T Cells from NOD-PerIg Mice Target Both Pancreatic and Neuronal Tissue.

It has become increasingly appreciated that autoimmune responses against neuronal components play an important role in type 1 diabetes (T1D) pathogenesis. In fact, a large proportion of islet-infiltrating B lymphocytes in the NOD mouse model of T1D produce Abs directed against the neuronal type III intermediate filament protein peripherin. NOD-PerIg mice are a previously developed BCR-transgenic model in which virtually all B lymphocytes express the H and L chain Ig molecules from the intra-islet-derived anti-peripherin-reactive hybridoma H280. NOD-PerIg mice have accelerated T1D development, and PerIg B lymphocytes actively proliferate within islets and expand cognitively interactive pathogenic T cells from a pool of naive precursors. We now report adoptively transferred T cells or whole splenocytes from NOD-PerIg mice expectedly induce T1D in NOD.scid recipients but, depending on the kinetics of disease development, can also elicit a peripheral neuritis (with secondary myositis). This neuritis was predominantly composed of CD4+ and CD8+ T cells. Ab depletion studies showed neuritis still developed in the absence of NOD-PerIg CD8+ T cells but required CD4+ T cells. Surprisingly, sciatic nerve-infiltrating CD4+ cells had an expansion of IFN-γ- and TNF-α- double-negative cells compared with those within both islets and spleen. Nerve and islet-infiltrating CD4+ T cells also differed by expression patterns of CD95, PD-1, and Tim-3. Further studies found transitory early B lymphocyte depletion delayed T1D onset in a portion of NOD-PerIg mice, allowing them to survive long enough to develop neuritis outside of the transfer setting. Together, this study presents a new model of peripherin-reactive B lymphocyte-dependent autoimmune neuritis.

Control of Innate Immunity by Sialic Acids in the Nervous Tissue.

Sialic acids (Sias) are the most abundant terminal sugar residues of glycoproteins and glycolipids on the surface of mammalian cells. The nervous tissue is the organ with the highest expression level of Sias. The 'sialylation' of glycoconjugates is performed via sialyltransferases, whereas 'desialylation' is done by sialidases or is a possible consequence of oxidative damage. Sialic acid residues on the neural cell surfaces inhibit complement and microglial activation, as well as phagocytosis of the underlying structures, via binding to (i) complement factor H (CFH) or (ii) sialic acid-binding immunoglobulin-like lectin (SIGLEC) receptors. In contrast, activated microglial cells show sialidase activity that desialylates both microglia and neurons, and further stimulates innate immunity via microglia and complement activation. The desialylation conveys neurons to become susceptible to phagocytosis, as well as triggers a microglial phagocytosis-associated oxidative burst and inflammation. Dysfunctions of the 'Sia-SIGLEC' and/or 'Sia-complement' axes often lead to neurological diseases. Thus, Sias on glycoconjugates of the intact glycocalyx and its desialylation are major regulators of neuroinflammation.

Manduca sexta hemolymph protease-2 (HP2) activated by HP14 generates prophenoloxidase-activating protease-2 (PAP2) in wandering larvae and pupae.

Melanization is a universal defense mechanism of insects against microbial infection. During this response, phenoloxidase (PO) is activated from its precursor by prophenoloxidase activating protease (PAP), the terminal enzyme of a serine protease (SP) cascade. In the tobacco hornworm Manduca sexta, hemolymph protease-14 (HP14) is autoactivated from proHP14 to initiate the protease cascade after host proteins recognize invading pathogens. HP14, HP21, proHP1*, HP6, HP8, PAP1-3, and non-catalytic serine protease homologs (SPH1 and SPH2) constitute a portion of the extracellular SP-SPH system to mediate melanization and other immune responses. Here we report the expression, purification, and functional characterization of M. sexta HP2. The HP2 precursor is synthesized in hemocytes, fat body, integument, nerve and trachea. Its mRNA level is low in fat body of 5th instar larvae before wandering stage; abundance of the protein in hemolymph displays a similar pattern. HP2 exists as an active enzyme in plasma of the wandering larvae and pupae in the absence of an infection. HP14 cleaves proHP2 to yield active HP2. After incubating active HP2 with larval hemolymph, we detected higher levels of PO activity, i.e. an enhancement of proPO activation. HP2 cleaved proPAP2 (but not proPAP3 or proPAP1) to yield active PAP2, responsible for a major increase in IEARpNA hydrolysis. PAP2 activates proPOs in the presence of a cofactor of SPH1 and SPH2. In summary, we have identified a new member of the proPO activation system and reconstituted a pathway of HP14-HP2-PAP2-PO. Since high levels of HP2 mRNA were present in integument and active HP2 in plasma of wandering larvae, HP2 likely plays a role in cuticle melanization during pupation and protects host from microbial infection in a soil environment.

Pentraxins CRP-I and CRP-II are post-translationally deiminated and differ in tissue specificity in cod (Gadus morhua L.) ontogeny.

