Comparison of the tolerability of 161Tb- and 177Lu-labeled somatostatin analogues in the preclinical setting.

PURPOSE: [177Lu]Lu-DOTATATE is an established somatostatin receptor (SSTR) agonist for the treatment of metastasized neuroendocrine neoplasms, while the SSTR antagonist [177Lu]Lu-DOTA-LM3 has only scarcely been employed in clinics. Impressive preclinical data obtained with [161Tb]Tb-DOTA-LM3 in tumor-bearing mice indicated the potential of terbium-161 as an alternative to lutetium-177. The aim of the present study was to compare the tolerability of 161Tb- and 177Lu-based DOTA-LM3 and DOTATATE in immunocompetent mice. METHODS: Dosimetry calculations were performed based on biodistribution data of the radiopeptides in immunocompetent mice. Treatment-related effects on blood cell counts were assessed on Days 10, 28 and 56 after application of [161Tb]Tb-DOTA-LM3 or [161Tb]Tb-DOTATATE at 20 MBq per mouse. These radiopeptides were also applied at 100 MBq per mouse and the effects compared to those observed after application of the 177Lu-labeled counterparts. Bone marrow smears, blood plasma parameters and organ histology were assessed at the end of the study. RESULTS: The absorbed organ dose was commonly higher for the SSTR antagonist than for the SSTR agonist and for terbium-161 over lutetium-177. Application of a therapeutic activity level of 20 MBq [161Tb]Tb-DOTA-LM3 or [161Tb]Tb-DOTATATE was well tolerated without major hematological changes. The injection of 100 MBq of the 161Tb- and 177Lu-based somatostatin analogues affected the blood cell counts, however. The lymphocytes were 40-50% lower in treated mice compared to the untreated controls on Day 10 irrespective of the radionuclide employed. At the same timepoint, thrombocyte and erythrocyte counts were 30-50% and 6-12% lower, respectively, after administration of the SSTR antagonist (p < 0.05) while changes were less pronounced in mice injected with the SSTR agonist. All blood cell counts were in the normal range on Day 56. Histological analyses revealed minimal abnormalities in the kidneys, liver and spleen of treated mice. No correlation was observed between the organ dose and frequency of the occurrence of abnormalities. CONCLUSION: Hematologic changes were more pronounced in mice treated with the SSTR antagonist than in those treated with the SSTR agonist. Despite the increased absorbed dose delivered by terbium-161 over lutetium-177, [161Tb]Tb-DOTA-LM3 and [161Tb]Tb-DOTATATE should be safe at activity levels that are recommended for their respective 177Lu-based analogues.

Natural outbreak of Mycobacterium caprae infection in imported laboratory cynomolgus macaques (Macaca fascicularis): diagnostic pitfalls and management of safety precautions.

Tuberculosis (TB) is a major health threat for humans and for non-human primates used for toxicology or research purposes. Emerging mycobacterial species represent a major challenge for diagnosis and surveillance programs. Here, we report a natural outbreak of Mycobacterium caprae in imported cynomolgus macaques (Macaca fascicularis) that occurred at AnaPath Research S.A.U. (APR). The macaques underwent repeated negative intradermal tuberculin tests (IDT) before importation and at the European quarantine station. Exhaustive TB screening was started at APR after confirmation of one positive case at another facility. The animal in question belonged to the same colony received at APR. Diagnostic approaches included clinical examination, PCR, culture, spoligotyping, IDT testing, interferon-γ release assay (IGRA), and thoracoabdominal ultrasound (US). Three regulatory toxicity studies and stock animals were affected. The macaques lacked clinical signs, except for one showing a fistulizing nodule in the right inguinal area, which tested positive for the Mycobacterium tuberculosis complex by PCR. All animals were necropsied and 10 macaques (n=114) showed gross and histologic findings compatible with TB confirmed by PCR and culture. M. caprae was identified as the etiological agent by Direct Variable Repeat spacer oligonucleotide typing (DVR spoligotyping). The infection was traced to Asia via the SB1622 spoligotype involved, confirming that the animals were infected prior to their import into Europe. Tuberculin skin test (TST), IGRA, and US were only sensitive in detecting advanced cases of M. caprae infection. One staff member showed a positive TST reaction, which was handled in accordance with the Spanish government's health regulations. All the sanitary measures implemented were effective in eradicating the disease.

Adherent-invasive Escherichia coli associated with granulomatous colitis and extraintestinal dissemination in a Sphynx cat.

