Cytokine responses in gnotobiotic pigs after infection with virulent or attenuated human rotavirus.

To understand the role of cytokines during rotavirus infection, we assessed the kinetics of tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) (proinflammatory), IL-12 (Th1 inducer), gamma interferon (IFN-gamma) (Th1), IL-4 and IL-10 (Th2), and transforming growth factor beta (Th3) cytokine responses by enzyme-linked immunosorbent assay in serum and intestinal contents of neonatal gnotobiotic pigs and IL-12, IFN-gamma, IL-4, and IL-10 cytokine-secreting cell (CSC) responses of mononuclear cells from ileum, spleen, and blood by ELISPOT. Pigs received the virulent Wa P1A[8]G1 strain of human rotavirus (HRV) (VirHRV), attenuated Wa HRV (AttHRV), or mock (controls). The TNF-alpha levels peaked earlier and remained elevated in serum of the VirHRV group but peaked later in the AttHRV group. In serum, IL-6 was significantly elevated at postinoculation day (PID) 1 in the VirHRV group and at PID 3 in both HRV groups. The IL-12 was detected in serum of all pigs including controls with significantly elevated peaks in both HRV-infected groups, indicating a role for IL-12 in the induction of immune responses to rotavirus infection. Only low and transient IFN-gamma responses occurred in serum and intestinal contents of the AttHRV-infected pigs, compared to significantly higher and prolonged IFN-gamma responses in the VirHRV-infected pigs. This observation coincides with the diarrhea and viremia induced by VirHRV. The number of IFN-gamma-secreting cells was significantly higher in the ileum of the VirHRV group than in that of the controls. The number of IL-4 CSCs was significantly higher in ileum of both HRV groups than in that of the controls. Significantly higher levels of IL-10 in the serum occurred early in the VirHRV group, compared to lower levels in the AttHRV group. However, the number of IL-10 CSCs was significantly higher later in ileum and spleen of the AttHRV than in the VirHRV group, suggesting a delayed initiation of a Th2 response induced by AttHRV. A significantly higher percentage of pigs had IFN-gamma and IL-10 responses in serum after VirHRV infection than after AttHRV infection or in controls. These data indicate a balanced Th1/Th2 response during rotavirus infection, with higher cytokine levels early after infection with VirHRV compared to that with AttHRV. Mapping the kinetics and patterns of cytokine responses after rotavirus infection has important implications for induction of protective immunity by HRV vaccines. Higher protection rates may be associated with more balanced Th1- and Th2-type responses, but induction of higher earlier IFN-gamma (Th1) and proinflammatory cytokines triggered by VirHRV may also play an important role in the higher intestinal immunoglobulin A responses and protection rates induced by VirHRV.

Viremia and nasal and rectal shedding of rotavirus in gnotobiotic pigs inoculated with Wa human rotavirus.

Respiratory symptoms with rotavirus shedding in nasopharyngeal secretions have been reported in children with and without gastrointestinal symptoms (Zheng et al., 1991, J. Med. Virol. 34:29-37). To investigate if attenuated and virulent human rotavirus (HRV) strains cause upper respiratory tract infections or viremia in gnotobiotic pigs, we inoculated them with attenuated or virulent HRV intranasally, intravenously, or orally or via feeding tube (gavage) and assayed virus shedding. After oral or intranasal inoculation with attenuated HRV, the pigs remained asymptomatic, but 79 to 95% shed virus nasally and 5 to 17% shed virus rectally. After inoculation by gavage, no pigs shed virus nasally or rectally, but all pigs seroconverted with antibodies to HRV. No viremia was detected through postinoculation day 10. Controls inoculated intranasally with nonreplicating rotavirus-like particles or mock inoculated did not shed virus. In contrast, 100% of pigs inoculated with virulent HRV (oral, intranasal, or gavage) developed diarrhea, shed virus nasally and rectally, and had viremia. The infectivity of sera from the viremic virulent HRV-inoculated pigs was confirmed by inoculating gnotobiotic pigs orally with pooled HRV-positive serum. Serum-inoculated pigs developed diarrhea and fecal and nasal virus shedding and seroconverted with serum and intestinal HRV antibodies. Pigs inoculated intravenously with serum or intestinal contents from the viremic virulent HRV-inoculated pigs developed diarrhea, virus shedding, and viremia, similar to the orally inoculated pigs. This study provides new evidence that virulent HRV causes transient viremia and upper respiratory tract infection in addition to gastrointestinal infection in gnotobiotic pigs, confirming previous reports of rotavirus antigenemia (Blutt et al., Lancet 362:1445-1449, 2003). Our data also suggest that intestinal infection might be initiated from the basolateral side of the epithelial cells via viremia. Additionally, virus shedding patterns indicate a different pathogenesis for attenuated versus virulent HRV.

