Evolutionarily conserved prefrontal-amygdalar dysfunction in early-life anxiety.

Some individuals are endowed with a biology that renders them more reactive to novelty and potential threat. When extreme, this anxious temperament (AT) confers elevated risk for the development of anxiety, depression and substance abuse. These disorders are highly prevalent, debilitating and can be challenging to treat. The high-risk AT phenotype is expressed similarly in children and young monkeys and mechanistic work demonstrates that the central (Ce) nucleus of the amygdala is an important substrate. Although it is widely believed that the flow of information across the structural network connecting the Ce nucleus to other brain regions underlies primates' capacity for flexibly regulating anxiety, the functional architecture of this network has remained poorly understood. Here we used functional magnetic resonance imaging (fMRI) in anesthetized young monkeys and quietly resting children with anxiety disorders to identify an evolutionarily conserved pattern of functional connectivity relevant to early-life anxiety. Across primate species and levels of awareness, reduced functional connectivity between the dorsolateral prefrontal cortex, a region thought to play a central role in the control of cognition and emotion, and the Ce nucleus was associated with increased anxiety assessed outside the scanner. Importantly, high-resolution 18-fluorodeoxyglucose positron emission tomography imaging provided evidence that elevated Ce nucleus metabolism statistically mediates the association between prefrontal-amygdalar connectivity and elevated anxiety. These results provide new clues about the brain network underlying extreme early-life anxiety and set the stage for mechanistic work aimed at developing improved interventions for pediatric anxiety.

Retrovirus mediated gene transfer of the self antigen MBP into the bone marrow of mice alters resistance to experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a prototypic model of organ specific autoimmunity. MHC class II restricted T-cells directed against myelin basic protein (MBP) have been shown to cause EAE in susceptible strains of mice. We have asked whether the introduction of a gene encoding an autoantigen (MBP) into the hematopoetic stem cells of mice would result in tolerance to that protein. We have introduced cDNA encoding the 21.5 kDa isoform of MBP into the hematopoetic stem cells of B10.PL (73NS), SJL, and B10 mice by retrovirus-mediated gene transfer. Our experiments show expression of proviral MBP in peripheral blood and thymus following transplantation of genetically modified stem cells. Such expression does not result in deletion of MBP-specific T cells or tolerance to MBP, nor does it alter susceptibility to MBP-induced EAE in susceptible strains B10.PL and SJL. However, retrovirus-mediated gene transfer resulted in resistant B10 mice developing mild EAE. This report demonstrates that autoreactive MBP-specific T cells can be selected in the presence of endogenous antigen or an MBP-encoding retrovirus.

Changes in the amount of diseased white matter over time in patients with relapsing-remitting multiple sclerosis.

MRI is a sensitive technique for assessing disease activity in MS. Diseased white matter (WM) can be identified on T2-weighted images, and active disease is reflected by abnormalities in the blood-brain barrier (BBB) shown on T1-weighted images after administration of paramagnetic contrast agents. Active disease may be demonstrated by contrast-enhanced MRI in patients with early, mild relapsing-remitting (RR) MS even during periods of clinical stability, which indicates that MS is an active process even during the early phase of the illness. To examine the amount of abnormal WM at frequent intervals over time, we studied seven mildly affected RRMS patients, all of whom had frequent contrast-enhancing lesions. These RRMS patients were imaged monthly for 26 to 36 months at 1.5 tesla; the area of abnormal increased WM signal was calculated by image-processing software that utilizes both the T2- and T1-weighted images. All patients showed fluctuations over time in amount of abnormal WM signal, which reflected factors such as the amount of BBB breakdown (measured by number or area of enhancing lesions) and measurement error. All seven RRMS patients, however, showed an overall increase in abnormal WM. Because of the fluctuations between individual measurements, the increase was most accurately reflected when the mean of the first 6 months' measurements was compared with the mean of the final 6 months' measurements, or when a linear regression model was applied.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequence of a cDNA encoding chicken vasoactive intestinal peptide (VIP).

Mammalian pre-pro-vasoactive intestinal peptide (pre-proVIP) gives rise to the neuropeptides vasoactive intestinal peptide (VIP) and peptide histidine isoleucine amide (PHI). The cDNA encoding chicken VIP was cloned and sequenced. The region of chicken pre-proVIP homologous to the mammalian PHI region is not followed by an amidation signal. This unusual feature suggests that processing of the precursor may be different in the chicken.

Retinoid treatment of experimental allergic encephalomyelitis. IL-4 production correlates with improved disease course.