This case report describes a case of granulomatous colitis (GC) associated with adherent-invasive Escherichia coli (AIEC) with extension to cecum and ileum and dissemination to multiple lymph nodes, the spleen, and brain in a 10-year-old, male Sphynx cat. The cat had an episode of diarrhea 4 months prior to consultation due to sudden blindness. Signs rapidly progressed to ataxia, seizures, and death. Gross and histologic findings were consistent with granulomatous inflammation in all affected organs. In situ hybridization confirmed the presence of intracellular E. coli within enterocytes and infiltrating macrophages, and whole genome sequencing identified virulence traits commonly linked to AIEC strain. This is the first characterization of GC in a cat associated to AIEC resembling the metastatic form of Crohn's disease in humans and GC of dogs. Extraintestinal involvement might provide evidence of the ability of AIEC to promote granulomatous inflammation beyond the gut.

Experimental Infection of Mink with SARS-COV-2 Omicron Variant and Subsequent Clinical Disease.

We report an experimental infection of American mink with SARS-CoV-2 Omicron variant and show that mink remain positive for viral RNA for days, experience clinical signs and histopathologic changes, and transmit the virus to uninfected recipients. Preparedness is crucial to avoid spread among mink and spillover to human populations.

Bronchioalveolar carcinoma in an adult alpaca (Vicugna pacos).

BACKGROUND: This report describes a case of a bronchiolar adenocarcinoma in a 6-year old alpaca mare. For the first time in an alpaca, neoplasia was classified by histopathology as a lepidic-predominant bronchiolar adenocarcinoma. CASE PRESENTATION: The mare was referred to the Clinic for Ruminants after a 6-week period of forced breathing and weight loss. The clinical examination included complete blood count, blood chemistry, ultrasound, radiographs and a CT-scan of the thorax. A bilateral pneumothorax and several, structures within the lung parenchyma were diagnosed. Differential diagnosis included neoplasia, tuberculosis and fungal granulomas. The owner requested euthanasia due to the mare's ongoing deterioration. At postmortem examination, the granulomatous changes in the lungs were histopathologically classified as lepidic dominant bronchiolar adenocarcinoma. CONCLUSIONS: Neoplastic diseases are more often seen in South American camelids compared to other farm animal species. The use of a CT scan was helpful in classifying the lung lesions and give a clear prognosis.

Amplicon sequencing of bacterial microbiota in abortion material from cattle.

Abortions in cattle have a significant economic impact on animal husbandry and require prompt diagnosis for surveillance of epizootic infectious agents. Since most abortions are not epizootic but sporadic with often undetected etiologies, this study examined the bacterial community present in the placenta (PL, n = 32) and fetal abomasal content (AC, n = 49) in 64 cases of bovine abortion by next generation sequencing (NGS) of the 16S rRNA gene. The PL and AC from three fetuses of dams that died from non-infectious reasons were included as controls. All samples were analyzed by bacterial culture, and 17 were examined by histopathology. We observed 922 OTUs overall and 267 taxa at the genus level. No detectable bacterial DNA was present in the control samples. The microbial profiles of the PL and AC differed significantly, both in their composition (PERMANOVA), species richness and Chao-1 (Mann-Whitney test). In both organs, Pseudomonas was the most abundant genus. The combination of NGS and culture identified opportunistic pathogens of interest in placentas with lesions, such as Vibrio metschnikovii, Streptococcus uberis, Lactococcus lactis and Escherichia coli. In placentas with lesions where culturing was unsuccessful, Pseudomonas and unidentified Aeromonadaceae were identified by NGS displaying high number of reads. Three cases with multiple possible etiologies and placentas presenting lesions were detected by NGS. Amplicon sequencing has the potential to uncover unknown etiological agents. These new insights on cattle abortion extend our focus to previously understudied opportunistic abortive bacteria.

Neglected zoonotic agents in cattle abortion: tackling the difficult to grow bacteria.