Pentraxins are fluid phase pattern recognition molecules that form an important part of the innate immune defence and are conserved between fish and human. In Atlantic cod (Gadus morhua L.), two pentraxin-like proteins have been described, CRP-I and CRP-II. Here we show for the first time that these two CRP forms are post-translationally deiminated (an irreversible conversion of arginine to citrulline) and differ with respect to tissue specific localisation in cod ontogeny from 3 to 84 days post hatching. While both forms are expressed in liver, albeit at temporally differing levels, CRP-I shows a strong association with nervous tissue while CRP-II is strongly associated to mucosal tissues of gut and skin. This indicates differing roles for the two pentraxin types in immune responses and tissue remodelling, also elucidating novel roles for CRP-I in the nervous system. The presence of deimination positive bands for cod CRPs varied somewhat between mucus and serum, possibly facilitating CRP protein moonlighting, allowing the same protein to exhibit a range of biological functions and thus meeting different functional requirements in different tissues. The presented findings may further current understanding of the diverse roles of pentraxins in teleost immune defences and tissue remodelling, as well as in various human pathologies, including autoimmune diseases, amyloidosis and cancer.

Access of protective antiviral antibody to neuronal tissues requires CD4 T-cell help.

Circulating antibodies can access most tissues to mediate surveillance and elimination of invading pathogens. Immunoprivileged tissues such as the brain and the peripheral nervous system are shielded from plasma proteins by the blood-brain barrier and blood-nerve barrier, respectively. Yet, circulating antibodies must somehow gain access to these tissues to mediate their antimicrobial functions. Here we examine the mechanism by which antibodies gain access to neuronal tissues to control infection. Using a mouse model of genital herpes infection, we demonstrate that both antibodies and CD4 T cells are required to protect the host after immunization at a distal site. We show that memory CD4 T cells migrate to the dorsal root ganglia and spinal cord in response to infection with herpes simplex virus type 2. Once inside these neuronal tissues, CD4 T cells secrete interferon-γ and mediate local increase in vascular permeability, enabling antibody access for viral control. A similar requirement for CD4 T cells for antibody access to the brain is observed after intranasal challenge with vesicular stomatitis virus. Our results reveal a previously unappreciated role of CD4 T cells in mobilizing antibodies to the peripheral sites of infection where they help to limit viral spread.

Anti-neural antibody reactivity in patients with a history of Lyme borreliosis and persistent symptoms.

Some Lyme disease patients report debilitating chronic symptoms of pain, fatigue, and cognitive deficits despite recommended courses of antibiotic treatment. The mechanisms responsible for these symptoms, collectively referred to as post-Lyme disease syndrome (PLS) or chronic Lyme disease, remain unclear. We investigated the presence of immune system abnormalities in PLS by assessing the levels of antibodies to neural proteins in patients and controls. Serum samples from PLS patients, post-Lyme disease healthy individuals, patients with systemic lupus erythematosus, and normal healthy individuals were analyzed for anti-neural antibodies by immunoblotting and immunohistochemistry. Anti-neural antibody reactivity was found to be significantly higher in the PLS group than in the post-Lyme healthy (p<0.01) and normal healthy (p<0.01) groups. The observed heightened antibody reactivity in PLS patients could not be attributed solely to the presence of cross-reactive anti-borrelia antibodies, as the borrelial seronegative patients also exhibited elevated anti-neural antibody levels. Immunohistochemical analysis of PLS serum antibody activity demonstrated binding to cells in the central and peripheral nervous systems. The results provide evidence for the existence of a differential immune system response in PLS, offering new clues about the etiopathogenesis of the disease that may prove useful in devising more effective treatment strategies.

[Limbic encephalitis in the course of testicular lymphoma].

The malignant tumor patient tends to develop various neuropathy by direct invasion, metastasis, secondary infectious disease of tumor, metabolic disorders, vascular damage and adverse drug reactions with a treatment, and, however, it rarely appear by mechanism of autoimmunization. Tumor tissue with paraneoplastic neurological syndrome (PNS) produces an antigen attacking nerve tissue by it's cross reaction, and many studies indicates that there are a few kinds of antineuritic antibodies occurred by the charactor of malignant diseases or the patterns of progression. There is no relationship between the symptoms and the progression of disease. We report a case of malignant testicular tumor presented the paraneoplastic limbic encephalitis which is one of paraneoplastic neurological syndrome.

Gangliosides play pivotal roles in the regulation of complement systems and in the maintenance of integrity in nerve tissues.

Gangliosides are considered to be essential in the maintenance and repair of nervous tissues; however, the mechanisms for neurodegeneration caused by ganglioside defects are unknown. We examined gene expression profiles in double knockout (DKO) mice of GM2/GD2 synthase and GD3 synthase genes and showed that the majority of complement genes and their receptors were up-regulated in cerebellum in DKO mice. Inflammatory reactions were demonstrated in those tissues by measuring up-regulated inflammatory cytokines, indicating the presence of complement activation and inflammation as reported in Alzheimer's disease. Immunoblotting of fractionated membrane extracts by sucrose density gradient revealed that complement-regulatory molecules such as decay-accelerating factor and CD59 were dispersed from glycolipid-enriched microdomain/rafts in DKO cerebellum. Immunohistostaining of these molecules showed disordered membrane localization. These results suggested that dysfunction of complement-regulatory molecules may be due to abnormal glycolipid-enriched microdomain/rafts that triggered complement activation, subsequent inflammation, and neurodegeneration in DKO mice. Generation of the triple KO mice lacking complement activity in addition to the two glycosyltransferases suggested that complement activation is involved in the inflammatory reactions and neurodegeneration caused by the ganglioside deficiency.

