Th17-skewed immune response and cluster of differentiation 40 ligand expression in canine steroid-responsive meningitis-arteritis, a large animal model for neutrophilic meningitis

Background: Steroid-responsive meningitis-arteritis (SRMA) is an immune-mediated disorder characterized by neutrophilic pleocytosis and an arteritis particularly in the cervical leptomeninges. Previous studies of the disease have shown increased levels of IL-6 and TGF-beta(1) in cerebrospinal fluid (CSF). In the presence of these cytokines, naive CD4+ cells differentiate into Th17 lymphocytes which synthesize interleukin 17 (IL-17). It has been shown that IL-17 plays an active role in autoimmune diseases, it induces and mediates inflammatory responses and has an important role in recruitment of neutrophils. The hypothesis of a Th17-skewed immune response in SRMA should be supported by evaluating IL-17 and CD40L, inducing the vasculitis. Methods: An enzyme-linked immunosorbent assay (ELISA) was performed to measure IL-17 and CD40L in serum and CSF from a total of 79 dogs. Measurements of patients suffering from SRMA in the acute state (SRMA A) were compared with levels of patients under treatment with steroids (SRMA T), recurrence of the disease (SRMA R), other neurological disorders, and healthy dogs, using the two-part test. Additionally, secretion of IL-17 and interferon gamma (IFN-gamma) from the peripheral blood mononuclear cells (PBMCs) was confirmed by an enzyme-linked immunospot (ELISpot) assay. Results: Significant higher levels of IL-17 were found in CSF of dogs with SRMA A compared with SRMA T, other neurological disorders and healthy dogs (p < 0.0001). In addition, levels of CD40L in CSF in dogs with SRMA A and SRMA R were significantly higher than in those with SRMA T (p = 0.0004) and healthy controls (p = 0.014). Furthermore, CSF concentrations of IL-17 and CD40L showed a strong positive correlation among each other (rSpear = 0.6601;p < 0.0001) and with the degree of pleocytosis (rSpear = 0.8842;p < 0.0001 and rSpear = 0.6649;p < 0.0001, respectively). IL-17 synthesis from PBMCs in SRMA patients was confirmed;however, IL-17 is mainly intrathecally produced. Conclusions: These results imply that Th17 cells are inducing the autoimmune response in SRMA and are involved in the severe neutrophilic pleocytosis and disruption of the blood-brain barrier (BBB). CD-40L intrathecal synthesis might be involved in the striking vasculitis. The investigation of the role of IL-17 in SRMA might elucidate important pathomechanism and open new therapeutic strategies

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

Imepitoin is well tolerated in healthy and epileptic cats

Abstract Background Epilepsy in the cat is a serious medical condition. To date there are no licensed treatments for feline epilepsy and no well-controlled clinical studies on the efficacy or safety of antiepileptic drugs in cats. The aim of this study was to collect tolerability data and first exploratory efficacy data of imepitoin in both healthy and epileptic cats. Results In two tolerability studies, 30 healthy cats received imepition twice daily in doses of 0, 30, 40 or 80 mg/kg bodyweight for 30 days. No serious adverse events were observed in any of the dose groups. In the imepitoin treated groups, emesis was observed in some animals temporarily and intermittently mainly in the second and third weeks of treatment. In a small, single-arm, open label, uncontrolled clinical trial eight cats suffering from idiopathic epilepsy were treated with imepitoin twice daily at doses of 30 mg/kg bodyweight for 30 days. Four of these cats (50%) achieved seizure freedom for at least 8 weeks under treatment. Adverse events, mostly lethargy, decreased appetite and emesis, were often mild and transient. Conclusion In summary, imepitoin was well tolerated in healthy and epileptic cats and showed in a pilot trial indication for efficacy in treating feline epilepsy

CB₁ immunoreactivity of the cerebellum and choclear nuclei.

<p>In figure A notice strong CB₁ immunoreactivity within the molecular layer of the cerebellar cortex in a six-month-old Beagle dog. Figure B depicting in detail immunonegative Purkinje cells surrounded by strong immunorreactive fibers particularly in the basal portion (arrow). In the ten-year-old dog, there is a slight immunoreactivity in the molecular layer of the cerebellar cortex (C). Purkinje cells surrounded by a dot-like immunoreactivity appear devoid of immunoreactivity in the ten-year-old dog (D; arrow). The cochlear nucleus in a six-month-old dog showing strong CB₁ immunoreactivity (E). In figure F detail of the cochlear nucleus with strong CB₁ immunoreactivity surrounding the unstained neuronal bodies (arrow). IHC was performed using the avidin-biotin-peroxidase complex (ABC) method.</p

CB₁ immunoreactivity of the Olfactory bulb.

<p>CB₁ immunoreactivity of a six-month-old Beagle dog (A, B, C) and four-week-old dog (D). Strong immunoreactivity of the glomerular layer (GL), lack of immunoreactivity in the external plexiform layer (EPL) and mitral cell layer (ML), while moderate immunoreactivity of the internal plexiform layer (IPL) are observed in the six-month-old Beagle dog (A). Detailed immunoreactivity of the GL (arrow) is depicted in B. In the six-month-old Beagle dog, a population of cells within the internal granule cell layer (arrow) is strongly CB₁ receptor positive (C). Contrary, the glomerular layer in the four-week-old dog was only slightly CB₁ receptor positive (D). IHC was performed using the avidin-biotin-peroxidase complex (ABC) method.</p

Double immunofluorescence staining of the sciatic nerve of a six-month-old dog.