BACKGROUND: Coxiella burnetii, Chlamydia abortus and Leptospira spp. are difficult to grow bacteria that play a role in bovine abortion, but their diagnosis is hampered by their obligate intracellular lifestyle (C. burnetii, C. abortus) or their lability (Leptospira spp.). Their importance is based on the contagious spread in food-producing animals, but also as zoonotic agents. In Switzerland, first-line routine bacteriological diagnostics in cattle abortions is regulated by national law and includes only basic screening by staining for C. burnetii due to the high costs associated with extended spectrum analysis. The aim of this study was to assess the true occurrence of these zoonotic pathogens in 249 cases of bovine abortion in Switzerland by serology (ELISA for anti-C. burnetii and C. abortus antibodies and microscopic agglutination test for anti-Leptospira spp. antibodies), molecular methods (real-time PCR and sequencing of PCR products of Chlamydiales-positive cases), Stamp's modification of the Ziehl-Neelsen (mod-ZN) stain and, upon availability of material, by histology and immunohistochemistry (IHC). RESULTS: After seroanalysis the prevalence was 15.9% for C. burnetii, 38.5% for C. abortus and 21.4% for Leptospira spp. By real-time PCR 12.1% and 16.9% of the cases were positive for C. burnetii and Chlamydiales, respectively, but only 2.4% were positive for C. burnetii or Chlamydiales by mod-ZN stain. Sequencing of PCR products of Chlamydiales-positive cases revealed C. abortus in 10% of cases and the presence of a mix of Chlamydiales-related bacteria in 5.2% of cases. Pathogenic Leptospira spp. were detected in 5.6% of cases. Inflammatory lesions were present histologically in all available samples which were real-time PCR-positive for Chlamydiales and Leptospira spp. One of 12 real-time PCR-positive cases for C. burnetii was devoid of histological lesions. None of the pathogens could be detected by IHC. CONCLUSION: Molecular detection by real-time PCR complemented by histopathological analysis is recommended to improve definitive diagnosis of bovine abortion cases and determine a more accurate prevalence of these zoonotic pathogens.

Transcriptional profiling predicts overwhelming homology of Schwann cells, olfactory ensheathing cells, and Schwann cell-like glia.

Schwann cells (SCs), olfactory ensheathing cells (OECs), and central nervous system Schwann cell-like glia (SG) represent a group of nerve growth factor receptor p75 (NGFR)-positive cells, originating from different tissues. Because of their pro-regenerative capacities, these cells are subjects in experimental transplantation-based therapies of spinal cord trauma. The objective of this study was to compare the transcriptomes of uninfected and canine distemper virus-infected OECs, SCs, SG and fibroblasts (FBs) derived from four beagle dogs and cultured under identical conditions in vitro, employing canine genome 2.0 arrays (Affymetrix). Here, we observed a complete lack of transcriptional differerences between OECs and SG, a high similarity of OECs/SG to SCs, and a marked difference of SCs and OECs/SG towards FBs. Differentially expressed genes possibly involved in the maintenance of cell type-specific identity included an up-regulation of HOXD8 and HOXC4 in SCs, and an up-regulation of CNTNAP2 and EFEMP1 in OECs/SG. We identified cell type-specific biomarkers employing supervised clustering with a K-nearest-neighbors algorithm and correlation-based feature selection. Thereby AQP1 and SCRG1 were predicted to be the most powerful biomarkers distinguishing SCs from OECs/SG. Immunofluorescence confirmed a higher expression of SCRG1 in OECs and SG, and conversely a higher expression of AQP1 in SCs in vitro. Furthermore, canine and murine olfactory nerves showed SCRG1-positive, AQP1-negative OECs and/or axons, whereas sciatic nerves displayed multifocal non-myelinated, AQP1-positive, SCRG1-negative cells. Conclusively, OECs/SG are suggested to be a uniform cell type differing only in the tissue of origin and highly related to SCs.

CNS Schwann cells display oligodendrocyte precursor-like potassium channel activation and antigenic expression in vitro.

Central nervous system (CNS) injury triggers production of myelinating Schwann cells from endogenous oligodendrocyte precursors (OLPs). These CNS Schwann cells may be attractive candidates for novel therapeutic strategies aiming to promote endogenous CNS repair. However, CNS Schwann cells have been so far mainly characterized in situ regarding morphology and marker expression, and it has remained enigmatic whether they display functional properties distinct from peripheral nervous system (PNS) Schwann cells. Potassium channels (K+) have been implicated in progenitor and glial cell proliferation after injury and may, therefore, represent a suitable pharmacological target. In the present study, we focused on the function and expression of voltage-gated K+ channels Kv(1-12) and accessory ß-subunits in purified adult canine CNS and PNS Schwann cell cultures using electrophysiology and microarray analysis and characterized their antigenic phenotype. We show here that K+ channels differed significantly in both cell types. While CNS Schwann cells displayed prominent K D-mediated K+ currents, PNS Schwann cells elicited K(D-) and K(A-type) K+ currents. Inhibition of K+ currents by TEA and Ba2+ was more effective in CNS Schwann cells. These functional differences were not paralleled by differential mRNA expression of Kv(1-12) and accessory ß-subunits. However, O4/A2B5 and GFAP expressions were significantly higher and lower, respectively, in CNS than in PNS Schwann cells. Taken together, this is the first evidence that CNS Schwann cells display specific properties not shared by their peripheral counterpart. Both Kv currents and increased O4/A2B5 expression were reminiscent of OLPs suggesting that CNS Schwann cells retain OLP features during maturation.

