Periodontitis is commonly observed in dogs. In human medicine, it is well documented that matrix metalloproteinases (MMPs) are involved in the destruction of the periodontium. Therefore, the aim of this prospective study was to investigate the impact of MMPs and their inhibitors, the TIMPs (tissue inhibitors of metalloproteinases), on canine periodontitis. The oral cavities of 57 dogs were examined clinically and radiologically. Gingival biopsies were obtained from the examined dogs and histologically analysed via haematoxylin and eosin stained sections. Immunohistological detection of MMP-2, MMP-3, MMP-8 and MMP-9 as well as TIMP-1 and TIMP-2 was performed by the avidin-biotin peroxidase complex technique. All sections were evaluated by light microscopy. Statistically significant positive correlations were detected between the histologically verified degree of inflammation and the expression of MMP-2, MMP-3, MMP-8 and MMP-9 as well as between changes in collagen fibre content and the occurrence of MMP-2, MMP-8 and MMP-9. Concerning TIMP-1 and TIMP-2, non-significant, generally negative correlations were observed. In summary, in canine periodontitis, an increased expression of the above mentioned MMPs and a tendentially decreased expression of TIMPs are present. In conclusion, in canine periodontitis, a MMP-TIMP imbalance is suggestive of contributing to the destruction of the periodontium.

CD44 is an important adhesion molecule for hyaluronan, a major component of the extracellular matrix (ECM). In human breast cancer, the interaction of tumour cells with the ECM via CD44 is favoured as a major candidate for tumour progression and metastasis. The present study was designed to investigate immunohistochemically the expression of the standard form of CD44 in normal, hyperplastic and neoplastic canine mammary tissue. CD44 was expressed in normal and hyperplastic mammary tissue predominantly by ductal and alveolar epithelial cells and to a minor extent by myoepithelial cells. Stromal cells and blood vessels displayed low to moderate CD44 expression. In simple and complex adenomas and benign mixed tumours there was significant up-regulation of CD44 expression in alveolar epithelial cells compared with adjacent non-neoplastic mammary tissue. Peripheral epithelial cells of simple and complex adenomas, benign mixed tumours and complex carcinomas expressed significantly more CD44 compared with adjacent non-neoplastic mammary tissue. Peripheral epithelial cells of simple adenomas revealed a significantly higher CD44 expression compared with simple carcinomas. A statistical trend to greater CD44 expression was found in peripheral epithelial cells of complex adenomas, benign mixed tumours and complex carcinomas compared with simple carcinomas. Up-regulation of CD44 therefore appears to be associated with benign or relatively benign biological behaviour of canine mammary tumours.

Canine distemper virus (CDV) belongs to the genus Morbillivirus of the Paramyxoviridae family. Due to the central nervous system (CNS) tropism of the virus and associated neuropathological changes, demyelinating canine distemper encephalitis (CDE) represents a relevant model for human demyelinating diseases like multiple sclerosis. The present review decribes the role of CD44 antigen (CD44), the principle cell surface receptor for hyaluronate and extracellular matrix (ECM) processing enzymes (matrix metalloproteinases [MMPs]) and their inhibitors (TIMPs) in the pathogenesis of demyelination. In acute and subacute CDE, a plaque-associated CD44 up-regulation is found that parallels astrocyte activation. Likewise, MMPs and TIMPs are prominently up-regulated in these lesions and are expressed mostly by astrocytes and microglia. In chronic lesions, CD44 expression declines together with the number of glial fibrillary acidic protein (GFAP) positive astrocytes. In addition, in this plaque type, CD44 is expressed on the cell membrane of perivascular mononuclear cells. In this phase, a decrease of MMP and TIMP expressions apart from MMP-11, -12, and -13 is obvious. In summary, CD44 and MMPs might be associated with the onset of demyelination and may interact to initiate ECM disturbances. Ligation of CD44 in the early phase may induce chemokines and cytokines and hence initiate and perpetuate the inflammatory process. In the chronic phase, it is conceivable that a MMP-TIMP imbalance may be the motor for lesion progression with a simultaneous influx of CD44-positive activated immune cells.

