Upregulation of virulence genes promotes Vibrio cholerae biofilm hyperinfectivity.

Vibrio cholerae remains a major global health threat, disproportionately impacting parts of the world without adequate infrastructure and sanitation resources. In aquatic environments, V. cholerae exists both as planktonic cells and as biofilms, which are held together by an extracellular matrix. V. cholerae biofilms have been shown to be hyperinfective, but the mechanism of hyperinfectivity is unclear. Here we show that biofilm-grown cells, irrespective of the surfaces on which they are formed, are able to markedly outcompete planktonic-grown cells in the infant mouse. Using an imaging technique designed to render intestinal tissue optically transparent and preserve the spatial integrity of infected intestines, we reveal and compare three-dimensional V. cholerae colonization patterns of planktonic-grown and biofilm-grown cells. Quantitative image analyses show that V. cholerae colonizes mainly the medial portion of the small intestine and that both the abundance and localization patterns of biofilm-grown cells differ from that of planktonic-grown cells. In vitro biofilm-grown cells activate expression of the virulence cascade, including the toxin coregulated pilus (TCP), and are able to acquire the cholera toxin-carrying CTXФ phage. Overall, virulence factor gene expression is also higher in vivo when infected with biofilm-grown cells, and modulation of their regulation is sufficient to cause the biofilm hyperinfectivity phenotype. Together, these results indicate that the altered biogeography of biofilm-grown cells and their enhanced production of virulence factors in the intestine underpin the biofilm hyperinfectivity phenotype.

The extrachromosomal elements of the Naegleria genus: How little we know.

There are currently 47 characterized species in the Naegleria genus of free-living amoebae. Each amoeba has thousands of extrachromosomal elements that are closed circular structures comprised of a single ribosomal DNA (rDNA) copy and a large non-rDNA sequence. Despite the presence of putative open reading frames and introns, ribosomal RNA is the only established transcript. A single origin of DNA replication (ori) has been mapped within the non-rDNA sequence for one species (N. gruberi), a finding that strongly indicates that these episomes replicate independently of the cell's chromosomal DNA component. This article reviews that which has been published about these interesting DNA elements and by analyzing available sequence data, discusses the possibility that different phylogenetically related clusters of Naegleria species individually conserve ori structures and suggests where the rRNA promoter and termination sites may be located.

Enteroviruses, type 1 diabetes and hygiene: a complex relationship.

Type 1 diabetes (T1D) is an autoimmune disease in which the immune system mounts an attack on the host's insulin-producing beta cells. Because most cases of T1D cannot be attributed only to individual genetics, it is strongly inferred that there is a significant environmental contribution, such as infection, impacting disease development. The human enteroviruses (HEV) are common picornaviruses often implicated as triggers of human T1D, although precisely which of the numerous HEV may be involved in human T1D development is unknown. Experiments using non-obese diabetic (NOD) mice, commonly used to model T1D, show that induction of T1D by HEV infection in NOD mice is a multifactorial process involving both the virus and the host. Interestingly, results demonstrate that HEV infection of NOD mice can also induce long-term protection from T1D under certain conditions, suggesting that a similar mechanism may occur in humans. Based upon both experimental animal and observational human studies, we postulate that HEV have a dual role in T1D development and can either cause or prevent autoimmune disease. Whichever outcome occurs depends upon multiple variables in the host-virus equation, many of which can be deduced from results obtained from NOD mouse studies. We propose that the background to the sharply rising T1D incidences observed in the 20th century correlates with increased levels of hygiene in human societies. Viewing T1D in this perspective suggests that potential preventative options could be developed.

Double dissociation of the effects of haloperidol and the dopamine D3 receptor antagonist ABT-127 on acquisition vs. expression of cocaine-conditioned activity in rats.