Equus caballus papillomavirus type 2 (EcPV2)-associated benign penile lesions and squamous cell carcinomas.

BACKGROUND: Squamous cell carcinoma is the most common genital, ocular and gastric tumour in horses. Equus caballus papillomavirus type 2 (EcPV2) DNA has been detected in several studies in equine penile squamous cell carcinomas (SCCs) and precursor lesions providing evidence of a causal role of EcPV2 in equine genital SCCs. Recently, EcPV2 E6/E7 nucleic acids were also detected in equine gastric SCCs, but further studies are required to determine the role of EcPV2 infection in the pathogenesis of gastric SCC. EcPV2 nucleic acids have been rarely described in ocular SCCs and precursor lesions. OBJECTIVES: To investigate the presence of EcPV2 nucleic acids with polymerase chain reaction (PCR) and in situ hybridisation (ISH) in penile hyperplasias, papillomas and SCCs in horses and to determine whether EcPV2 nucleic acids can be detected in SCCs affecting other locations, including the stomach, ocular tissues and larynx. METHODS: Twenty-one archival formalin-fixed paraffin embedded (FFPE) tissue samples, including 12 genital lesions comprising penile hyperplasias, papillomas and SCCs, 6 ocular SCCs, 2 gastric SCCs and 1 laryngeal SCC, were screened by PCR and ISH for EcPV2 E6/E7 DNA and mRNA. Archival FFPE tissue samples (eyelid and penile mucosa and preputium) from six horses without a diagnosis or history of neoplastic or papillomavirus-associated disease were included as controls. RESULTS: EcPV2 nucleic acids were detected by PCR and ISH in all genital lesions (12/12) and gastric SCCs (2/2), in two ocular SCCs (2/6) and in one laryngeal SCC (1/1). In control horses, one eyelid sample was positive in PCR but not in ISH. The remaining control samples were negative for EcPV2 E6/E7 nucleic acids in PCR and ISH. CONCLUSIONS: These results further support the role of EcPV2 infection in the development of equine genital SCCs and suggest that EcPV2 infection may also act as a predisposing factor for other SCCs in horses, including gastric, ocular and laryngeal SCCs.

Onset and progression of postmortem histological changes in the central nervous system of RccHan™: WIST rats.

Death initiates a cascade of physiological and biochemical alterations in organs and tissues, resulting in microscopic changes that challenge the histopathological evaluation. Moreover, the brain is particularly susceptible to artifacts owing to its unique composition and its location within the cranial vault. The aim of this study was to compile and illustrate the microscopic changes in the central nervous system (CNS) of rats subjected to delayed postmortem fixation. It also scrutinizes the influence of exsanguination and cooling methods on the initiation and progression of these alterations. Twenty-four Wistar Han outbred rats (RccHan™: WIST) were sacrificed and stored either at room temperature (18-22°C) or under refrigeration (2-4°C). Necropsies were conducted at different time points postmortem (i.e., 0.5 h, 1 h, 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, 7 days and 14 days). Brain sections underwent simultaneous digital evaluation by 14 pathologists until a consensus was reached on terminology, key findings, and intensity levels. Microscopic observations varied among cell types. Glial cells were similarly affected throughout the CNS and showed pericellular halo, chromatin condensation and nuclear shrinkage. Neurons showed two types of postmortem changes as most of them showed progressive shrinkage, cytoplasmic dissolution and karyorrhexis whereas others acquired a dark-neuron-like appearance. Neuronal changes showed marked differences among neuroanatomical locations. Additional postmortem changes encompassed: granulation and microcavitation in neuropil and white matter; retraction spaces; detachment of ependyma, choroid plexus, and leptomeninges. Severity of findings after 48 h at room temperature was higher than after seven days under refrigeration and similar to or slightly lower than after 14 days under refrigeration. No clear differences were observed related to the sex or weight of the animals or their exsanguination status. This work elucidates the onset and progression of autolytic changes in the brains of Wistar Han rats, offering insights to accurately identify and enhance the histopathological evaluation.