Gene products of immediate early genes (IEGs) interact with specific binding sites in promoter regions of inducible and constitutively expressed genes. Thereby, they control transcription of down-stream targets, like pro- and anti-apoptotic genes and matrix-metalloproteinases (MMPs), known to play an important role in development, plasticity, response to injury and repair of the central nervous system (CNS). A real-time quantitative RT-PCR and immunohistochemical investigation was performed to study mRNA expression levels and protein distribution patterns of IEGs in cerebrum, cerebellum, and spinal cord of SJL/J mice between postnatal weeks 1 and 40. A down-regulation of c-jun, NF-κB1, Max, Ets-1, and p53 mRNA, and an up-regulation of c-fos mRNA was noticed. Down-regulations of Ets-1 and p53 were most prominent between week 1 and 3. The prominent role in CNS development for c-jun, Ets-1 and Max was supported by immunohistochemistry. One-week-old mice were strongly positive for all three proteins in cerebral cortex, medulla oblongata, and gray matter of the spinal cord. A high staining intensity was detected in the developing granule cell layer of the cerebellum for c-jun and Ets-1, and in the Purkinje cell layer of the cerebellum for Max. In addition to the general down-regulation of most mRNAs, minor up-regulations of all IEG proteins could be detected in restricted parts of the CNS indicating regional variations and differential expression and translation during development. Apoptosis was demonstrated using immunohistochemistry for active caspase-3. The expression patterns of IEGs might represent the key to understand the balance of proteolytic activities by MMPs, myelination, and the induction of apoptosis during the development of the CNS.

Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are essential for coordinated extracellular matrix turnover during central nervous system development. Reverse transcription quantitative polymerase chain reaction was employed to evaluate the mRNA expression of MMP-2, -3, -7, -9, -10, -11, -12, -13, -14, -15, and -24, and TIMP-1, -2, -3, and -4 in the prosencephalon, rhombencephalon, and spinal cord of 1- to 40-week-old mice. The molecular data were interpreted in the context of morphological observations. Significantly higher expression levels of MMP-2, -11, -13, -14, -15, and -24, and TIMP-1 and -3 were found in the brain and spinal cord 1 week after birth compared to later time points, while MMP-9 and TIMP-2 upregulation was restricted to the brain. This upregulation coincided with the maximal extension of the transient cerebellar external granular layer, a marker of neuronal progenitor proliferation and migration. MMP-12 was significantly upregulated at later time points and found to be positively correlated with myelination in the rhombencephalon and spinal cord. MMP-3, -7, and -10 mRNA expressions remained unchanged or were negligible. In summary, while most of the MMPs and TIMPs studied seem to be involved in cell proliferation and migration, MMP-12 might be decisive for myelination.

Tissue section replicates from lymphoid tissues and livers of gnotobiotic swine were examined by immunohistochemistry for the colocalization of porcine circovirus-2 (PCV-2) nucleocapsid and terminal deoxynucleotidyl transferase (Td T)-mediated incorporation of biotinylated nucleotides ( UTP) onto the 3′-exposed hydroxyl groups ( nick end labeling) nuclear deoxyribonucleic acid (TUNEL), a marker for apoptosis. Single- and dually stained replicates from uninfected controls, subclinically affected PCV-2-infected gnotobiotic pigs, PCV-2-infected piglets immunosuppressed with cyclosporine (Cys), and PCV-2-infected piglets with postweaning multisystemic wasting syndrome (PMWS) were evaluated. Thymuses were used as positive controls for apoptosis absent PCV-2, tissue sections from dogs given hyperthermic stress were examined as positive controls for induced TUNEL. Tissues from heat-stressed dogs contained TUNEL-positive cell nuclei in both lymphoid tissues and liver, TUNEL was greatest shortly after the delivery of the hyperthermic insult. In uninfected control and subclinically affected PCV-2-infected gnotobiotic pigs, rare hepatocytes and lymphoid cells were TUNEL positive, the frequency of these was similar to that seen in uninfected controls. In PMWS-affected and Cys-treated PCV-2 piglets, the only consistent strongly positive TUNEL signal was contained within the cytoplasm of virus-positive phagocytic mononuclear cells. In phagocytes, some PCV-2 inclusions were TUNEL positive. Collectively, these data indicate that apoptosis is not the primary mechanism of lymphoid depletion and hepatocyte loss in PMWS. Apoptosis associated with systemic viral diseases may be attributable to pyrexia rather than direct or indirect effects of viruses on target cells.