NG2 immunoreactivity on human brain endothelial cells.

In this study, we evaluated the expression of NG2 on human brain endothelial cells derived from temporal lobe tissue resected as a treatment for intractable epilepsy. Using dissociated cell cultures, we found expression of NG2 on both proliferating and non-proliferating cells, at the mRNA level by reverse transcription-polymerase chain reaction analyses, and at the protein level by immunocytochemistry and immunoblotting. We further observed that human cerebral microvessels in nonmalignant CNS tissues immunoreacted with NG2. NG2 protein was detected using both a rabbit antibody raised against the rodent NG2 and a monoclonal antibody raised against the human NG2 (9.2.27). Our findings further define the range of resident cells of the CNS that can express NG2 and indicate that expression of NG2 by endothelial cells is not restricted to proliferating CNS endothelial cells or to endothelial cells found in brain tumors.

Remyelination in vitro following protein kinase C activator-induced demyelination.

In previous work we found that mezerein, a C kinase activator, as well as basic fibroblast growth factor (FGF-2) induce demyelination and partial oligodendrocyte dedifferentiation in highly differentiated aggregating brain cell cultures. Here we show that following protein kinase C activator-induced demyelination, effective remyelination occurs. We found that mezerein or FGF-2 caused a transient increase in DNA synthesis following a pronounced decrease of the myelin markers myelin basic protein and 2',3'-cyclic nucleotide 3'-phosphohydrolase. Both oligodendrocytes and astrocytes were involved in this mitogenic response. Within 17 days after demyelination, myelin was restored to the level of the untreated controls. Transient mitotic activity was indispensable for remyelination. The present results suggest that myelinating oligodendrocytes retain the capacity to reenter the cell cycle, and that this plasticity is important for the regeneration of the oligodendrocyte lineage and remyelination. Although it cannot be excluded that a quiescent population of oligodendrocyte precursor cells was present in the aggregates and able to proliferate, differentiate and remyelinate, we could not find evidence supporting this view.

Neuregulins and erbB receptor expression in adult human oligodendrocytes.

We have previously reported that neonatal rat oligodendrocytes (OLGs) express and secrete neuregulins (NRGs) (Raabe et al., 1997). This laboratory has also shown that NRGs stimulate the differentiation of neonatal rat OLGs and that these cells express the erbB receptors for NRGs (Raabe et al., 1997). In this study, we have characterized NRG expression in adult human OLG cultures isolated from the temporal lobe resection of intractable epilepsy patients. Using immunocytochemistry and Western blotting, we find that adult human OLGs contain both the alpha and beta isoforms of NRGs. In addition, Western blots show that the adult human OLGs secrete both isoforms as N-glycosylated molecules. These cells also express all four erbB receptor subtypes, and possibly an activated erbB receptor. The observation that these cells synthesize and secrete their own NRGs, and possibly express a tyrosine-phosphorylated erbB receptor, is consistent with autocrine and/or paracrine signaling. Amplification of this signaling may provide a useful mechanism to stimulate differentiation of adult human OLGs in demyelinating disease.

Mechanisms of tissue injury in multiple sclerosis: opportunities for neuroprotective therapy.