Escherichia coli-associated follicular cystitis in dogs: Clinical and pathologic characterization.

BACKGROUND: Follicular cystitis is an uncommon inflammatory change in the urinary bladder wall characterized by the formation of tertiary lymphoid structures (TLSs) in the submucosa. OBJECTIVES: To characterize clinical and pathologic features of follicular cystitis in dogs and to explore in situ distribution and possible role of Escherichia coli as an associated cause. ANIMALS: Eight dogs diagnosed with follicular cystitis and 2 control dogs. METHODS: Retrospective descriptive study. Dogs diagnosed with follicular cystitis (macroscopic follicular lesions in the urinary bladder mucosa and histopathologic detection of TLSs in bladder wall biopsies) were identified from medical records. Paraffin embedded bladder wall biopsies were subject to in situ hybridization for E. coli 16SrRNA identification. RESULTS: Follicular cystitis was diagnosed in large breed (median weight 24.9 kg, interquartile range [IQR] 18.8-35.4 kg) female dogs with a history of chronic recurrent urinary tract infections (UTIs; median duration of clinical signs 7 months, IQR 3-17 months; median number of previous UTIs 5, IQR 4-6). Positive E. coli 16SrRNA signal was detected within developing, immature and mature TLSs in 7/8 dogs, through submucosal stroma in 8/8 dogs and within the urothelium in 3/8 dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: Chronic inflammation associated with an intramural E. coli infection in the urinary bladder wall represents a possible triggering factor for the development of follicular cystitis.

Aberrant cortical projections to amygdala GABAergic neurons contribute to developmental circuit dysfunction following early life stress.

Early life stress (ELS) results in enduring dysfunction of the corticolimbic circuitry, underlying emotional and social behavior. However, the neurobiological mechanisms involved remain elusive. Here, we have combined viral tracing and electrophysiological techniques to study the effects of maternal separation (MS) on frontolimbic connectivity and function in young (P14-21) rats. We report that aberrant prefrontal inputs to basolateral amygdala (BLA) GABAergic interneurons transiently increase the strength of feed-forward inhibition in the BLA, which raises LTP induction threshold in MS treated male rats. The enhanced GABAergic activity after MS exposure associates with lower functional synchronization within prefrontal-amygdala networks in vivo. Intriguingly, no differences in these parameters were detected in females, which were also resistant to MS dependent changes in anxiety-like behaviors. Impaired plasticity and synchronization during the sensitive period of circuit refinement may contribute to long-lasting functional changes in the prefrontal-amygdaloid circuitry that predispose to neuropsychiatric conditions later on in life.

Fatal infection with emerging apicomplexan parasite Hepatozoon silvestris in a domestic cat.

BACKGROUND: Hepatozoon silvestris is an emerging apicomplexan parasite discovered in European wild cats from Bosnia and Herzegovina and blood samples of a domestic cat from Southern Italy in 2017. It has also been identified in Ixodes ricinus collected from a domestic cat in Wales, UK, in 2018. The clinical relevance, pathogenesis and epidemiology of this novel Hepatozoon species are not yet understood. Thus, the objective of this paper was to report and describe the first fatal case of an H. silvestris infection in a domestic cat. RESULTS: The cat, which originated from Switzerland, died shortly after presenting clinical signs of lethargy, weakness and anorexia. At necropsy, no specific lesions were observed. Histopathology of the heart revealed a severe lympho-plasmacytic and histiocytic myocarditis. Mature and developing protozoal meronts morphologically compatible with Hepatozoon species were observed associated with the myocardial inflammation. No other lesions were present in any other organ evaluated, and the cat tested negative for retroviral and other immunosuppressive infectious agents. Polymerase chain reaction from the myocardium resulted in a specific amplicon of the Hepatozoon 18S rRNA gene. Sequencing and BLAST analysis revealed 100% sequence identity with H. silvestris. CONCLUSIONS: The severity of the infection with fatal outcome in an otherwise healthy animal suggests a high virulence of H. silvestris for domestic cats. The presence of this emerging parasite in a domestic cat in Switzerland with no travel history provides further evidence for a geographical distribution throughout Europe.