Canine distemper is caused by the canine distemper virus (CDV), a RNA virus belonging to the genus Morbillivirus of the family Paramyxoviridae. The genus Morbillivirus includes measles virus, Rinderpest virus and peste-des-petits-ruminants virus. The host spectrum of CDV, which includes numerous families of Carnivores, has been changed in the last years and distemper-like diseases have been observed in numerous other species. These include epidemics in large felids, marine mammals and javelinas. Different viruses have been isolated from pinnipeds including a seal-specific isolate, termed phocine distemper virus 1, PDV-1, and a CDV strain, named PDV-2. Retrospective analysis of previous epidemics among marine mammals in various regions of the world provide evidence for the occurrence of so far unrecognized morbillivirus epidemics. In some including the mass mortalities of Baikal and Caspian seals and of large felids in the Serengeti, terrestrial carnivores including dogs and wolves have been suspected as a vector for the infectious agent. However, in other epidemics among marine mammals the source of infection remains unknown including both seal epidemics in northwestern Europe in 1988 and 2002. It remains to be determined whether a morbillivirus from other marine mammals or terrestrial carnivores caused the infection in this unprotected seal populations.

Pasteurella haemolytica is a cattle pathogen of significant economic impact. An effective vaccine against bovine pneumonic pasteurellosis is therefore of high importance. Apart from economic concerns, pasteurellosis caused by P. haemolytica is a serious disease leading to death in cattle if it remains untreated. In this study P. haemolytica-ghosts are presented as a promising vaccine candidate in cattle. To obtain sufficient vaccination material a fermentation protocol for P. haemolytica-ghost production was established. With the obtained experimental P. haemolytica-ghost vaccine, cattle immunization studies were performed based on a Pasteurella cattle challenge model developed specifically for vaccine validation. It was shown that protective immunization of cattle against homologous challenge was induced by adjuvanted P. haemolytica-ghosts. The level of protection was similar to a commercially available vaccine.

Hyperthermia above a critical threshold results in multisystemic changes that include neurological manifestations of heat stroke. It is unknown if the latter represents an intrinsic thermal sensitivity of the CNS or whether injury is secondary to physiological responses of non-CNS origin. To address this issue, the present work examined functional, structural, and biochemical changes in the CNS of dogs subjected to a thermal dosage immediately below that which induces disseminated intravascular coagulation with secondary multiple organ injury. The experimental approach is previously reported, inducing a 42.5degreesC, 90 min, whole body hyperthermia while preventing other physiological responses to treatment, including respiratory alkalosis and significant reductions in mean arterial pressure. Functional analyses included neurologic examinations and brainstem auditory evoked potential recordings in the post-treatment interval in both hyperthermic and euthermic control populations. Biochemical and structural analyses examined the expression of 70-kDa heat shock proteins, cytokines, markers of astroglial and microglial injury/activation, evidence of vascular endothelial damage, and evidence of neuronal and axonal injury in brain between 0.5 h and 8 days from the end of the treatment. The only significant change associated with treatment was induction of the major inducible 70-kDa heat shock protein, this being most prominent in the cerebellum with maximal expression at 6 h and a return to baseline by 8 days.Collectively, from these results we suggest that the canine brain is intrinsically resistant to sublethal hyperthermia such that when CNS lesions occur, they do so in the presence of other physiological derangements. (C) 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved.

Three Alaskan Huskies, two females and one male, were diagnosed with GM(1)-gangliosidosis. Clinically, diseased;animals exhibited proportional dwarfism and developed progressive neurologic impairment with signs of cerebellar dysfunction at the age of 5-7 months. Skeletal lesions characterized by retarded enchondral ossification of vertebral epiphyses were revealed by radiographs of the male dog at 5.5 months of age. Histologic examination of the central nervous system (CNS) revealed that most neurons were enlarged with a foamy to granular cytoplasm due to tightly packed vacuoles that displaced the Nissl substance. Vacuoles in paraffin-embedded sections stained positively with Luxol fast blue and Grocott's method, and in frozen sections vacuoles were periodic acid-Schiff positive. Foamy vacuolation also occurred within neurons of the autonomic ganglia. Extracerebral cells such as macrophages and peripheral lymphocytes also displayed foamy cytoplasm and vacuolation. In the CNS of diseased animals, a mild demyelination and axonal degeneration was accompanied by a significant astrogliosis (P < 0.05) in the gray matter as compared with age- and sex-matched control dogs. There was also a significant loss (P < 0.05) of oligodendrocytes in the gray and white matter of affected animals as compared with controls. Ultrastructurally, the neuronal storage material consisted of numerous circular to concentric whorls of lamellated membranes or stacks of membranes in parallel arrays. GM(1)-gangliosidosis in Alaskan Huskies resembles beta -galactosidase deficiency in other canine breeds, and these CNS disorders may be a consequence of neuronal storage and disturbed myelin processing.