Dopamine receptors play a critical role in reward-related learning, but receptor subtypes may be differentially involved. D2-preferring receptor antagonists, e.g., haloperidol, attenuate acquisition of cocaine-conditioned motor activity at doses that fail to block expression. We compared haloperidol [4-[4-(4-chlorophenyl)-4-hydroxy-1-piperidyl]-1-(4-fluorophenyl)-butan-1-one] with the D3 receptor-preferring antagonist 2,3-di-tert-butyl-6-{4-[3-(4,5-dimethyl-4H-[1,2,4] triazol-3-yisulfanyl)-propyl]-piperazin-1-y1}-pyrimidine hydrochloride (ABT-127), given at D3 receptor-selective doses [i.e., no displacement of [(3)H]3,5-dichloro-N-[[(2S)-1-ethyl-2-pyrrolidinyl]methyl]-2-hydroxy-6-methoxybenzamide binding, no effects on γ-butyrolactone-induced striatal l-3,4-dihydroxyphenylalanine; haloperidol accumulation; no attenuation of apomorphine-induced stereotypy]. We hypothesized that haloperidol and ABT-127 will produce a doubly dissociable effect on acquisition versus expression of cocaine-conditioned activity. Rats received three 1-h habituation sessions to activity monitors followed by three 1-h cocaine (10 mg/kg) conditioning sessions. The expression phase (no cocaine injections) took place 48 h later. Haloperidol (50 µ/kg) given during the conditioning phase blocked the acquisition of conditioned activity but failed to block the expression of conditioning when given on the test day. In contrast, ABT-127 (1.0 mg/kg), when given during conditioning, failed to block the acquisition of conditioned activity but blocked the expression of conditioning when administered on the test day. Results suggest that D2 receptors are more critically involved in acquisition than initial expression and D3 receptors are more critically involved in expression than acquisition of conditioned activity based on cocaine.

The CVB and etiology of type 1 diabetes.

The origins of type 1 diabetes (T1D) are largely unknown. Fewer than 50% of the cases of the disease are attributable to host genetics, indicating that environmental factors are involved in disease development. The most often cited environmental agents implicated as initiators of T1D are the human enteroviruses, in particular the group B coxsackieviruses (CVB). Although the connection between the CVB and T1D has not been firmly established, significant' evidence supports the role of these pathogens in T1D development.

Chemoattraction and chemotaxis in Dictyostelium discoideum: myxamoeba cannot read spatial gradients of cyclic adenosine monophosphate.

Myxamoebae of the morphogenetic cellular slime mold Dictyostelium discoideum are thought to be able to accurately read and respond to directional information in spatial gradients of cyclic AMP. We examined the spatial and temporal mechanisms proposed for chemotaxis by comparing the behavior of spreading or evenly distributed cell populations after exposure to well-defined spatial gradients. The effects of gradient generation on cells were avoided by using predeveloped gradients. Qualitatively different responses were obtained using (a) isotropic, (b) static spatial, or (c) temporal (impulse) gradients in a simple chamber of penetrable micropore filters. We simulated models of chemotaxis and chemokinesis to aid our interpretations. The attractive and locomotory responses of populations were maximally stimulated by 0.05 microM cyclic AMP, provided that cellular phosphodiesterase was inhibited. But a single impulse of cyclic AMP during gradient development caused a greater and qualitatively different attraction. Attraction in spatial gradients was only transient, in that populations eventually developed a random distribution when confined to a narrow territory. Populations never accumulated nor lost their random distribution even in extremely steep spatial gradients. Attraction in spatial gradients was inducible only in spreading populations, not randomly distributed ones. Thus, spatial gradients effect biased-random locomotion: i.e., chemokinesis without adaptation. Cells cannot read gradients; the reaction of the cells is stochastic. Spatial gradients do not cause chemotaxis, which probably requires a sharp stimulant concentration increase (a temporal gradient) as a pulse or impulse. The results also bear on concepts of how embryonic cells might be able to decipher the positional information in a morphogen spatial gradient during development.

Expression of the human histocompatibility leukocyte antigen DR3 transgene reduces the severity of demyelination in a murine model of multiple sclerosis.