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease characterized by central nervous system inflammation and demyelination. Retinoids regulate cell differentiation and growth by binding to and activating retinoic acid receptors, which seem to be nuclear transcription factors. The effect of retinoids on chronic relapsing EAE produced by the transfer of myelin basic protein (MBP)-specific lymph node cells (LNC) was studied. All-trans-retinoic acid (tRA) inhibited the proliferation of MBP-specific LNC in vitro. However, the capacity of these cells to transfer EAE was markedly reduced by concentrations of tRA that only mildly inhibited T cell proliferation. The presence of tRA during in vitro MBP-specific LNC activation resulted in a considerable increase in IL-4 mRNA, whereas mRNA for IL-2, TNF-alpha, and IFN-gamma was decreased. Increased IL-4 also was detected in culture supernatants. However, the presence of a neutralizing Ab to IL-4 (11B11) during MBP-specific LNC activation in vitro did not reverse the inhibition of encephalitogenicity caused by tRA. The administration of retinoids in vivo resulted in an improved clinical course, even when given after disease onset. These findings suggest that T cell activation in the presence of tRA results in the development of T cells of the Th2 phenotype, which, in turn, might be responsible for the decrease in the encephalitogenicity of MBP-specific T cells. The modulation by retinoids of an immune response dominated by Th1-like T cells to one in which the protective cytokines of Th2-like cells predominate may have potential relevance for human demyelinating diseases such as multiple sclerosis.

Mapping the origin of the avian enteric nervous system with a retroviral marker.

The enteric nervous system is largely formed from the vagal neural crest which arises from the neuroaxis between somites 1-7. In order to evaluate the contribution of different regions of the vagal crest to the enteric nervous system, we marked crest cells by injecting somites 1-10 with a replication-defective spleen necrosis virus vector which contains the marker gene lacZ. After incubation in X-gal, lacZ-positive blue cells were found in the wall of the gut in three locations. Most were found at the peripheral edge of the developing circular muscle and within the developing submucosa, sites characteristic of developing ganglia. LacZ-positive cells in these ganglionic sites were always surrounded by HNK-1 immunostained cells, confirming their neural crest origin. LacZ-positive cells were also seen in a third location, the circular muscle layer of the esophagus and crop, and were separated from the HNK-1 positive ganglionic elements. These cells in the circular muscle are probably muscle cells derived from labeled mesodermal cells of the somite. Injection of somites 3, 4, 5, and 6 resulted in the largest percentage of preparations with lacZ-positive crest-derived cells and in the largest number of positive cells in the gut. After injection of these somites, lacZ-positive crest-derived cells were found in all regions of the gut from the proventriculus to the rectum. Very few positive crest-derived cells were found in the esophagus.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental allergic encephalomyelitis induced by the peptide encoded by exon 2 of the MBP gene, a peptide implicated in remyelination.

The discovery of T lymphocytes reactive to the peptide encoded by exon 2 of the myelin basic protein (MBP) gene in multiple sclerosis (MS) patients has drawn attention to MBP isoforms harboring that peptide as candidate autoantigens. Previously, immunological studies in MS had almost exclusively used the more abundant 18.5 kDa isoform of MBP, which does not contain the exon 2 peptide. Investigations of experimental allergic encephalomyelitis (EAE) have also focussed on the 18.5 kDa MBP isoform and its peptides. Since EAE is an animal model widely used to study MS, we examined the encephalitogenic potential of exon 2 peptide in the SJL/J mouse. Evidence for increased expression of exon 2-containing isoforms during remyelination in mouse CNS suggested that exon 2-sensitized T cells, with encephalitogenic capacity, might be important in the perpetuation of relapsing EAE (rEAE). Our experiments have demonstrated that exon 2 peptide is inherently immunogenic in SJL mice and that EAE could be induced by the adoptive transfer of exon 2-sensitized lymphocytes. Furthermore, the disease could be accentuated by the transfer of short-term exon 2-reactive lines or by a combination of adoptive transfer and antigenic challenge with exon 2 peptide. The immunodominant epitope(s) appeared to localize to the segment bordered by amino acids 59-85.

Superantigen modulation of experimental allergic encephalomyelitis: activation of anergy determines outcome.

