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A morbillivirus infection of tumour cells is known to exert oncolytic activity, but the mechanism of this inhibitory action has not been well defined. Matrix metalloproteinases (MMPs) are important enzymes degrading the extracellular matrix and are often upregulated in malignant neoplasms. Recent studies have demonstrated that RECK may potently suppress MMP-2 and -9 activity, thus inhibiting angiogenesis and metastasis. In this study, real time quantitative polymerase chain reaction (RT-qPCR) was used to determine the effect of persistent infection with canine distemper virus (CDV) infection on the expression of MMPs and their inhibitors (TIMPS) in a canine macrophage/monocytic tumour cell line (DH82). The activity of proMMP-2 and proMMP-9 was also verified zymographically. Following CDV infection, MMP-2, TIMP-1 and TIMP-2 were down-regulated, while RECK was upregulated. These findings suggest that CDV infection restores RECK expression in tumour cells and may interfere with the intracellular processing of MMPs and TIMPs, thus possibly influencing tumour cell behaviour beneficially for the host. However, this needs to be verified in in vivo studies.

The BeAn strain of Theiler's murine encephalomyelitis virus (TMEV) induces demyelinating disease in susceptible mice comparable to human multiple sclerosis. Recent in vivo studies showed that matrix metalloproteinases (MMPs) and their inhibitors (tissue inhibitors of MMPs, TIMPs) are associated with demyelination in Theiler's murine encephalomyelitis. The present study was performed to evaluate the in vitro MMP and TIMP expression in astrocytes and microglia following TMEV infection. Brain cell cultures from SJL/J mice were infected with the BeAn strain of TMEV and the expressions of 11 MMPs and 4 TIMPs were evaluated by reverse-transcription quantitative polymerase chain reaction (RT-qPCR) at different time points post infection (p.i.). In control astrocytes and microglia, a constitutive expression of MMP-2, -3, -9, -10, -12, -13, -14, -15, -24 and TIMP-2 to -4 was detected. In addition, TIMP-1 and MMP-11 was found in astrocytes only, and MMP-7 was absent in both cells cultures. RT-qPCR demonstrated high virus RNA copy numbers in astrocytes and a low amount in microglia. In accordance, TMEV antigen was detected in astrocytes, whereas it was below the limit of detection in microglia. MMP-3, -9, -10, -12, and -13 as well as TIMP-1 were the enzymes most prominently up-regulated in TMEV-infected astrocytes. In contrast, TMEV infection was associated with a down-regulation of MMPs and TIMPs in microglia. Conclusively, in addition to inflammatory infiltrates, TMEV-induced astrocytic MMPs might trigger a proteolysis cascade leading to an opening of the blood-brain barrier and demyelination in vivo.

Theiler murine encephalomyelitis (TME) represents an important mouse model of multiple sclerosis. Activator protein and nuclear factor-kappaB proteins are interacting transcription factors controlling the expression of cytokines involved in the demyelination process. However, specific expression patterns of these transcription factors in susceptible and resistant mouse strains and their relationship to demyelination remains to be determined. The expression of activator protein-1 (c-fos and c-jun) and nuclear factor-kappaB (p50 and p65) genes, TME virus, tumor necrosis factor-alpha, and interferon-gamma was investigated in the spinal cord of TME virus (BeAn strain)-infected SJL/J and C57BL/6 mice until 196 days postinfection (dpi) using reverse transcription-quantitative polymerase chain reaction. Additionally, c-fos, c-jun, and p50 expression was examined by applying immunohistochemistry. In susceptible SJL/J mice, in contrast to resistant C57BL/6 mice, all investigated mRNA transcripts were upregulated in the early (0-7 days dpi) and late phases (28-196 days dpi) of TME. In addition, white matter lesions of SL/J mice were characterized by c-jun-positive astrocytes and p50-positive mononuclear immune cells. Upregulation of activator protein-1 and nuclear factor-kappaB in resident glial cells in the early phase followed by strong downstream tumor necrosis factor-alpha production might account for disease development in susceptible SJL/J mice. In the late phase, the formation of JUN/JUN homodimers in intralesional astrocytes might contribute to the sustained release of proinflammatory cytokines, thereby promoting disease progression.

Demyelination of Theiler's murine encephalomyelitis (TME) depends on viral persistence and on the mouse genotype. Ets-1 expression, a transcription factor involved in T cell activation and cytokine expression, was investigated in the spinal cord during TME using RT-qPCR and immunohistochemistry. Resistant C57BL/6 mice lacking virus persistence and demyelination demonstrated a stronger upregulation of Ets-1 mRNA transcripts in the early phase of TME compared to susceptible SJL/J mice probably linked to viral clearance. Though strong Ets-1 expression in resident glial cells such as astrocytes might inhibit lesion development, delayed Ets-1 activation in inflammatory cells seemed to promote demyelination in the late phase of TME in SJL/J mice.