The role of various MHC genes in determining the progression of multiple sclerosis (MS) remains controversial. The HLA-DR3 gene has been associated with benign relapsing MS in some genetic epidemiologic studies, but with disease progression in others. We induced demyelination in highly susceptible B10.M and B10.Q mice expressing the DR3 (HLA-DRB1*0301) transgene to determine directly the effects of a human transgene by infecting them with Theiler's murine encephalomyelitis virus (TMEV). DR3+ mice experienced a dramatic reduction in the extent and severity of demyelination compared with DR3- littermate controls, whereas anti-TMEV antibody titers, delayed-type hypersensitivity responses, and levels of infectious virus, virus antigen, and virus RNA were similar in both groups. To address a possible mechanism of how the human transgene is reducing virus-induced demyelination, we analyzed cytokine expression in the lesions and also determined whether B10.M mice can respond to peptides derived from the DR3 molecule. Intense staining for IFN-gamma and IL-4, T helper (TH) 1 and TH2 cytokines, respectively, was found in the lesions of TMEV-infected DR3- mice but not in the DR3+ transgenic mice at day 21 after infection. DR3 peptides elicited strong proliferative responses in B10.M mice but not in B10.M (DR3+) mice. These experiments are the first to demonstrate that a human class II DR gene can alter the severity of demyelination in an animal model of MS without influencing viral load. These experiments are consistent with a mechanism by which DR3 reduces demyelination by altering the cytokine expression in the lesions, possibly by deleting T cells involved in virus-induced pathology.

Evolution of virulence in picornaviruses.

The Picornaviridae encompass many positive-strand RNA viruses, all of which share a generally similar genome design and capsid structure, but which induce quite diverse diseases in humans and other animals. Picornavirus strains of the same serotype have been shown to express different virulence (or pathogenic) phenotypes when studied in animal models, demonstrating that key elements of pathogenesis reside in the viral genome. However, the genetics that determine the virulence phenotype of any picornavirus are poorly understood. Picornaviruses do not have virulence genes per se, but the design ofthe capsid andhow it interacts with the virus receptor expressed on the host cell surface, specific sequences within the nontranslated regions of the viral genome, as well as coding sequences that result in different protein sequences may all have a part in determining the virulence phenotype. Virulence may be better understood as a continuum from an apparent inability to induce disease to the ability to cause severe pathogenic changes. Ultimately, the ability of a picornavirus to induce disease depends upon viral genetics and how they are modulated by the host environment.

The effect of short-term prophylactic methylprednisolone on the incidence and severity of postpericardiotomy syndrome in children undergoing cardiac surgery with cardiopulmonary bypass.

OBJECTIVE: The aim of this study was to determine the effect of prophylactic immune suppression on the incidence and severity ofpostpericardiotomy syndrome (PPS) in children after cardiac surgery with cardiopulmonary bypass (CPB). BACKGROUND: Prophylactic suppression of the inflammatory response has an unknown effect on the incidence and severity of PPS in children undergoing surgery with CPB. METHODS: This randomized double-blind placebo controlled trial included two study groups. Group A received pre-CPB intravenous methylprednisolone (1 mg/kg) plus four additional intravenous doses over 24 h, and Group B received intravenous saline placebo at identical intervals. Data included patient demographics, cardiac diagnosis/operation, CPB time, incidence and severity of PPS. Noncomplicated PPS--temperature >100.5 degrees F, pericardial friction rub, patient irritability, small pericardial +/- pleural effusion. Complicated PPS--noncomplicated PPS plus hospital readmission +/- pericardiocentesis or thoracentesis. RESULTS: We randomized 266 children: 20 exclusions (6 perioperative deaths, 14 reasons unrelated to treatment) leaving Group A (n = 126) and Group B (n = 120). There were no significant group differences in gender, cardiac diagnosis or CPB time. Group mean age differed (p = 0.05) and was treated as a covariate with no substantive outcome effect. In total, 39/246 children (16%) developed PPS (noncomplicated: n = 30, complicated: n = 9). There was no inter-group difference in overall PPS incidence (p = 0.73). However, Group A had a marginally significant increase in complicated PPS (p = 0.05). CONCLUSIONS: Intravenous methylprednisolone at a standard anti-inflammatory dose administered pre-CPB and early post-CPB neither prevents nor attenuates PPS in children. Short-term pre-CPB and post-CPB methylprednisolone treatment may complicate PPS.

Influence of age on the effect of bidirectional cavopulmonary anastomosis on left ventricular volume, mass and ejection fraction.

