S. Schwender

Fibronectin mediates Opc-dependent internalization of Neisseria meningitidis in human brain microvascular endothelial cells

A central step in the pathogenesis of bacterial meningitis caused by Neisseria meningitidis (the meningococcus) is the interaction of the bacteria with cells of the blood–brain barrier. In the present study, we analysed the invasive potential of two strains representing hypervirulent meningococcal lineages of the ET‐5 and ET‐37 complex in human brain‐derived endothelial cells (HBEMCs). In contrast to previous observations made with epithelial cells and human umbilical vein‐derived endothelial cells (HUVECs), significant internalization of encapsulated meningococci by HBMECs was observed. However, this uptake was found only for the ET‐5 complex isolate MC58, and not for an ET‐37 complex strain. Furthermore, the uptake of meningococci by HBMECs depended on the presence of human serum, whereas serum of bovine origin did not promote the internalization of meningococci in HBMECs. By mutagenesis experiments, we demonstrate that internalization depended on the expression of the opc gene, which is present in meningococci of the ET‐5 complex, but absent in ET‐37 complex meningococci. Chromatographic separation of human serum proteins revealed fibronectin as the uptake‐promoting serum factor, which binds to HBMECs via α5β1 integrin receptors. These data provide evidence for unique molecular mechanisms of the interaction of meningococci with endothelial cells of the blood–brain barrier and contribute to our understanding of the pathogenesis of meningitis caused by meningococci of different clonal lineages.

CBS 844ins68, MTHFR TT677 and EPCR 4031ins23 genotypes in patients with deep-vein thrombosis

The role of methylenetetrahydrofolate reductase (MTHFR) TT677 genotype, cystathionine beta-synthase (CBS) 844ins68 mutation and endothelial cell protein C receptor (EPCR) 4031ins23 in the development of deep-vein thrombosis (DVT) was investigated in 300 consecutive DVT patients and 410 healthy blood donors. MTHFR TT677 was found in 40 (13.3%) patients and in 59 (14.4%) controls (OR 0.92; 95% C.I. 0.54-1.41); CBS 844ins68 in 20 (6.7%) patients and in 56 (13.7%) control subjects (OR 0.45; 95% C.I. 0.27-0.77); and the combination of MTHFR TT677 with CBS 844ins68 in 4 (1.3%) patients and in 7 (1.7%) controls (OR 0.78; 95% C.I. 0.23-2.68). Logistic regression analysis did not show a further increase of risk for MTHFR TT677 or CBS 844ins68 in combination with the factor V Leiden or the prothrombin gene G20210A mutations. The EPCR 4031ins23 was observed in 2 patients (0.66%) and none of the controls. In conclusion, MTHFR TT677 does not appear to be an important risk factor for DVT, EPCR 403ins23 seems to be very rare, its role in the development of DVT unclear. A putative protective effect of CBS 844ins68 should be further investigated.

Cervical lymphoid tissue but not the central nervous system supports proliferation of virus-specific T lymphocytes during coronavirus-induced encephalitis in rats

Abstract The CD4+ T lymphocyte response in the central nervous system (CNS) and cervical lymph nodes (CLNs) of rats with different susceptibility to coronavirus-induced encephalitis was investigated. The majority of CD4+ T lymphocytes entering the virus-infected CNS in the course of the infection are primed cells that neither proliferate ex vivo nor can be stimulated to proliferation by viral antigens or mitogen in vitro. In contrast, T lymphocytes taken from CLNs of the same animals revealed a strong proliferative response. Restimulation of CLN lymphocytes by viral antigens disclosed a striking difference between the disease-resistant rat strain Brown Norway (BN) and the susceptible Lewis (LEW) strain. Whereas BN lymphocytes responded as early as 5 days post infection, it took more than 11 days until a comparable proliferation was detectable in LEW lymphocytes. From these data we postulate that the majority of T lymphocytes entering the virus-infected brain after sensitisation and expansion in cervical lymph nodes is unresponsive to further proliferation signals and that the kinetics and magnitude of T lymphocyte stimulation in CLNs play an important role in the clinical course of the infection.