<p>P0, a marker for myelinating Schwann cells (red, B) and CB₁ (green, A) co-localize in about 100% of Schwann cells (C). p75^NTR (red, E) and CB₁ (green, D) do not co-localize (F), suggesting the absence of CB₁ receptors in non-myelinating Schwann cells. Nuclear staining (blue) with bisbenzimid.</p

Double immunofluorescence staining of the cerebral white matter of a six-month-old Beagle dog.

<p>Double immunofluorescence staining of CB₁ (green, A) with GFAP (red, B) reveals co-localization in about 20% astrocytes (C). CNPase expression (red, E) and CB₁ (green, D) do not co-localize, suggesting a lack of expression of CB₁ receptors by mature oligodendrocytes (F). Nuclear staining (blue) with bisbenzimide.</p

CB₁ immunoreactivity in the spinal cord, dorsal root ganglia and peripheral nerve.

<p>In figure A, strong CB₁ immunoreactivity is shown in the grey matter of the cervical spinal cord of a six-month-old dog and the cytoplasm of ependymal cells lining the central canal (A; arrow). Within the dorsal horn, CB₁ immunoreactivity appears surrounding unstained neuronal bodies (B; arrow). In the cervical spinal cord of a ten-year-old dog notice slight immunoreactivity of the grey matter (C). Figure D showing the cervical dorsal root ganglia of a six-month-old dog with slight immunoreactivity of large neurons and strong CB₁ immunoreactivity of small dark neurons (arrows) and satellite cells (arrowheads). The thoracic dorsal root ganglia of a ten-year-old dog with moderate CB₁ immunoreactivity of small dark neurons and satellite cells, large neurons show slight immunoreactivity (E; arrow). The cervical dorsal root ganglia of a four-week-old dog depicting scattered large and small neurons and satellite cells with slight CB₁ immunoreactivity (F; arrow). In figure G the cervical spinal nerve of a six-month-old dog shows strong CB₁ expression in Schwann cells ensheating axons (arrow). Few Schwann cells show moderate CB₁ immunoreactivity (arrow) in a thoracic spinal nerve of a ten-year-old dog (H). The cervical spinal nerve in the four-week-old dog shows moderate CB₁ immunoreactivity of scattered Schwann cells (I; arrow). IHC was performed using the avidin-biotin-peroxidase complex (ABC) method.</p

The endocannabinoid system in canine Steroid-Responsive Meningitis-Arteritis and Intraspinal Spirocercosis.

Endocannabinoids (ECs) are involved in immunomodulation, neuroprotection and control of inflammation in the central nervous system (CNS). Activation of cannabinoid type 2 receptors (CB2) is known to diminish the release of pro-inflammatory factors and enhance the secretion of anti-inflammatory cytokines. Furthermore, the endocannabinoid 2-arachidonoyl glycerol (2-AG) has been proved to induce the migration of eosinophils in a CB2 receptor-dependent manner in peripheral blood and activate neutrophils independent of CB activation in humans. The aim of the current study was to investigate the influence of the endocannabinoid system in two different CNS inflammatory diseases of the dog, i.e. Steroid-Responsive Meningitis-Arteritis (SRMA) and Intraspinal Spirocercosis (IS). The two main endocannabinoids, anandamide (AEA) and 2-AG, were quantified by mass spectrometry in CSF and serum samples of dogs affected with Steroid- Responsive Meningitis-Arteritis in the acute phase (SRMA A), SRMA under treatment with prednisolone (SRMA Tr), intraspinal Spirocercosis and healthy dogs. Moreover, expression of the CB2 receptor was evaluated in inflammatory lesions of SRMA and IS and compared to healthy controls using immunohistochemistry (IHC). Dogs with SRMA A showed significantly higher concentrations of total AG and AEA in serum in comparison to healthy controls and in CSF compared to SRMA Tr (p<0.05). Furthermore, dogs with IS displayed the highest ECs concentrations in CSF, being significantly higher than in CSF samples of dogs with SRMA A (p<0.05). CSF samples that demonstrated an eosinophilic pleocytosis had the highest levels of ECs, exceeding those with neutrophilic pleocytosis, suggesting that ECs have a major effect on migration of eosinophils in the CSF. Furthermore, CB2 receptor expression was found in glial cells in the spinal cord of healthy dogs, whereas in dogs with SRMA and IS, CB2 was strongly expressed not only in glial cells but also on the cellular surface of infiltrating leukocytes (i.e. neutrophils, eosinophils, lymphocytes, plasma cells, and macrophages) at lesion sites. The present study revealed an upregulated endocannabinoid system in dogs with inflammatory CNS diseases, highlighting the endocannabinoid system as a potential target for treatment of inflammatory CNS diseases

Number of CSF and serum samples analysed for AEA and total AG.

<p>Number of CSF and serum samples analysed for AEA and total AG.</p