OBJECTIVES: We sought to identify age-related differences in the ventricular response of patients after bidirectional cavopulmonary anastomosis (CPA) and to compare changes in the ventricular response among children < 3 years of age who underwent CPA with that of age-matched control subjects who had a systemic to pulmonary artery shunt alone. BACKGROUND: Pre-Fontan CPA has been advocated over a systemic to pulmonary artery shunt alone in patients with a single ventricle to facilitate ventricular volume unloading and minimize risk of the Fontan operation. METHODS: Our study evaluated 23 patients who initially received a systemic to pulmonary artery shunt as an initial procedure before subsequent Fontan palliation. In eight of these patients (group I), bidirectional CPA was performed before age 3 years, and in four (group II), it was performed after age 10 years. The remaining 11 patients (group III, age and weight control group for group I) were maintained with their initial shunt until they underwent Fontan palliation. Serial echocardiographic analysis was used retrospectively to evaluate left ventricular volume and mass and systolic pump function (ejection fraction) before and after bidirectional CPA. RESULTS: Through 10 months of follow-up, group I patients showed significant decreases in indexed end-diastolic volume both after CPA (120 ml/m1.5 body surface area vs. 78 ml/m1.5, p = 0.001) and in comparison with values in patients in group II and III, who showed no changes in end-diastolic volume (p < 0.001). Indexed ventricular mass decreased moderately after bidirectional CPA in group I (from 228 g/m1.5 body surface area to 148 g/m1.5) but remained unchanged in groups II and III. The differences in trends between groups I and III were significant (p = 0.03). Ejection fraction decreased significantly in group II versus group I patients (0.48 to 0.27 vs. 0.51 to 0.52, p < 0.05) after CPA. Oxygen saturation measurements before and after bidirectional CPA revealed a significant increase in group I (73% to 86%, p < 0.001) and a decrease in group II (82% to 73%, p < 0.01). CONCLUSIONS: Bidirectional CPA facilitates ventricular volume unloading and promotes regression of left ventricular mass in younger children (< 3 years) in preparation for a Fontan operation. In contrast, bidirectional CPA is of questionable value in older children as a staging procedure for Fontan palliation.

Chromosome 14 contains determinants that regulate susceptibility to Theiler's virus-induced demyelination in the mouse.

Theiler's murine encephalomyelitis virus causes a chronic demyelinating disease in susceptible strains of mice that is similar to human multiple sclerosis. Several nonmajor histocompatibility complex-linked genes have been implicated as determinants of susceptibility or resistance to either demyelination or virus persistence. In this study, we used linkage analysis of major histocompatibility complex identical H-2d (DBA/2J x B10.D2) F2 intercross mice to identify loci associated with susceptibility to virus-induced demyelinating disease. In a 20-cM region on chromosome 14, we identified four markers, D14Mit54, D14Mit60, D14Mit61, and D14Mit90 that are significantly associated with demyelination. Because two peaks were identified, one near D14Mit54 and one near D14Mit90, it is possible that two loci in this region are involved in controlling demyelination.

Absence of spontaneous central nervous system remyelination in class II-deficient mice infected with Theiler's virus.

We previously showed that Theiler's murine encephalomyelitis virus (TMEV)-infected major histocompatibility complex (MHC) class II-deficient mice develop both demyelination and neurologic deficits, whereas MHC class I-deficient mice develop demyelination but no neurologic deficits. The absence of neurologic deficits in the class I-deficient mice was associated with preserved sodium channel densities in demyelinated lesions, a relative preservation of axons, and extensive spontaneous remyelination. In this study, we investigated whether TMEV-infected class II-deficient mice, which have an identical genetic background (C57BL/6 x 129) as the class I-deficient mice, have preserved axons and spontaneous myelin repair following chronic TMEV-infection. Both class I- and class II-deficient mice showed similar extents of demyelination of the spinal cord white matter 4 months after TMEV infection. However, the class I-deficient mice demonstrated remyelination by oligodendrocytes, whereas class II-deficient mice showed minimal if any myelin repair. Demyelinated lesions, characterized by inflammatory infiltrates in both mutants, revealed disruption of axons in class II- but not class I-deficient mice. Further characterization revealed that even though class II-deficient mice lacked TMEV-specific IgG, they had virus-specific IgM, which, however, did not neutralize TMEV in vitro. In addition, class II-deficient mice developed TMEV-specific cytotoxic T-lymphocytes in the CNS during the acute (7 days) disease, but these cytotoxic lymphocytes were not present in the chronic stage of disease, despite a high titer of infectious virus throughout the disease. We envision that the presence of demyelination, high virus titer, absence of remyelination, and axonal disruption in chronically infected class II-deficient mice contributes to the development of paralytic disease.

CD40L is critical for protection from demyelinating disease and development of spontaneous remyelination in a mouse model of multiple sclerosis.