Theiler murine encephalomyelitis (TME) represents a highly relevant viral model for multiple sclerosis. Matrix metalloproteinases (MMPs) degrade extracellular matrix molecules and are involved in demyelination processes. To elucidate their impact on demyelination in TME, spinal cords of TME virus (TMEV)-infected SJL/J mice were taken at 9 different time points postinfection (pi) ranging from 1 hour to 196 days pi and investigated for the expression of TMEV, MMP-2, -3, -7, -9, -10, -11, -12, -13, -14, -15, -24, and TIMP-1 to -4 by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). High TMEV RNA levels were detectable throughout the observation period using RT-qPCR. In addition, TMEV RNA was visualized within demyelinated lesions by in situ hybridization. MMP-3 mRNA was significantly upregulated at 1 day pi and again in the late phase of infection. TIMP-1 mRNA was significantly elevated throughout the observation period. MMP-12 mRNA was most prominently upregulated in the late phase of infection and MMP-12 protein was localized in intralesional microglia/macrophages and astrocytes by immunohistochemistry. In summary, in early TMEV infection, MMP-3 and TIMP-1 mRNA upregulation might be directly virus-induced, whereas persistent TMEV infection directly or indirectly stimulated MMP-12 production in microglia/macrophages and astrocytes and might account for ongoing demyelination in TME.

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-kappaB1, 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) comprise a family of proteolytic zinc- and calcium-dependent enzymes that are capable of disrupting the blood-brain barrier and mediating the destruction of extracellular matrix and myelin components. MMPs are also involved in facilitating leukocyte migration into inflammatory sites of the central nervous system. To determine the cellular localization and the amount of mRNA for MMP-9, MMP-14 and a tissue inhibitor of metalloproteinases (TIMP-1) in dogs with spontaneous demyelinating distemper encephalitis, formalin-fixed paraffin-embedded cerebella were investigated by in situ hybridization using specific digoxigenin-labeled RNA probes. Additionally, immunohistochemistry was performed to characterize the different types of plaques of demyelinating leukoencephalitis. Furthermore, virus antigen and mRNA were detected by immunohistochemistry and in situ hybridization. Healthy control dogs revealed a weak signal for mRNA for MMP-9, MMP-14, and TIMP-1 in various numbers of neurons, astrocytes, microglial cells and oligodendrocytes. In the cerebella of dogs with distemper, a strong increase of both number and staining intensity of MMP-9, MMP-14, and TIMP-1 mRNA-expressing cells, mainly in subacute inflammatory lesions and chronic plaques, was observed. The number of cells expressing mRNA for MMP-9 and MMP-14 increased about two- to threefold compared to TIMP-1 mRNA-expressing cells, whereas staining intensity of individual cells was similar. In early lesions, especially astrocytes and activated macrophages/microglial cells displayed a positive signal for MMPs and TIMP-1, whereas in older lesions activated microglia/macrophages and infiltrating lymphocytes represented the main source for MMP-9, MMP-14, and TIMP-1 mRNA synthesis as revealed by double-labeling techniques. In summary, the proportionally higher increase of MMP mRNA-expressing cells might indicate an MMP/TIMP imbalance as a cause for lesion initiation and progression in demyelinating canine distemper leukoencephalitis.

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., (Copyright (c) 2005 S. Karger AG, Basel.)

Matrix metalloproteinases (MMPs) are zinc- and calcium-dependent enzymes that cleave molecules of the extracellular matrix, and thus are able to open the blood-brain-barrier and affect myelin. Their inhibitors (TIMPs) are important candidates for the therapy of demyelinating diseases. To establish an immunohistochemical profile of MMP and TIMP expression in plaque variants in dogs with spontaneous demyelinating distemper encephalitis, paraffin-embedded cerebella were studied employing the avidin-biotin-peroxidase complex method with a panel of nine polyclonal (anti-MMP-1, -3, -7, -9, -12, -13, -14, -TIMP-1, and -2) and two monoclonal antibodies (anti-latent MMP-2, and -MMP-11). All MMPs and TIMPs were prominently up-regulated in acute and subacute non-inflammatory lesions, and double-labeling techniques showed that they were mainly expressed by astrocytes and brain macrophages/microglia. In subacute inflammatory and chronic plaques, a moderate to strong decrease of MMP and TIMP expression compared to acute lesions was observed. In these phases MMP-11, -12, and -13 were still moderately present. In addition to astro- and microglia, invading perivascular mononuclear cells were positive for MMPs and TIMPs. In summary, there seems to be a phase-dependent expression of MMPs and TIMPs in demyelinating canine distemper encephalitis, and an MMP-TIMP imbalance might account for the lesion progression in this disease.

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.

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.

