The flagellum protein flagellin of Listeria monocytogenes is encoded by the flaA gene. Immediately downstream of flaA, two genes, cheY and cheA, encoding products with homology to chemotaxis proteins of other bacteria, are located. In this study we constructed deletion mutants with mutations in flaA, cheY, and cheA to elucidate their role in the biology of infection with L. monocytogenes. The ⌬cheY, ⌬cheA, and double-mutant ⌬cheYA mutants, but not ⌬flaA mutant, were motile in liquid media. However, the ⌬cheA mutant had impaired swarming and the ⌬cheY and ⌬cheYA mutants were unable to swarm on soft agar plates, suggesting that cheY and cheA genes encode proteins involved in chemotaxis. The ⌬flaA, ⌬cheY, ⌬cheA, and ⌬cheYA mutants (grown at 24°C) showed reduced association with and invasion of Caco-2 cells compared to the wild-type strain. However, spleens from intragastrically infected BALB/c and C57BL/6 mice showed larger and similar numbers of the ⌬flaA and ⌬cheYA mutants, respectively, compared to the wild-type controls. Such a discrepancy could be explained by the fact that tumor necrosis factor receptor p55 deficient mice showed dramatically exacerbated susceptibility to the wild-type but unchanged or only slightly increased levels of the ⌬flaA or ⌬cheYA mutant. In summary, we show that listerial flaA, cheY, and cheA gene products facilitate the initial contact with epithelial cells and contribute to effective invasion but that flaA could also be involved in the triggering of immune responses.

Background and Purpose-Perinatal hypoxia-ischemia (HI) produces acute and prolonged inflammation of the brain.Mast cells (MCs), numerous in the pia and CNS of neonatal rats, can initiate inflammation attributable to preformed mediators. MCs contribute to HI brain damage in the neonatal rat; MC stabilization protects through 48 hours of reperfusion. Here we hypothesize that HI induces early MC migration, activation, and release of proinflammatory molecules. Methods-HI

Perinatal hypoxic-ischemic (HI) brain damage is a major cause of mortality and neurological morbidity in infants and children. Using an established model of unilateral hypoxia-ischemia in neonatal rats, the present study focused on mast cells (MCs), important regulators of inflammatory processes, as potential contributors to HI damage. MCs are present in the pia of the neonatal rat, entering the central nervous system (CNS) during cerebral development along penetrating blood vessels. Following hypoxia-ischemia, MC numbers increased dramatically in the ipsilateral (ischemic) hemisphere (p < 0.01). In animals exposed to hypoxia only, the numbers of MCs were elevated in both hemispheres to an extent equal to that observed in the contralateral hemisphere of HI animals (p < 0.05 vs. control). Within damaged areas (ipsilateral only), MCs were observed in regions of activated microglia and astroglia that characterize the ischemic hemisphere. Using a triple-label paradigm, MCs were observed along elongating blood vessels, some of which express the GLUT1 isoform of the glucose transporter protein, indicative of blood-brain barrier vessels. To determine whether MC activation has a role in HI brain damage, rat pups were treated with the MCs stabilizer, disodium cromoglycate (cromolyn),prior to and/or following hypoxia-ischemia. The cromolyn treatment inhibited MC migration into the CNS (p < 0.05) and limited brain damage more than 50% (p < 0.01) vs. saline controls. These data support the hypothesis that MCs are key contributors to the extent of brain damage due to hypoxia-ischemia in the immature animal.

The aim of this study was to analyse cognitive and motor function in a population-based sample of people with multiple sclerosis (PwMS), taking into account both disease-related data and sociodemographic factors. Data were collected from 166 PwMS during home visits. Cognitive function was assessed by the Mini-Mental State Examination (MMSE), the Free Recall and Recognition of 12 Random Words Test (FRR12RWT), and the Symbol Digit Modalities Test (SDMT); manual dexterity by the Nine-Hole Peg Test (NHPT); global motor capacity by the Lindmark Motor Capacity Assessment; and walking capacity by a timed 10-metre walk. On cognitive tests, 55% (MMSE), 84% (FRR12RWT), and 45% (SDMT) of PwMS scored within the normal range; 27% of PwMS displayed normal manual dexterity, 9% had a maximal motorcapacity score, and 8% walked at normal speed. Factors associated with normal cognitive function were lower disability and higher education; lower disability and current employment were predictive of capacity to perform the NHPT and to walk 10 metres. In conclusion, cognitive function was normal in approximately half of the PwMS investigated, while a minority displayed normal manual dexterity and normal walking capacity. Thus, both disease severity and sociodemographic factors appear to influence cognitive and motor function in MS.