Theiler's murine encephalomyelitis virus (TMEV) induces acute neuronal disease followed by chronic demyelination in susceptible strains of mice. In this study we examined the role of a limited immune defect (deletion or blocking of CD40 ligand [CD40L]) on the extent of brain disease, susceptibility to demyelination, and the ability of demyelinated mice to spontaneously remyelinate following TMEV infection. We demonstrated that CD40L-dependent immune responses participate in pathogenesis in the cerebellum and the spinal cord white matter but protect the striatum of susceptible SJL/J mice. In mice on a background resistant to TMEV-induced demyelination (C57BL/6), the lack of CD40L resulted in increased striatal disease and meningeal inflammation. In addition, CD40L was required to maintain resistance to demyelination and clinical deficits in H-2b mice. CD40L-mediated interactions were also necessary for development of protective H-2b-restricted cytotoxic T cell responses directed against the VP2 region of TMEV as well as for spontaneous remyelination of the spinal cord white matter. The data presented here demonstrated the critical role of this molecule in both antibody- and cell-mediated protective immune responses in distinct phases of TMEV-mediated pathology.

CNS cell populations are protected from virus-induced pathology by distinct arms of the immune system.

The basis for the distinct patterns of brain pathology in individuals experiencing virus-induced encephalitis may be related to either the tropism of the virus or the host's response to virus infection of the central nervous system (CNS). In these studies we used Theiler's murine encephalomyelitis virus (TMEV) and a series of mice deficient in various immune system components (alpha/beta T cells, antibody, Class I MHC, and Class II MHC) to examine the hypothesis that discrete populations of CNS cells are protected differentially from virus infection by distinct arms of the immune response. Here we demonstrate that the Class I-mediated immune response provided more protection from areas of the brain (brainstem, corpus callosum and cerebellum) with abundant white matter as there was significantly more disease in these areas in beta2m -/- (Class I-deficient) mice as compared to A beta(0) (Class II-deficient) mice. In contrast, the striatum, with an abundance of neurons, was protected from virus-induced pathology primarily by antibody. In addition, we determined that antibody and alpha/beta T cells provided protection from severe deficits and death during the acute phase of the disease. The data presented here support the hypothesis that distinct immune system components function to protect discrete areas of the CNS from virus-induced pathology.

Influence of antipsychotics and serotonin antagonists on presynaptic receptors modulating the release of serotonin in synaptosomes of the nucleus accumbens of rats.

In the nucleus accumbens of rats the release of [3H]serotonin (5-HT) from superfused synaptosomes stimulated by 30 mM K+ was investigated. In the presence of 40 microM of the uptake inhibitor cocaine the release of [3H]5-HT was inhibited by 5-HT in a concentration-dependent manner (IC50 = 0.45 microM). The maximum inhibitory effect of 5-HT was 54% of controls. The inhibition of K+-stimulated release of [3H]5-HT induced by 5-HT was antagonized completely by methiothepine and clozapine, respectively, whereas methysergide had only a weak antagonizing effect in a concentration of 20 microM or less, haloperidol was ineffective. Furthermore, the synaptosomal K+-stimulated release of [3H]5-HT was also inhibited by dopamine (DA) in a concentration-dependent manner (IC50 = 0.1 microM). This inhibitory effect was antagonized by antipsychotic drugs, the rank order of antagonistic potencies was sulpiride greater than haloperidol greater than clozapine; methiothepine was ineffective. The experimental system (the K+-stimulated synaptosomal release of [3H]5-HT seems to be a suitable model for differentiating dopaminergic and/or serotonergic components of antipsychotics or other drugs on presynaptic receptors.

Influence of antipsychotics on presynaptic receptors modulating the release of dopamine in synaptosomes of the nucleus accumbens of rats.

The release of preloaded [3H]dopamine (DA) from superfused synaptosomes stimulated by 30 mM K+ was investigated in the nucleus accumbens of rats. Under conditions preventing the uptake of DA (presence of 40 microM cocaine) release of [3H]DA was inhibited by DA and apomorphine in a concentration-dependent manner (IC50s 0.65 and 0.3 microM, respectively). The maximal inhibitory effects of DA, as well as of apomorphine, were about 50% of the controls. The DA-induced inhibition was antagonized by antipsychotics completely; the rank order of antagonistic potencies was haloperidol greater than clozapine greater than sulpiride; methiothepine was ineffective. Furthermore, the K+-stimulated release of [3H]DA was inhibited by serotonin in a concentration-dependent manner (IC50 = 0.9 microM). This inhibitory effect was antagonized by methiothepine with a high efficiency, by clozapine and methysergide with moderate efficiencies; haloperidol and sulpiride were ineffective. The experimental system demonstrated appears to be suitable for characterizing the DA- and serotonin-antagonistic potencies of antipsychotics and other drugs on presynaptic autoreceptors as well as receptors modulating release of DA in the nucleus accumbens.