Spatial distribution of cannabinoid receptor type 1 (CB1) in normal canine central and peripheral nervous system.

The endocannabinoid system is a regulatory pathway consisting of two main types of cannabinoid receptors (CB1 and CB2) and their endogenous ligands, the endocannabinoids. The CB1 receptor is highly expressed in the central and peripheral nervous systems (PNS) in mammalians and is involved in neuromodulatory functions. Since endocannabinoids were shown to be elevated in cerebrospinal fluid of epileptic dogs, knowledge about the species specific CB receptor expression in the nervous system is required. Therefore, we assessed the spatial distribution of CB1 receptors in the normal canine CNS and PNS. Immunohistochemistry of several regions of the brain, spinal cord and peripheral nerves from a healthy four-week-old puppy, three six-month-old dogs, and one ten-year-old dog revealed strong dot-like immunoreactivity in the neuropil of the cerebral cortex, Cornu Ammonis (CA) and dentate gyrus of the hippocampus, midbrain, cerebellum, medulla oblongata and grey matter of the spinal cord. Dense CB1 expression was found in fibres of the globus pallidus and substantia nigra surrounding immunonegative neurons. Astrocytes were constantly positive in all examined regions. CB1 labelled neurons and satellite cells of the dorsal root ganglia, and myelinating Schwann cells in the PNS. These results demonstrate for the first time the spatial distribution of CB1 receptors in the healthy canine CNS and PNS. These results can be used as a basis for further studies aiming to elucidate the physiological consequences of this particular anatomical and cellular distribution.

Morphologic, phenotypic, and transcriptomic characterization of classically and alternatively activated canine blood-derived macrophages in vitro.

Macrophages are a heterogeneous cell population playing a pivotal role in tissue homeostasis and inflammation, and their phenotype strongly depends on the micromilieu. Despite its increasing importance as a translational animal model for human diseases, there is a considerable gap of knowledge with respect to macrophage polarization in dogs. The present study comprehensively investigated the morphologic, phenotypic, and transcriptomic characteristics of unstimulated (M0), M1- (GM-CSF, LPS, IFNγ-stimulated) and M2- (M-CSF, IL-4-stimulated)-polarized canine blood-derived macrophages in vitro. Scanning electron microscopy revealed distinct morphologies of polarized macrophages with formation of multinucleated cells in M2-macrophages, while immunofluorescence employing literature-based prototype-antibodies against CD16, CD32, iNOS, MHC class II (M1-markers), CD163, CD206, and arginase-1 (M2-markers) demonstrated that only CD206 was able to discriminate M2-macrophages from both other phenotypes, highlighting this molecule as a promising marker for canine M2-macrophages. Global microarray analysis revealed profound changes in the transcriptome of polarized canine macrophages. Functional analysis pointed out that M1-polarization was associated with biological processes such as "respiratory burst", whereas M2-polarization was associated with processes such as "mitosis". Literature-based marker gene selection revealed only minor overlaps in the gene sets of the dog compared to prototype markers of murine and human macrophages. Biomarker selection using supervised clustering suggested latexin (LXN) and membrane-spanning 4-domains, subfamily A, member 2 (MS4A2) to be the most powerful predicting biomarkers for canine M1- and M2-macrophages, respectively. Immunofluorescence for both markers demonstrated expression of both proteins by macrophages in vitro but failed to reveal differences between canine M1 and M2-macrophages. The present study provides a solid basis for future studies upon the role of macrophage polarization in spontaneous diseases of the dog, a species that has emerging importance for translational research.

Immunohistochemical and transcriptome analyses indicate complex breakdown of axonal transport mechanisms in canine distemper leukoencephalitis.

INTRODUCTION: CDV-DL (Canine distemper virus-induced demyelinating leukoencephalitis) represents a spontaneously occurring animal model for demyelinating disorders. Axonopathy represents a key pathomechanism in this disease; however, its underlying pathogenesis has not been addressed in detail so far. This study aimed at the characterization of axonal cytoskeletal, transport, and potential regenerative changes with a parallel focus upon Schwann cell remyelination. METHODS: Immunohistochemistry of canine cerebellar tissue as well as a comparative analysis of genes from an independent microarray study were performed. RESULTS: Increased axonal immunoreactivity for nonphosphorylated neurofilament was followed by loss of cytoskeletal and motor proteins. Interestingly, a subset of genes encoding for neurofilament subunits and motor proteins was up-regulated in the chronic stage compared to dogs with subacute CDV-DL. However, immunohistochemically, hints for axonal regeneration were restricted to up-regulated axonal positivity of hypoxia-inducible factor 1 alpha, while growth-associated protein 43, erythropoietin and its receptor were not or even down-regulated. Periaxin-positive structures, indicative of Schwann cell remyelination, were only detected within few advanced lesions. CONCLUSIONS: The present findings demonstrate a complex sequence of axonal cytoskeletal breakdown mechanisms. Moreover, though sparse, this is the first report of Schwann cell remyelination in CDV-DL. Facilitation of these very limited endogenous regenerative responses represents an important topic for future research.