Phenotypic and functional characterization of CD8+ T lymphocytes from the central nervous system of rats with coronavirus JHM induced demyelinating encephalomyelitis

Intracerebral infection of Lewis (LEW) inbred rats with the neurotropic strain of the murine coronavirus JHM (JHMV) frequently results in a monophasic paralytic disease. In contrast, infection of Brown Norway (BN) inbred rats does not lead to clinical disease. Previous findings indicated that in both rat strains brain-infiltrating leukocytes consisted mainly of CD8+ T lymphocytes. Here, we phenotypically as well as functionally characterised this T cell subset after isolation from the central nervous system (CNS). Using JHMV-infected target cells, MHC class I restricted, cytotoxic T lymphocytes were demonstrated to be present in the leukocyte fraction from the CNS of both, susceptible LEW and disease-resistant BN rats. However, compared to infected, but healthy BN rats, diseased LEW rats generated an enhanced cytotoxic immune response which became most prominent at the maximum of neurological disease. Recently published observations from our laboratory demonstrated a strong virus-specific antibody response in the CNS of BN rats. In LEW rats, however, the response was delayed and of low magnitude. This suggests, that consequences of cytotoxic T lymphocyte action in JHMV-infected CNS tissue largely depend on the efficacy of an accompanying virus-specific humoral immune response.

Modulation of acute coronavirus-induced encephalomyelitis in γ-irradiated rats by transfer of naive lymphocyte subsets before infection

Clinical course, recovery of infectious virus from brain tissue and histopathology of the central nervous system were examined in gamma-irradiated Lewis rats reconstituted by naive lymphocytes before infection with coronavirus MHV-4 (strain JHM). Up to 9 days past infection, no differences were seen between immunologically competent and immuno-deficient animals in terms of onset and progression of neurological disease. However, in the latter animals neurological symptoms were dominated by signs of encephalitis instead of paralytic disease as usually seen in immunocompetent animals. Nevertheless, despite high titers of infectious virus in the CNS of immunodeficient animals only mild histopathological changes were noticeable. In contrast, infectious virus in the CNS of immunologically competent animals was below the detection limit of the assay. Paralytic disease and tissue destruction were T lymphocyte mediated because gamma-irradiated rats that were reconstituted by CD4+ or CD8+ T lymphocyte enriched cells in the absence of B lymphocytes revealed an earlier onset of clinical symptoms and a more rapid deterioration of their clinical state compared to fully competent animals. Whereas in CD4+ T cell reconstituted animals infectious virus was moderately reduced and tissue destruction as well as inflammatory changes in the CNS were focal, in CD8+ T cell reconstituted animals vacuolizing white matter inflammation was diffuse without reduction of infectious virus in brain tissue. From the presented data we conclude that in the acute stage of JHMV-induced encephalomyelitis of Lewis rats: (i) tissue destruction and paralytic clinical symptomatology are mainly T cell-mediated; (ii) CD4+ T lymphocytes can directly contribute to reduction of viral load in the brain and (iii) only coordinated action of both, the T and the B cell compartment enables animals to survive the infection and recover from disease.

On the Role of Different Lymphocyte Subpopulations in the Course of Coronavirus MHV IV (JHM)-Induced Encephalitis in Lewis Rats

The neurotropic strain of the mouse hepatitis virus (MHV), MHV-IV or JHM virus, causes neurological disorders in rats and mice when given intracerebrally 1,2. Depending on the rat strain and the age of the infected animal different courses of disease have been observed. Up to 2 weeks post partum Lewis (LEW) rats develop an acute fatal encephalitis, whereas animals older than 3 weeks usually do not succumb to the infection 3 but suffer from a subacute paralytic disease accompanied by multiple neurological disorders. However, the majority of them recovers completely roughly 3 weeks past infection 4. Since rats are expected to he immunologically competent at the age of 3 weeks, it is conceivable that maturity of the immune system probably plays a major role in the clinical course of the infection. We have recently shown, that T- and B-lymphocytes infiltrate into the central nervous system of these rats 4 and that the humoral immune response most likely contributes significantly to convalescence 5.

Coronavirus-JHM-induced demyelinating encephalomyelitis in rats. Analysis of the intrathecal immune response.