Development of neuroprotective therapies for multiple sclerosis is dependent on defining the precise mechanisms whereby immune effector cells and molecules are able to induce relatively selective injury of oligodendrocytes (OLs) and their myelin membranes. The selectivity of this injury could be conferred either by the properties of the effectors or the targets. The former would involve antigen specific recognition by either antibody or T cell receptor of the adaptive immune system. OLs are also susceptible to non antigen restricted injury mediated by components of the innate immune system including macrophages/microglia and NK cells. Target related selectivity could reflect the expression of death inducing surface receptors (such as Fas or TNFR-1) required for interaction with effector mediators and subsequent intracellular signaling pathways, including the caspase cascade. Development of therapeutic delivery systems, which would reach the site of disease activity within the CNS, will permit the administration of inhibitors either of the cell death pathway or of effector target interaction and opens new avenues to neuroprotection approach.

Kinin B1 receptor expression and function on human brain endothelial cells.

The kinin B1 receptor is an inducible receptor expressed in response to inflammatory mediators. We sought to determine whether kinin B1 receptor can be expressed on human brain endothelial cells (HBECs) in vitro and whether signaling via this receptor can regulate permeability and chemokine production properties of these cells. Multiplex RT-PCR amplification and western blot techniques were used to evaluate B1 receptor expression by HBECs. Although B1 receptor mRNA and protein could not be detected on resting HBECs, interferon-gamma induced a dose- and time-dependent up-regulation of B1 receptor mRNA and protein on HBECs. Stimulation of interferon-gamma-treated HBECs with the selective B1 agonist R-838 (Sar [D-Phe8] des Arg9-BK) induced a dose- and time-dependent increase in the production of inositol 3,4,5 tri-phosphate and nitric oxide. Permeability of the HBECs monolayer, as measured by BSA diffusion, was significantly increased by application of the B1 agonist. This biological effect of R-838 could be prevented by R-715, a B1 receptor antagonist and by L-NAME, a nitric oxide synthase blocker. R-838 also inhibited interleukin-8 release from HBECs. We demonstrate that B1 receptors can be up regulated on the surface of HBECs by molecules released during inflammatory response and that signaling via this receptor can regulate BBB permeability and chemokine production in vitro.

Characterization of T cell lines derived from glatiramer-acetate-treated multiple sclerosis patients.

We analyzed the effects of glatiramer acetate (GA) therapy on in vitro proliferative responses and cytokine production by lymphocytes derived from multiple sclerosis patients receiving this therapy. We confirmed that lymphocytes derived from GA naïve patients show a high frequency of response when initially exposed to GA in vitro; this frequency decreased following GA therapy. The frequency of lymphocytes responding to whole MBP stimulation did not change with GA therapy. GA- and MBP-specific T cell lines generated from these patients by repeated cycles of in vitro stimulation did not cross react. Some (23%) whole MBP-reactive T cell lines did cross react with MBP peptide 83-99. The mean levels of interferon (IFN) gamma secretion and the mean ratio of IFN-gamma/IL-5 were lower for GA-reactive cell lines, derived from patients both prior to and during GA therapy, compared to MBP-reactive T cell lines. The proportion of IFN-gamma(+) cells in unfractionated lymphocyte preparations derived from the GA-treated patients did not differ from that found for healthy controls. Our findings indicate that GA-reactive T cell lines derived from GA-treated MS patients continue to show a relative Th2 cytokine bias consistent with a bystander suppressor function. GA treatment is not associated with a cytokine phenotype shift in the total T cell or MBP-reactive T cell populations.

Interferon-gamma modulates human oligodendrocyte susceptibility to Fas-mediated apoptosis.

Interferon gamma (IFN-gamma) has been shown to be produced within multiple sclerosis (MS) lesions by infiltrating lymphocytes; systemic administration of this cytokine induces exacerbation of the disease. The aim of the current study was to establish the contribution of IFN-gamma to oligodendrocyte (OL) injury. Our studies utilized cultured human OLs, obtained by dissociation of surgically derived non-MS adult brain tissue. Neither cell survival nor myelin basic protein (MBP) gene expression were affected after 96 hours of treatment with IFN-gamma (100 U/ml), as assessed by LDH release, nucleosome enrichment assay, and RT-PCR. Expression of the death receptor Fas (CD95, APO-1) was, however, significantly increased. Furthermore, IFN-gamma-treated OLs became susceptible to Fas-mediated apoptosis when compared with untreated cells, and were protected by pretreatment with the caspase inhibitor ZVAD. TNF-alpha augmented the IFN-gamma-induced effect. Our results thus indicate that IFN-gamma is not directly cytotoxic for human OLs in culture, but could indirectly modulate functional injury-related responses by upregulating Fas on the cell surface.