Canine distemper virus infection in dogs is commonly associated with demyelinating central nervous system lesions. Investigations on viral protein expression by studying mRNA and protein distribution together with the characterization of CD4 and CD8 inflammatory cells and MHC class II up-regulation revealed a biphasic disease process. To further investigate the cellular interactions in the different plaque types the cerebella of 14 dogs with confirmed distemper infection were investigated for expression of interleukin (IL)-1β, -6, -12 and tumor necrosis factor-α (TNF) by immunohistochemistry using rabbit polyclonal anti-cytokine antibodies. T-cells and astrocytes were identified with rabbit anti CD3- and GFAP-monoclonal and polyclonal antibodies, respectively; and microglia/macrophages were characterized by their ability to bind lectin from Bandeiraea simplicifolia (BS-1). To further name the cytokine expressing cells immunoenzymatic double staining using DAB and New Fuchsin was performed. White matter lesions were classified according to histopathological criteria into acute, subacute and chronic. Canine distemper virus nucleoprotein antigen was demonstrated in nearly all plaques, except in older plaques where virus was not present within the plaque but adjacent to the lesion. IL-1 expression was observed to varying degrees in all types of lesions. Most often IL-1 was present in CD3 and BS-1 positive cells in the brain parenchyma in earlier plaques and comprising perivascular cuffs found in chronic plaques. IL-6 expression was present in all lesions, and followed a similar distribution pattern as IL-1. IL-12 displayed very often a granular extracellular pattern of immunoreactivity, especially in the brain parenchyma, and was found only in individual perivascular cells. TNF staining, predominantly found in astrocytes, was present in lesions of various types; however, staining appeared to be stronger in acute lesions and decreased in chronic plaques. In the latter, TNF seemed to be more prominent in areas adjacent to the plaques. Summarizing, in early non-demyelinating lesions without overt inflammation TNF seemed to be important, whereas in distemper lesions with inflammatory infiltrates IL-1 and to a lesser degree IL-6 were more prominent. These results imply that TNF may be involved in the pathogenesis of early demyelination in nervous distemper.

CD44 antigen (CD44), the principle cell surface receptor for hyaluronate, is up-regulated in the human demyelinating disease multiple sclerosis on fibrous astrocytes. As astrocytes are the main target cell of canine distemper virus (CDV), the consequences of a CDV infection on the CD44 expression and distribution in brains with spontaneous demyelinating canine distemper encephalitis (CDE) were of interest. Thirteen acute, 35 subacute, and 11 chronic plaques of nine dogs with immunohistologically confirmed CDE and brains of control dogs were included in the study. For light microscopy, 5-μm-thick serial sections were stained with H & E and incubated with monoclonal antibodies (mAbs) against CD44 and canine distemper virus nucleoprotein and polyclonal antibodies (pAbs) against glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP). For immunoelectron microscopy, 90-nm-thick sections were double stained with anti-GFAP and anti-CD44 mAbs to specify CD44-expressing structures. In controls, CD44 was diffusely distributed in the white matter and single meningeal cells exhibited a marginal expression of the antigen. In acute and more prominently in subacute demyelinating encephalitis, there was a plaque-associated up-regulation of CD44 which paralleled GFAP. In chronic demyelinating lesions, a reduction of CD44 associated with a loss of GFAP-positive astrocytes was noted. Additionally, in chronic plaques, CD44 was expressed on the cell membrane of perivascular mononuclear cells. Immunoelectron microscopically, in controls, CD44 was rarely demonstrated on astrocytic cell processes. In contrast, in brains with CDE CD44 was found on the cell membrane of broadened astrocytic cell processes. In summary, CD44 is up-regulated on astrocytes in the early phase of CDE and seems to represent a marker for the activation of immune cells in the late phase of the infection.

Monoclonal antibodies (mAbs) were generated against a CD44 mRNA expressing (RT-PCR) macrophage/monocyte cell line (DH82) from a dog with malignant histiocytosis. The mAbs, that reacted with DH82 cells by FACS analysis were tested on formalin-fixed, paraffin-embedded tissues. Exclusively the incubation of DH82 cell pellets with mAbs from clone 2D10 resulted in a cell membrane associated immunoreaction. Immunoelectron microscopy specified, that the antibody bound exclusively to the cell membrane and processes of DH82 cells. The mAb was tested on a variety of normal canine tissues, including lymphoid, urinary, alimentary, respiratory, and endocrine organs, nervous tissues, liver, pancreas, skin, and muscles. Furthermore, tumour and inflamed tissues were tested for immunoreaction with the mAb. Immunohistologically, the 2D10 mAb reacted with macrophages/monocytes, subsets of lymphocytes, epithelial cells, and central nervous system white matter. FACS analysis of canine peripheral blood leukocytes showed, that a high proportion of lymphocytes and granulocytes were positive with this mAb. Western blot analysis revealed, that the 2D10 mAb bound to a protein with a molecular weight of about 85 kDa. The results of FACS and Western blot analyses, RT-PCR, immunohistology and immunoelectron microscopy strongly suggest that the antigen detected by the 2D10 mAb is most likely the canine equivalent of human CD44, a cell bound hyaluronan binding proteoglycan.