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease that can be induced by the adoptive transfer of CD4, myelin basic protein (MBP)-specific T cells. Superantigens activate T cells expressing appropriate TCR V genes. In this study, MBP-specific T cells activated in vitro with a superantigen, staphylococcal enterotoxin B (SEB), could adoptively transfer a severe form of EAE in (PLxSJL)F1 mice, but did not transfer disease in PL/J or SJL/J mice. SEB treatment of donor mice anergized MBP-specific T cells using V beta 8 in (PLxSJL)F1 mice, because subsequent in vitro activation with SEB resulted in a marked decrease in proliferation to SEB and inability to transfer EAE. However, donor cells from (PLxSJL)F1 mice immunized with MBP/CFA that had been exposed to SEB in vivo before MBP stimulation in vitro still produced EAE in recipient mice. To confirm that non-V beta 8 T cells could transfer disease, donor mice were treated with antibody that eliminated V beta 8 T cells; MBP-activated T cells from these mice could still transfer EAE. Finally, EAE induced by SEB-activated T cells was substantially reduced in mice receiving anti-V beta 8 therapy in vivo. The ability of superantigens to activate encephalitogenic T cells may have relevance to human diseases such as multiple sclerosis.

Demonstration of human T-cell lymphotropic virus type I (HTLV-I) from an HTLV-I seronegative south Indian patient with chronic, progressive spastic paraparesis.

Here we describe a human T-cell lymphotropic virus type I (HTLV-I) seronegative patient from South India with a chronic, progressive spastic paraparesis from which HTLV-I has been isolated from peripheral blood lymphocytes. HTLV-I pol and tax viral sequences were detected in DNA from fresh peripheral blood lymphocytes (PBL) by polymerase chain reaction (PCR) and liquid hybridization techniques. Southern blot analysis of the PCR products demonstrated a low copy number of HTLV-I at the level of one viral copy per 10,000 fresh PBL. A long-term CD4+ T-cell line was established from PBL of this patient using recombinant interleukin-2, OKT3, and feeder cells. DNA from these cultured lines was amplified and portions of the HTLV-I long terminal repeat (U3), pol, env, and tax regions were sequenced (a total of 1,115 bp). The sequence data showed that the HTLV-I associated with this patient was 98.8% homologous to prototype HTLV-I. Southern blot analysis also confirmed the presence of full-length HTLV-I. These results indicate that HTLV-I can be demonstrated in an HTLV-I seronegative patient from South India with a chronic progressive neurological disorder.

Sequence comparisons of HTLV-I from HAM/TSP patients and their asymptomatic spouses.

We amplified and sequenced portions of the human T-lymphotropic virus type I (HTLV-I) (U3), pol, env, and pX provirus regions (1212 bp per person) from peripheral blood lymphocytes (PBL) of two married couples (case 1 and case 2). Both husbands are patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and the wives are asymptomatic HTLV-I carriers. We selected these regions because the LTR and env regions of murine retrovirus models have been involved in determining tissue tropism. In addition, the predominant immunogenic epitope for HTLV-I-specific cytotoxic T cells obtained from circulating PBL of HAM/TSP patients was localized in the HTLV-I pX region. Our aim was to examine variations in these HTLV-I regions between affected and asymptomatic spouses. In the HTLV-I regions studied, we detected no sequence variation between each couple. These data do not favor the hypothesis that neurotropic mutants of HTLV-I are involved in the pathogenesis of HAM/TSP.

Correlation between magnetic resonance imaging findings and lesion development in chronic, active multiple sclerosis.

Magnetic resonance imaging is a highly sensitive method for the detection of the lesions of multiple sclerosis and renders possible the study and the evolution of early lesions. Previous reports on magnetic resonance imaging following gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) injection demonstrated that new lesions can be recognized by contrast enhancement. The pathological basis of these observations is uncertain. We have had the opportunity to study at autopsy the brain of a patient with chronic progressive multiple sclerosis who suffered acute worsening leading to death. Magnetic resonance imaging performed 10 days and 4 weeks prior to death showed new Gd-DTPA-enhanced lesions in the posterior hemispheric white matter adjacent to the lateral ventricles. Light microscopic examination of these areas demonstrated them to be fresh lesions comprising intense inflammatory activity and dense perivascular cuffs within an edematous lesion center and a striking parenchymal mononuclear cell infiltration at the margins of the lesions. Lesions that were demonstrated by increased signal on T2-weighted images, but were not enhanced following administration of Gd-DTPA, were all of the chronic type, either inactive or active. None of these showed the intense inflammatory activity of the acute lesions and most displayed fibrous astrogliosis.

Myelin basic protein-specific T-cell responses in identical twins discordant or concordant for multiple sclerosis.

