schwann cells. Whether these differences are either related to strain-specific distemper pathogenesis or support a role of OECs during CDV infection and virus spread needs to be addressed in future studies.

Canine dirofilariasis and concurrent tick-borne transmitted diseases in Bangkok, Thailand

A retrospective study in dogs presented to the Chulalongkorn Small Animal Teaching Hospital, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand, from January 2001 to December 2003 was carried out. A total of 917 dogs were diagnosed with canine dirofilariasis and other concurrent tick-borne transmitted diseases by the Veterinary Diagnostic Laboratory, Faculty of Veterinary Science, Chulalongkorn University. The highest occurrence within each year was observed in November 2001 (40 cases), April 2002 (41 cases), and July 2003 (36 cases), respectively. Of the total 917 positive cases, a single infection of dirofilariasis was detected in 869 dogs (94.8%; group 1), while 37 dogs (4.0%; group 2) were diagnosed with dirofilariasis and ehrlichiosis, 4 dogs (0.4%; group 3) with dirofilariasis and hepatozoonosis, and 7 dogs (0.8%; group 4) with dirofilariasis, ehrlichiosis, and babesiosis, respectively. Laboratory data comprising hematological and blood chemistry profiles were evaluated and compared among groups. Group 4 was defined as moderate microcytic anemia and was significantly different with regard to hematological profiles from others (P < 0.05). In addition, severe thrombocytopenia was observed in both groups 2 and 4 (65.4 × 103 and 59.0 × 103 cells/μl, respectively). The hepatobiliary profiles in all groups revealed increases in serum alanine aminotransferase and serum alkaline phosphatase activities indicative of hepatocellular damage. These epidemiological results serve as baseline information for preventive strategies against blood parasites in the endemic area. Moreover, both biochemical and hematological abnormalities should be considered as appropriate monitors during disease interventions.