CD4 and CD8 antigen expression of T cells as well as B cell and canine distemper virus (CDV) antigen distribution were immunohistologically examined in the cerebellum of dogs with spontaneous distemper encephalitis. Cellular and viral antigen expression were evaluated at intralesional and extralesional sites and in the perivascular space. Histologically, acute and subacute non-inflammatory encephalitis and subacute inflammatory and chronic plaques were distinguished. Demyelination was a feature of all subacute and chronic lesions, although the majority of plaques exhibited no or only a low level of active demyelination as demonstrated by single macrophages with luxol fast blue positive material in their cytoplasm. CDV antigen expression, observed in all distemper brains, was reduced in chronic plaques. CD4+, CD8+, and B cells were absent in controls and in some brains with acute encephalitis. A mild infiltration of CD8+ cells was noticed in the neuropil of the remaining brains with acute and all brains with subacute non-inflammatory encephalitis. Single CD4+ cells were found in two brains with acute and in all brains with subacute non-inflammatory encephalitis. Numerous CD8+ and CD4+ cells and few B cells, with a preponderance of CD8+ cells, were detected in subacute inflammatory and chronic lesions. In contrast, in perivascular infiltrates (PVI) of subacute and chronic lesions a dominance of CD4+ cells was detected. The dominating CD8+ cells in acute and subacute non-inflammatory encephalitis might be involved in viral clearance or contribute as antibody-independent cytotoxic T cells to early lesion development. In subacute inflammatory and chronic lesions CD8+ cells may function as cytotoxic effector cells and CD4+ cells by initiating a delayed-type hypersensitivity reaction. The simultaneous occurrence of perivascular B and CD4+ cells indicated that an antibody-mediated cytotoxicity could synergistically enhance demyelination. Summarized, temporal and spatial distribution of CD4+, CD8+ and B cells and virus antigen in early and late lesions support the hypothesis of a heterogeneous in part immune-mediated plaque pathogenesis in distemper demyelination.

Major histocompatibility complex class II (MHC II) and canine distemper virus (CDV) antigen expression were compared by immunohistochemistry in the cerebellar white matter of ten dogs with naturally occurring canine distemper encephalitis. In addition, infiltrating mononuclear cells were characterized by employing poly- and monoclonal antibodies directed against human CD3, canine MHC II, CD5, B cell antigen and CDV-specific nucleoprotein. Positive antigen-antibody reaction was visualized by the avidin-biotin-peroxidase complex method on frozen sections. Histologically, neuropathological changes were categorized into acute, subacute, and chronic. In control brains, MHC II expression was weak and predominantly detected on resident microglia of the white matter and on endothelial, perivascular and intravascular cells. In CDV antigen-positive brains, MHC II was mainly found on microglia and to a lesser extent on endothelial, meningeal, choroid plexus epithelial, ependymal and intravascular cells. In addition, virtually all of the perivascular cells expressed MHC II antigen. CDV antigen was demonstrated most frequently in astrocytes. Of the perivascular lymphocytes, the majority were CD3-positive cells, followed by B cells. Only a small proportion of perivascular cells expressed the CD5 antigen. In addition, B cells and CD3 and CD5 antigen-positive cells were found occasionally in subacute and frequently in chronic demyelinating plaques. In acute encephalitis, CDV antigen exhibited a multifocal or diffuse distribution, and MHC II was moderately up-regulated throughout the white matter and accentuated in CDV antigen-positive plaques. In subacute encephalitis, moderate multifocal CDV antigen and moderate to strong diffuse MHC II-specific staining, especially prominent in CDV antigen-positive lesions, were observed. In chronic encephalitis, CDV antigen expression was restricted to single astrocytes at the edge of the lesions or was absent, while MHC II expression, especially prominent on microglia, was strongly up-regulated throughout the white matter, most pronounced in demyelinated plaques. In summary, in acute and subacute lesions without perivascular cuffs, MHC II expression correlated with the presence of CDV antigen. In contrast, in chronic lesions, MHC II expression on microglial cells was the most prominent despite a few CDV antigen-positive astrocytes, indicating that nonviral antigens may play an important role as triggering molecules for the process of demyelination.