Disease processes of the central nervous system (CNS) accompanied by demyelination may be the result of a viral infection or the consequence of an immunopathological reaction directed against myelin (ter Meulen and Hall, 1978 ; Wisniewski, 1977; Weiner and Stohlman, 1978). In acute viral infections it has been assumed that the infection of oligodendroglial cells, leading to cell destruction, may be the main mechanism for inducing this neuropathological lesion. In the case of a persistent virus infection in oligodendroglia cells, however, it is conceivable that functional impairment of oligodendroglia cells, andlor the induction of an immune reaction to the agent which may cross-react with brain antigens, could eventually cause demyelination. Therefore, pathogenic studies on subacute or chronic demyelinating encephalomyelitides in association with viral infections may provide information on the mechanisms involved in demyelination. In connection with this, infections by murine coronaviruses are of increasing interest (Wege et al., 1982). Strain JHM is known for its ability to cause demyelinating encephalomyelitis in different animal species (Cheever et al., 1949 ; Bailey et al., 1949 ; Weiner, 1973 ; Powell and Lampert, 1975; Fleury, 1980). Additionally, the virus has a tendency to cause chronic infections accompanied by demyelination (Herndon et al., 1975 ; Stohlman and Weiner, 1981). In rats, depending on the biological property of the virus material used, the genetic background and immune response of the host, a subacute or late demyelinating encephalomyelitis can be induced, accompanied by primary demyelination (Nagashima et al., 1978, 1979; Sorensen et al., 1980). This provides a model for analysis of the virus and host factors which interact in the pathogenesis of these diseases. In this chapter the results of our studies are summarized.

Phenotypic and Functional Characterization of CD4+ T-Cells Infiltrating the Central Nervous System of Rats Infected with Coronavirus MHV IV

Mouse hepatitis virus strain JHM, a neurotropic member of coronaviridae, causes neurological diseases in rats after intracerebral inoculation. The course of the infection depends on the rat strain, the age of the animals and the type of virus used. After infection at the age of 3 weeks BN rats remain clinically healthy. In contrast, 40% of LEW rats die within the first week past infection, when inoculated at the same age. Most of the surviving animals develop neurological signs with increasing severity up to the second week past infection, followed by complete convalescence 1,2,3.

Functional Characterization of CD8+ Lymphocytes during Coronavirus MHV IV Induced Encephalitides in Rats

Intracerebral infection of rodents with the murine coronavirus JHM is a well established animal model to study the pathology of virus-induced primary demyelination of the central nervous system (CNS). Although it was assumed by Weinerl that cytopathogenic effects of the virus play the dominant role in this axonal loss of myelin sheaths, a growing body of evidence suggests now a significant contribution of the virus-specific immune response to the histopathological changes in the central nervous system as well as to the clinical course of the infection. In this context, in mice action of CD8+ T-lymphocytes appears to be a two-edged sword. On the one hand, they are necessary to clear JHM virus from infected brain tissue,2,3 on the other hand, in vivo depletion of this lymphoid subset reduces drastically the appearance of white matter destruction4,5 and adoptive transfer of either viral-specific6 or naive syngeneic CD8+ splenocytes7 in immunosuppressed animals fully reconstitutes neurological disease. This suggests that cytotoxic T-lymphocytes may cause demyelination by killing of virus-infected oligodendrocytes and thereby contribute to the clinical symptomatology of the infection.

Quantitation, Phenotypic Characterization and In Situ Localization of Lymphoid Cells in the Brain Parenchyma of Rats with Differing Susceptibility to Coronavirus JHM-Induced Encephalomyelitis

Intracerebral infection of rats with Coronavirus JHM may cause multiple neurological syndromes ranging from acute lethal encephalitis to subclinical demyelination1,2. The outcome of the infection is determined by both the type of virus used for inoculation as well as the genetic background of the host animal1,2,3,4. Data accumulated from different inbred strains of rats suggest that important host factors which influence the course of the disease include the maturation of viral target cells5 and the state of immunocompetence of the animal6,7. Although virus-specific immunity in the cerebrospinal fluid of affected animals has been examined repeatedly in the past8,9,10, little information has been collected about the dynamics of immune reactions taking place in the brain parenchyma. However, these local immunological events are almost certainly decisive for the clinical course of the infection. Therefore, we attempted to analyse the kinetics of the lymphoid cell infiltration following intracerebral inoculation of two rat strains with coronvirus JHM. In order to detect relationships between neurological symptomatology and local immune reactions in the brain tissue we selected two inbred strains which are known to behave differently after intracerebral infection. Whereas Lewis rats often develop a subacute demyelinating encephalomyelitis accompanied by severe paralytic signs of disease, Brown Norway (BN) rats reveal no signs whatsoever, although small foci of nodular demyelination can be detected in periventricular areas of the brain2.