Although multiple sclerosis (MS) is thought to be an autoimmune disease, the target antigen of the immune response is unknown. Both myelin basic protein (MBP) and proteolipid protein (PLP) have been considered candidate autoantigens. Because the immune response to either foreign or self antigens is influenced by the genetic background of the host, the importance of these candidate antigens has been difficult to establish in humans because of genetic diversity. To eliminate genetic differences in MS patients and healthy controls, we have studied the MBP-specific T-cell response in 6 sets of identical twins, 3 of which were concordant and 3 discordant for MS. A total of 638 short-term T-cell lines were established and characterized for MBP-specific proliferative and cytotoxic activity, fine specificity, and human leukocyte antigen (HLA) restriction. Similar frequencies of MBP-specific T cells were observed in affected and unaffected individuals. A slightly higher percentage of cytotoxic T-cell lines was found in affected individuals. For most of the cell lines, the restriction elements were the HLA class II antigens that have been reported previously to be associated with MS; no important differences with respect to HLA restriction were found between the patients and healthy individuals. The peptide epitopes of MBP that were recognized most frequently by the T-cell lines were those previously shown to be immunodominant. Differences in specificity were seen in some discordant twins indicating that, despite genetic identity, the MBP-specific T-cell repertoire may be shaped differently. These findings indicate that differences in frequency, peptide specificity, or HLA restriction are not sufficient to implicate MBP-specific T cells in the pathogenesis of MS. However, the T-cell response to MBP may still represent one necessary component with disease occurring when this response is combined with other host characteristics such as regulation of cytokine-, adhesion molecule-, or HLA-antigen expression in the nervous system or immunoregulatory mechanisms.

Cell type-specific regulation of major histocompatibility complex (MHC) class I gene expression in astrocytes, oligodendrocytes, and neurons.

Mechanisms of major histocompatibility complex (MHC) class I gene regulation in cells of the CNS have been studied in vitro. Astrocytes in primary cultures, but neither oligodendrocytes nor neurons, constitutively expressed cell surface MHC class I molecules. Interferon-gamma (IFN-gamma) treatment led to induction of MHC class I expression in astrocytes and oligodendrocytes but not in neurons. The conserved upstream sequence containing the juxtaposed nuclear factor (NF)-kappa B-like region I and IFN-response consensus sequence (ICS) constitutively enhanced MHC class I gene promoter activity in astrocytes, but not in oligodendrocytes or in neurons. Nuclear extracts from astrocytes, but not from oligodendrocytes and neurons, had a binding activity specific for the NF-kappa B-like region I sequence, indicating that constitutive expression of MHC class I genes is governed by the upstream region I enhancer and its binding factor. IFN-gamma treatment led to induction of MHC class I promoter activity in astrocytes and oligodendrocytes, but not in neurons. In accordance with this observation, a nuclear factor that binds to the ICS was induced in astrocytes and oligodendrocytes but not in neurons following IFN-gamma treatment. This study illustrates cell type-specific regulation of MHC class I genes in the CNS that correlates with the expression of DNA binding factors relevant to MHC class I gene transcription.

Skewed T-cell receptor repertoire in genetically identical twins correlates with multiple sclerosis.

Although the cause of multiple sclerosis (MS) is unknown, it is thought to involve a T cell-mediated autoimmune mechanism. Susceptibility to the disease is influenced by genetic factors such as genes of the HLA and T-cell receptor (TCR) complex. Other evidence for a genetic influence includes the low incidence in certain ethnic groups, the increased risk if there are affected family members and the increased concordance rate for disease in monozygotic twin pairs (26%), compared to dizygotic twins. Epidemiological studies indicate that there may be an additional role for environmental factors. Although the target antigen(s) are not yet identified, several myelin or myelin-associated proteins have been suspected, among them myelin basic protein. A lack of genetically comparable controls has impaired the analysis of the T-cell response in MS patients and caused disagreement on TCR usage in the disease. Here we analyse the role of TCR genes in MS by comparing TCR usage in discordant versus concordant monozygotic twins in response to self and foreign antigens. We find that after stimulation with myelin basic protein or tetanus toxoid, control twin sets as well as concordant twin sets select similar V alpha chains. Only the discordant twin sets select different TCRs after stimulation with antigens. Thus exogenous factors or the disease shape the TCR repertoire in MS patients, as seen by comparison with unaffected genetically identical individuals. This skewing of the TCR repertoire could contribute to the pathogenesis of MS and other T-cell-mediated diseases.

A novel candidate autoantigen in a multiplex family with multiple sclerosis: prevalence of T-lymphocytes specific for an MBP epitope unique to myelination.