Modulation of immune-associated surface markers and cytokine production by murine retinal glial cells.

Murine retinal glia are normally negative for major histocompatibility complex (MHC) Class II antigens and express low levels of MHC Class I and intercellular adhesion molecule-1 (ICAM-1) as detected by avidin-biotin-peroxidase immunohistochemistry. These surface molecules associated with immune function were either induced (Class II) or upregulated (Class I and ICAM-1) on cultured retinal glial cells in a dose- and time-dependent manner following exposure to recombinant interferon-gamma (rIFN-gamma). MHC Class I and II expression by passaged and primary cells was maximal (> 90% positive) after incubation with 100 U/m1 of rIFN-gamma for 48 h. ICAM-1 expression by primary and passaged cells tripled between 48 and 72 h after exposure to 25 or 50 U/m1 of rIFN-gamma. By 72 h after exposure to 100 U/m1 of rIFN-gamma, 62% of the retinal glia were positive for ICAM-1, whereas under normal culture conditions these molecules were detected on < 3% of the retinal glia. Bacterial lipopolysaccharide (LPS), a known stimulator of central nervous system (CNS) astrocytes, increased ICAM-1 expression only 3-fold to 9% of cells staining positively, but neither MHC Class I nor Class II expression was altered from baseline levels. Surface expression of ICAM-1, MHC Class I, and MHC Class II was unaffected by exposure to either rTNF-alpha (1000 U/m1) or rIL-6 (100 U/m1) for 24 h. Under normal culture conditions, intracellular interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) were detected immunohistochemically. Exposure to either rIFN-gamma or LPS induced more intense staining which correlated with increased secreted levels of both cytokines in culture supernatants. Levels of secreted TNF-alpha increased 6-fold after stimulation with LPS for 24 h, while secreted IL-6 increased over 9-fold. These results support the hypothesis that retinal glia may participate in intraretinal immune processes following stimulation during inflammatory and infections processes via either cell surface-or soluble mediator-dependent mechanisms or a combination of both.

Failure of treatment with Linomide or oral myelin tolerization to ameliorate demyelination in a viral model of multiple sclerosis.

Both Linomide (quinoline-3-carboxamide) and tolerization with self-antigens have been demonstrated to successfully ameliorate demyelinating disease in experimental autoimmune encephalomyelitis (EAE). Based on the autoimmune hypothesis of multiple sclerosis (MS), both agents have been tested in clinical trials but have been found to be toxic or not efficacious. We investigated the efficacy of these immunomodulators in an alternative experimental model of MS, a virus-induced demyelinating disease. Oral administration of Linomide to Theiler's virus-infected mice beginning either at time of infection or at day 15 post-infection (p.i.) resulted in an increased percentage of spinal cord quadrants with demyelination. Administration of Linomide beginning at day 15 p.i. increased lesion size as compared to infected control-treated mice. Treatment with 80 mg kg(-1) day(-1) of Linomide beginning at the time of infection significantly increased the number of Theiler's murine encephalomyelitis virus (TMEV)-positive cells mm(-2) of spinal cord white matter. There were no differences in the amount of remyelination between mice treated with Linomide or water. However, chronically infected mice treated with Linomide had severely reduced spontaneous vertical activity as measured using a activity wheel. Oral tolerization of mice with mouse or bovine myelin had no effect on virus-induced demyelination or virus antigen expression. The contrasting results obtained between the TMEV model and the autoimmune model of demyelination do not support recent reports suggesting that the underlying mechanism of demyelination in the Theiler's model is autoimmune.

Herpes simplex virus type 1 alters transcript levels of tumor necrosis factor-alpha and interleukin-6 in retinal glial cells.