Multiple sclerosis and central nervous system demyelination.

Multiple sclerosis (MS) is characterized by multifocal areas within the CNS of demyelination with relative but not absolute axonal sparing. Initial lesion development appears dependent on T cell infiltration into the CNS; however, lesion expansion may reflect tissue injury induced by additional effector mechanisms derived from cells of the immune system and endogenous CNS cells (glial cells). This relative susceptibility to injury in MS of myelin and its cell of origin, the oligodendrocyte (OL), could reflect either the properties of the effectors or the targets. Effector-determined susceptibility could relate to presence of OL/myelin-restricted T cells or antibody. OLs, at least in vitro, express MHC class I molecules and are susceptible to CD8(+)T cell-mediated cytotoxicity. OL/myelin-specific antibodies are identified in MS lesions and could induce injury via complement- or ADCC-dependent mechanisms. OLs are susceptible to injury-mediated by non-specific cell effectors including NK cells, NK-like T cells (CD56(+)), and gamma/delta T cells via perforin/granzyme-dependent mechanisms. In vitro studies of OL injury mediated via tumor necrosis factor (TNF) and CD95 indicate that differential glial cell susceptibility to injury can depend on cell surface receptor expression and intracellular signaling pathways that are activated. These target-determined susceptibility factors may be amenable to neuroprotective therapies.

Expression of a homologue of rat NG2 on human microglia.

The expression of NG2 chondroitin sulfate has been widely associated with oligodendrocyte precursors in rodents. We used a monoclonal antibody (9.2.27) against the human homologue of the rat NG2 to determine whether expression of this molecule was associated with a specific glial cell population present in dissociated cell preparations derived from adult and fetal human brain tissue. Our data, derived using FACS and immunocytochemical analyses of immediately ex vivo or cultured glial cells, indicate that the large majority of NG2 expressing cells belonged to the microglial lineage (CD68, CD11c) rather than to the oligodendrocyte lineage (O4, A2B5, GalC). In situ immunohistochemistry performed on non-fixed normal spinal cord tissue confirmed the observation that NG2 is expressed by mononuclear phagocytes of the CNS. In contrast, peripheral blood-derived monocytes were NG2(-). Cells from fetal brain tissue showed only small numbers of NG2(+) cells, which was consistent with the number of microglial cells in this preparation. In absence of additional markers, we cannot exclude that this anti-NG2 mAb might also recognize human oligodendrocyte progenitor cells.

Demyelination induced by protein kinase C-activating tumor promoters in aggregating brain cell cultures.

The plasticity of mature oligodendrocytes was studied in aggregating brain cell cultures at the period of maximal expression of myelin marker proteins. The protein kinase C (PKC)-activating tumor promoters mezerein and phorbol 12-myristate 13-acetate (PMA), but not the inactive phorbol ester analog 4alpha-PMA, caused a pronounced decrease of myelin basic protein (MBP) content and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) activity. In contrast, myelin/oligodendrocyte protein (MOG) content was affected relatively little. Northern blot analyses showed a rapid reduction of MBP and PLP gene expression induced by mezerein, and both morphological and biochemical findings indicate a drastic loss of compact myelin. During the acute phase of demyelination, only a relatively small increase in cell death was perceptible by in situ end labeling and in situ nick translation. Basic fibroblast growth factor (bFGF) also reduced the levels of the oligodendroglial differentiation markers and enhanced the demyelinating effects of the tumor promoters. The present results suggest that PKC activation resulted in severe demyelination and partial loss of the oligodendrocyte-differentiated phenotype.