Although the major isoform of myelin basic protein (MBP) in the healthy adult CNS is the 18.5-kDa protein, other isoforms containing exon 2 encoded protein (21.5 kDa and 20.2 kDa) exist and are expressed primarily during myelin formation. Since remyelination is a prominent feature in MS lesions, we examined the frequencies of T cell lines (TCLs) specific for epitopes within exon 2 encoded MBP (X2MBP), and also within 18.5-kDa MBP, in members of a multiplex family with MS. TCLs specific for X2MBP were as prevalent as TCLs specific for immunodominant epitopes within 18.5-kDa MBP. In addition, while frequencies of TCLs specific for 18.5-kDa MBP were no different between the affected and unaffected, the frequency of X2MBP-specific TCLs correlated with disease.

Long-term treatment of chronic relapsing experimental allergic encephalomyelitis by transforming growth factor-beta 2.

It had been demonstrated previously that the administration of transforming growth factor-beta 1 (TGF-beta 1) reduced the clinical severity of experimental allergic encephalomyelitis (EAE). Treatment with the related immunosuppressive molecule, TGF-beta 2, resulted in similar inhibition of T cell activation and proliferation in vitro. Long-term treatment was effective in reducing clinical severity of EAE and the number of relapses in mice receiving either myelin basic protein- or peptide-91-103-specific T cell lines. When examined histologically, mice that had received TGF-beta 2 demonstrated significantly less inflammation and demyelination in the central nervous system. Examination of other organs demonstrated no pathology or deleterious side effects from long-term TGF-beta 2 therapy. These findings have relevance for the use of TGF-beta 2 as a therapeutic agent for the human demyelinating disease, multiple sclerosis.

Differential effect of interleukin-1 beta on Ia expression in astrocytes and microglia.

In an earlier study we demonstrated the inhibitory effect of interleukin-1 beta (IL-1 beta) on human leukocyte antigens (HLA) class II enhancement by interferon-gamma (IFN-gamma) in a human glioblastoma multiforme cell line. In this study we have examined the effect of IL-1 beta on IFN-gamma induced major histocompatibility complex (MHC) class II (Ia) in primary cultures of newborn murine astrocytes and microglial cells. Astrocytes expressed very low levels of Ia molecules under basal culture conditions but these molecules could be induced with IFN-gamma. IL-1 beta in doses ranging from 1 to 100 units/ml inhibited the level of IFN-gamma induced Ia expression on astrocytes, and this inhibition was dose-dependent (mean maximum inhibition of 53 +/- 5% in number of positive cells and 53 +/- 2.6% in mean fluorescence intensity in four separate experiments). IL-1 beta treatment had no effect on MHC class I induction by IFN-gamma in the astrocytes. In contrast, microglial cells expressed Ia molecules under basal culture conditions, and this expression was enhanced by IFN-gamma treatment. Both basal and IFN-gamma induced Ia expression on microglia were resistant to IL-1 beta treatment in doses ranging from 1 to 100 units/ml. These results indicate that Ia expression is differentially regulated on astrocytes and microglial cells and that IL-1 beta may have an important immune regulatory function in the central nervous system.

Susceptibility for multiple sclerosis is determined, in part, by inheritance of a 175-kb region of the TcR V beta chain locus and HLA class II genes.

Genetic makeup thought to affect susceptibility to multiple sclerosis (MS) and current evidence suggests that multiple genes may be involved. We have mapped a potential susceptibility gene or genes in the germ-line T cell receptor (TcR) V beta region of multiple sclerosis (MS) patients. Six restriction fragment length polymorphisms (RFLPs) spanning approximately 600 kb of the TcR V beta region were used to define TcR haplotypes in 197 Caucasian controls and 83 Caucasian MS patients in the chronic progressive stage of the disease. The distribution of TcR subhaplotype frequencies was significantly different only in the approx. 175-kb region between RFLPs defined by V beta 8.1 and V beta 11. Stratification of the MS patients into HLA-DR2+ (n = 51) and HLA-DR2- (n = 32) populations demonstrated that the subhaplotype frequencies differed from the control population significantly only in the HLA-DR2+ (corrected P = 0.00007) and not in the HLA-Dr2- (corrected P = 0.46) population. Subhaplotypes which are rare in the normal population are overrepresented in the HLA-DR2+ MS patient population and confer a relative risk of 4.06. These results indicate the existence of an MS susceptibility gene within the TcR V beta region, and provide new evidence for gene complementation between a HLA class II gene and TcR V beta gene(s) in conferring susceptibility to MS.