PURPOSE: Studies were performed to determine whether retinal Müller cells transcribe genes for the proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF alpha). Isolated murine retinas were used to test whether these cytokines were upregulated in the retina in vivo after anterior chamber inoculation of herpes simplex virus type 1 (HSV-1). The effects of exposure to HSV-1 or interferon-gamma (IFN gamma) on transcript levels of these cytokines in cultured retinal glia also were examined. METHODS: In situ hybridization (ISH) using digoxigenin (DIG)-labeled RNA probes was used to localize mRNA for IL-6 and TNF alpha in cultured retinal glial cells. Changes in IL-6 and TNF alpha relative transcript levels were assessed in cultured retinal glial cells using a semiquantitative approach comprised of reverse transcription-polymerase chain reaction (RT-PCR) assay at low amplification cycle number followed by slot blotting and hybridization with DIG-labeled internal sequence probes. In the murine model of herpetic retinitis, the same methods were used to compare temporal changes in relative cytokine transcript levels in retinas isolated from eyes 1 to 7 days after anterior chamber injection of live HSV-1 (KOS strain; 2 x 10(4) pfu/eye) or buffer with levels in retinas isolated from normal, uninjected eyes. Densitometry was used to quantify relative signal changes obtained with serial diluted samples in slot blot assays. Cytokine signal was normalized to hypoxanthine phosphoribosyl transferase signal obtained from the same cDNA samples. RESULTS: Under baseline culture conditions, ISH and RT-PCR indicated that both IL-6 and TNF alpha were transcribed by cultured retinal glia. In vitro exposure to either viral (HSV-1) or inflammatory (IFN gamma) stimulants increased levels of these transcripts in a time-dependent manner. Peak TNF alpha mRNA levels were detected 4 hours after exposure to HSV, whereas IL-6 peaked 4 hours later (increases of 10.3 and 8.7 times over baseline, respectively). Differential increases in TNF alpha and IL-6 transcript levels were detected in retinas isolated from BALB/c mice that received anterior chamber injections of either HSV-1 or Hanks' balanced salt solution (HBSS). By day 3 after HSV-1 injection, increases of 4.5-fold in TNF alpha and 17-fold in IL-6 were detected, whereas substantially smaller changes in TNF alpha and IL-6 (1.5-fold and 6.3-fold, respectively) were observed in HBSS-injected eyes Virus-induced changes in TNF alpha mRNA levels occurred slightly earlier than for IL-6 because maximal levels of TNF alpha were detected 2 to 3 days after infection, but IL-6 peaked at day 3. CONCLUSIONS: Cultured retinal glial cells exhibit upregulated TNF alpha and IL-6 transcript levels after exposure to virus or inflammatory mediators. HSV-1 infection of the anterior segment of the mouse eye markedly upregulates TNF alpha and IL-6 mRNA levels compared to smaller responses to nonspecific inflammation. Taken together, these results identify retinal Müller cells as an intraretinal source of TNF alpha and IL-6 and support the potential of these resident cells to act as intraretinal modulators of immune and inflammatory responses.

Complementation between specific HLA-DR and HLA-DQ genes in transgenic mice determines susceptibility to experimental autoimmune encephalomyelitis.

To investigate the contribution of human leukocyte antigen (HLA) class II molecules in susceptibility to inflammatory demyelination, we induced experimental autoimmune encephalomyelitis (EAE) in transgenic (tg) mice expressing the HLA-DR3, HLA-DQ8 and HLA-DQ6 molecules in the absence of endogenous class II (Ab(o)). Following immunization with mouse myelin, HLA-DR3 tg mice mounted strong T-cell proliferative responses, and developed inflammatory lesions and demyelination in the central nervous system with mild to moderate clinical symptoms of EAE. HLA-DQ8 and HLA-DQ6 tg mice elicited weak T-cell proliferative responses and did not develop clinical symptoms of EAE. HLA-DR3/DQ6 double tg mice immunized with mouse myelin experienced clinical disease similar to the single tg HLA-DR3 tg mice, indicating that expression of DQ6 in this line had no effect on disease. In contrast, HLA-DR3/DQ8 double tg mice developed severe inflammatory lesions and clinical disease in response to immunization with mouse myelin. Our data suggest that in the presence of two susceptible class II alleles, namely HLA-DR3 and DQ8, there is additional selection and expansion of potential autoreactive T cells, resulting in enhanced severity of disease.