Contribution of Schwann Cells to Remyelination in a Naturally Occurring Canine Model of CNS Neuroinflammation.

Gliogenesis under pathophysiological conditions is of particular clinical relevance since it may provide evidence for regeneration promoting cells recruitable for therapeutic purposes. There is evidence that neurotrophin receptor p75 (p75NTR)-expressing cells emerge in the lesioned CNS. However, the phenotype and identity of these cells, and signals triggering their in situ generation under normal conditions and certain pathological situations has remained enigmatic. In the present study, we used a spontaneous, idiopathic and inflammatory CNS condition in dogs with prominent lympho-histiocytic infiltration as a model to study the phenotype of Schwann cells and their relation to Schwann cell remyelination within the CNS. Furthermore, the phenotype of p75NTR-expressing cells within the injured CNS was compared to their counter-part in control sciatic nerve and after peripheral nerve injury. In addition, organotypic slice cultures were used to further elucidate the origin of p75NTR-positive cells. In cerebral and cerebellar white and grey matter lesions as well as in the brain stem, p75NTR-positive cells co-expressed the transcription factor Sox2, but not GAP-43, GFAP, Egr2/Krox20, periaxin and PDGFR-α. Interestingly, and contrary to the findings in control sciatic nerves, p75NTR-expressing cells only co-localized with Sox2 in degenerative neuropathy, thus suggesting that such cells might represent dedifferentiated Schwann cells both in the injured CNS and PNS. Moreover, effective Schwann cell remyelination represented by periaxin- and P0-positive mature myelinating Schwann cells, was strikingly associated with the presence of p75NTR/Sox2-expressing Schwann cells. Intriguingly, the emergence of dedifferentiated Schwann cells was not affected by astrocytes, and a macrophage-dominated inflammatory response provided an adequate environment for Schwann cells plasticity within the injured CNS. Furthermore, axonal damage was reduced in brain stem areas with p75NTR/Sox2-positive cells. This study provides novel insights into the involvement of Schwann cells in CNS remyelination under natural occurring CNS inflammation. Targeting p75NTR/Sox2-expressing Schwann cells to enhance their differentiation into competent remyelinating cells appears to be a promising therapeutic approach for inflammatory/demyelinating CNS diseases.

Dynamic Changes of Microglia/Macrophage M1 and M2 Polarization in Theiler's Murine Encephalomyelitis.

Microglia and macrophages play a central role for demyelination in Theiler's murine encephalomyelitis (TME) virus infection, a commonly used infectious model for chronic-progressive multiple sclerosis. In order to determine the dynamic changes of microglia/macrophage polarization in TME, the spinal cord of Swiss Jim Lambert (SJL) mice was investigated by gene expression profiling and immunofluorescence. Virus persistence and demyelinating leukomyelitis were confirmed by immunohistochemistry and histology. Electron microscopy revealed continuous myelin loss together with abortive myelin repair during the late chronic infection phase indicative of incomplete remyelination. A total of 59 genes out of 151 M1- and M2-related genes were differentially expressed in TME virus-infected mice over the study period. The onset of virus-induced demyelination was associated with a dominating M1 polarization, while mounting M2 polarization of macrophages/microglia together with sustained prominent M1-related gene expression was present during the chronic-progressive phase. Molecular results were confirmed by immunofluorescence, showing an increased spinal cord accumulation of CD16/32(+) M1-, arginase-1(+) M2- and Ym1(+) M2-type cells associated with progressive demyelination. The present study provides a comprehensive database of M1-/M2-related gene expression involved in the initiation and progression of demyelination supporting the hypothesis that perpetuating interaction between virus and macrophages/microglia induces a vicious circle with persistent inflammation and impaired myelin repair in TME.

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.