Brain tissue from 236 wild carnivores, 146 mustelids and 90 foxes, originating from the same geographical area in southwest Germany was collected over a 2 year period between May 1989 and May 1991 and studied for the presence of canine distemper virus (CDV) antigen by immunohistochemistry. CDV antigen was found in the brains of 54 (37%) mustelids, predominantly in the cerebellar grey matter. Interestingly, no CDV infection was observed in foxes. An increasing number of CDV infections among mustelids was noted between November 1989 and November 1990, peaking in summer 1990. Histological brain lesions, demonstrated only in 45% of the CDV positive mustelids, were characterized by non-purulent encephalitis predominantly in the cerebrum and focal vacuolation of the cerebellar white matter, whereas demyelination was only rarely observed. Histological and immunocytochemical CNS findings indicate an early stage of distemper infection in these mustelids and the high percentage of CDV positive animals together with the seasonal prevalence are suggestive of a CDV epizootic among mustelids.

Bone lesions, restricted to the metaphyses of long bones, were observed in young dogs with systemic distemper following experimental and spontaneous infection. Canine distemper virus (CDV) antigen was found immunocytochemically in hematopoietic marrow cells, osteoclasts, osteoblasts and rarely in osteocytes. In experimentally infected dogs, viral antigen was demonstrated in the metaphysis between 5 and 36 days after infection. Associated lesions, characterized by necrosis of osteoclasts, persistence of primary spongiosa and atrophy and necrosis of osteoblasts and marrow cells, were mild and most prominent between 8 and 32 days postinfection. Metaphyseal osteosclerosis (MO) of the long bones, varying from mild to severe, was observed macroscopically in 8 (19%) out of 42 dogs with spontaneous distemper. Affected animals were between 3 and 6 months of age and belonged mainly to the large breeds. In these animals, MO was characterized histologically by persistence of primary spongiosa, loss of bone marrow cells and necrosis of osteoclasts and bone marrow cells varying from mild to severe. Summarized, CDV-associated bone lesions were only transient and there were no indications of viral persistence in bones of dogs experimentally infected with CDV. Although no clinical signs related to the bones were observed, the present study reveals that infection of metaphyseal bone cells is common in young dogs with systemic distemper and occurrence of viral antigen in these cells results in defects in bone modelling.

A digoxigenin-labelled dsDNA-probe of 287 basepairs length complementary to the nucleoprotein-gene of canine distemper virus (CDV) was generated by the polymerase-chain-reaction. The dsDNA-probe hybridized specifically with base sequences of 8 different CDV strains, whereas no hybridization was observed with a porpoise and a canine parainfluenza virus and only a weak signal was obtained with measles virus. In formalin-fixed, paraffin-embedded brain sections of 35 immunohistologically CDV antigen positive dogs with spontaneous distemper encephalitis CDV-RNA could be detected in 25 cases by in situ hybridization. The reason for the lack of RNA detection in some immunohistologically positive dogs may be due to the low stability of DNA-RNA-hybrids. Degradation of RNA by RNAses or diffusion out of autolysed cells can not be excluded.

Sixteen dogs with naturally occurring acute and chronic canine distemper virus (CDV) encephalitis were examined immunohistochemically for the presence of the five major CDV-specific proteins in the central nervous system. Monoclonal antibodies (mAbs) directed against two, three, four and five epitopes of the nucleo- (N), phospho- (P), fusion (F), and hemagglutinin (H) protein, respectively, and a polyclonal monospecific antibody recognizing the matrix (M) protein were used. Both core proteins and their epitopes, three F protein epitopes and the M protein were demonstrated in all animals examined. A fourth F protein epitope was found only in 13 animals. The H-2 and H-3 epitope of the H protein were detected in 15, the H-1 and H-5 epitope in 14, and the H-4 epitope in 3 animals. All viral proteins were observed in the same types of brain cells including neurons and astrocytes. The N and P protein were demonstrated in nucleus, cytoplasm and cell processes, whereas M, H and F protein were observed in the cytoplasm only and rarely in cell processes. In addition, the M protein was detected occasionally in the nucleus of neurons and reactive astrocytes. Intralesional distribution of CDV-specific proteins varied between core and surface proteins. In acute and subacute lesions without associated inflammation, expression of the M, H and F protein was only slightly diminished compared to the N and P protein. However, plaques with severe inflammation were either devoid of viral antigen or exhibited N- and P protein-specific immunoreactivity exclusively at the periphery, whereas expression of surface proteins was severely reduced or absent. These results are suggestive of restricted synthesis of CDV envelope proteins in acute, and more prominent in chronic, distemper encephalitis.

The occurrence of the nucleo-, phospho-, matrix, fusion, and hemagglutinin proteins of the canine distemper virus (CDV) was investigated immunocytochemically in the brains of 3 dogs, 6 stone martens, 1 polecat, and 1 weasel. In addition, viral protein expression was studied in primary brain cell cultures of the 3 dogs after co-cultivation with Vero cells. Immunohistochemically, only minor differences, restricted to the H-4 epitope, were noted between the various species and CDV isolates. The data presented indicate that the mustelid virus is antigenically not distinct from the canine morbillivirus.