The pathologic features of cerebral Listeria monocytogenes infection strongly suggest that besides hematogenous spread, bacteria might also spread via a neural route. We propose that after snout infection of recombination activating gene 1 (RAG- Listeria monocytogenes is a gram-positive facultative intracellular bacterium which can cause severe infections in the nervous system of humans as well as domestic animals. L. monocytogenes can cause meningitis in immunocompromised humans, but after the first description by Eck (13), a number of cases of L. monocytogenes brain stem encephalitis (rhombencephalitis) also were reported. L. monocytogenes infection of trigeminal ganglia (TG) and nerves and inflammation in the brain stem that is most severe on the side of the affected trigeminal nerve in sheep have been described (10).1The pathways by which L. monocytogenes reaches the brain stem to cause rhombencephalitis have not been clarified. In general, hematogenous spread of L. monocytogenes to the nervous system via crossing of the mucosal barrier in the intestines is the accepted theory. The opinion that L. monocytogenes can pass through the blood-brain barrier is supported by experimental studies in vivo (7) and by the ability of L. monocytogenes to penetrate human endothelial cells in vitro (17, 37). However, the asymmetric bacterial load and pathology in the TG of infected sheep, goats, rabbits, and mice have indicated that the bacteria may spread along cranial nerves (5). Moreover, human patients have shown signs of progressive unilateral cranial nerve palsies followed by inflammation and the appearance of abscesses in the brain stem (2, 34). The hypothesis of a neural route of neuroinvasion was supported by the ultrastructural finding of L. monocytogenes in myelinated axons in naturally infected sheep (26) and by our previous observations of L. monocytogenes infections of neurons of dorsal root ganglia (DRG) in vitro (11).We report here that L. monocytogenes spreads along the trigeminal nerve to the brain stem in genetically immunodeficient mice. We hypothesized that the immune responses controlling the neuronal dissemination of listerial infection might be qualitatively different from those active in the control of hematogenous infection with the bacteria. By using different knockout mice, we found that both innate and T-and/or B-celldependent immune mechanisms control the neural spread of bacteria. Innate gamma interferon (IFN-␥), apparently released by NK cells, but not NK-cell cytotoxicity, inhibited the spread of bacteria along this cranial nerve route. Inducible nitric oxide synthase (iNOS) activity accounted only partially for the IFN-␥-dependent protection. A direct bacteriostatic effect of IFN-␥-activated neural tissue on the protective effect of the cytokine is suggested. MATERIALS AND METHODSMice. C57BL/6 mice were bred under specific-pathogen-free conditions. Mutant mouse strains without recombination activating gene 1 (RAG-1) or the genes for IFN-␥ receptor (IFN-␥R) (18), perforin (19), iNOS (23), an...

Neurons can be targets for microbes, which could kill the neurons. Just in reverse, we, in this study, report that bacteria can be killed when entering a neuron. Primary cultures of foetal mouse hippocampal neurons and a neuronal cell line derived from mouse hypothalamus were infected by Listeria monocytogenes. Treatment with interferon-g (IFN-g) did not affect bacterial uptake, but resulted in increased killing of intracellular bacteria, whereas the neuronal cell remained intact. The IFN-g-mediated bacterial killing was mapped to the neuronal cytosol, before listerial actin tail formation. Treatment with IFN-g induced phosphorylation of the transcription factor STAT-1 in neurons and IFN-g-mediated listerial killing was not observed in STAT-1 -/-neurons or neurons treated with IFN regulatory factor-1 antisense oligonucleotides. IFN-g-treated neuronal cells showed increased levels of inducible nitric oxide synthase (iNOS) mRNA, and antisense iNOS oligonucleotides hampered the bacterial killing by neurons upon IFN-g treatment. This novel neuronal function -i.e., that of a microbe killer -could play a crucial role in the control of infections in the immuno-privileged nervous system.

Neurotropism of Listeria monocytogenes was studied in rat dorsal root ganglia (DRG) and hippocampal neurons in culture. Using a system in which the DRG neurons can grow relatively free from other cells, it was observed that such DRG neurons, in contrast to hippocampal neurons, can be effectively infected by L. monocytogenes. The bacteria aligned along DRG axons, but not along hippocampal neurites. A mutant deficient in internalin, a protein required for entry into E-cadherin-expressing cells, did not interact with DRG neurons. Axonal migration of bacteria was studied in the DRG neurons grown in a double-chamber system, where either the neurites or the nerve cell bodies were exposed to the bacteria. The data suggest that L. monocytogenes can infect both axons and DRG nerve cell bodies, and that the bacteria can migrate in a retrograde as well as anterograde direction. These results support the notion that L. monocytogenes can spread via primary sensory neurons to the central nervous system. Infection of DRG primary sensory neurons, as employed in the present study, provides a model for analysis of bacterial and neuronal factors of importance for neurovirulence of L. monocytogenes.

SUMMARYMultiple sclerosis (MS) is assumed to result from autoaggressive T cell-mediated immune responses, in which T helper type 1 (Th1) cells producing cytokines, e.g. IFN-g and lymphotoxin promote damage of oligodendrocyte-myelin units. Dendritic cells (DCs) as potent antigen presenting cells initiate and orchestrate immune responses. Whether phenotype and function of DCs with respect to Th1 cell promotion are altered in MS, are not known. This study revealed that blood-derived DCs from MS patients expressed low levels of the costimulatory molecule CD86. In addition, production of IFN-g by blood mononuclear cells (MNCs) was strongly enhanced by DCs derived from MS patients. IFNb and IL-10 inhibited the costimulatory capacity of DCs in mixed lymphocyte reaction (MLR) and showed additive effects on suppression of IL-12 production by DCs. Correspondingly, DCs pretreated with IFN-b and IL-10 significantly suppressed IFN-g production by MNCs. IFN-b in vitro also upregulated CD80 and, in particular, CD86 expression on DCs. In vitro, anti-CD80 antibody remarkably increased, while anti-CD86 antibody inhibited DC-induced IL-4 production in MLR. We conclude that DC phenotype and function are altered in MS, implying Th1-biased responses with enhanced capacity to induce Th1 cytokine production. In vitro modification of MS patients' DCs by IFN-b and IL-10 could represent a novel way of immunomodulation and of possible usefulness for future immunotherapy of MS.