Increased prevalence of antibodies to central nervous system tissue and gangliosides in Hashimoto's thyroiditis compared to other thyroid illnesses.

BACKGROUND: Previous studies point to central nervous system (CNS) alterations in euthyroid patients with Hashimoto's thyroiditis (HT). The aim of the present study was to investigate the prevalence and clinical significance of antibodies (Abs) against CNS tissue and gangliosides in female patients with HT compared to patients with other non-autoimmune thyroid disorders, comprising diffuse or nodular goitre and thyroid surgery for goitre. METHODS: 58 HT patients (mean age: 46+/-17 years) and 89 patients with other thyroid disorders (mean age: 51+/-15 years) were recruited consecutively from our endocrine outpatient clinic. Serum Abs against CNS tissue and gangliosides were determined using an enzyme-linked immunosorbent assay (ELISA). In a subgroup of 23 HT patients, neurocognitive function was studied using established neuropsychological tests. RESULTS: In HT patients, the prevalence of serum anti-ganglioside-Abs and anti-CNS-Abs were significantly higher compared to patients with other thyroid disorders (p<0.05 and p<0.005, respectively). In both cases, the number of Ab-positive HT patients was twice that of Ab-positive controls. Reactivity of IgM-Abs to gangliosides and IgG-Abs to CNS tissue was significantly higher in HT patients than in controls (p<0.05 and p<0.01, respectively). However, prevalence and reactivity of Abs to gangliosides and CNS tissue were associated neither with the prevalence of depression nor with impairment of neurocognitive function in HT patients. CONCLUSION: Ab reactivity towards CNS tissue and gangliosides is markedly enhanced in patients with HT as compared to patients with other thyroid disorders. Whether these Abs could be of prognostic value to evaluate the risk of future neurocognitive impairment has to be investigated in longitudinal studies.

Neurometabolic changes in normal white matter may predict appearance of hyperintense lesions in systemic lupus erythematosus.

To determine if neurometabolic changes in the white matter (WM) of systemic lupus erythematosus (SLE) patients may predict the appearance of small hyperintense lesions on T2-weighted magnetic resonance imaging (MRI) inside the magnetic resonance spectroscopy (MRS) region of interest (ROI). We included 30 SLE patients and 23 controls. We performed single voxel proton MRS over the superior-posterior region of the corpus callosum. We measured signals from N-acetyl-compounds (NAA), choline (Cho) and creatine-phosphocreatin (Cr) and determined NAA/Cr and Cho/Cr ratios. After a minimum of 12 months, MRI and MRS were repeated in all patients and nine volunteers. Twenty patients had normal MRI and 10 patients had MRI hyperintense lesions in the MRS ROI at baseline. All patients had hyperintense lesions in the MRS ROI in follow-up MRIs. All SLE patients had increased Cho/Cr values at both MRS when compared with normal controls (P = 0.001). In addition, there was an increase in Cho/Cr values when patients' baseline and follow-up MRS were compared (P = 0.001). We observed a correlation between Cho/Cr ratios and number of WM lesions (r = 0.69; P = 0.001). Increased Cho/Cr in normal appearing WM may be indicative of future appearance of hyperintense T2-weighted MRI lesions in SLE patients.

Stromal derived growth factor-1alpha: another mediator in neural-emerging immune system through Tac1 expression in bone marrow stromal cells.

Stromal cell-derived growth factor-1alpha (SDF-1alpha) is a member of the CXC chemokines and interacts with the G protein, seven-transmembrane CXCR4 receptor. SDF-1alpha acts as a chemoattractant for immune and hemopoietic cells. The Tac1 gene encodes peptides belonging to the tachykinin family with substance P being the predominant member. Both SDF-1alpha and Tac1 peptides are relevant hemopoietic regulators. This study investigated the effects of SDF-1alpha on Tac1 expression in the major hemopoietic supporting cells, the bone marrow stroma, and addresses the consequence to hemopoiesis. Reporter gene assays with the 5' flanking region of Tac1 showed a bell-shaped effect of SDF-1alpha on luciferase activity with 20 ng/ml SDF-1alpha acting as stimulator, whereas 50 and 100 ng/ml SDF-1alpha acted as inhibitors. Gel shift assays and transfection with wild-type and mutant IkappaB indicate NF-kappaB as a mediator in the repressive effects at 50 and 100 ng/ml SDF-1alpha. Northern analyses and ELISA showed correlations among reporter gene activities, mRNA (beta-preprotachykinin I), and protein levels for substance P. Of relevance is the novel finding by long-term culture-initiating cell assays that showed an indirect effect of SDF-1alpha on hemopoiesis through substance P production. The results also showed neurokinin 1 and not neurokinin 2 as the relevant receptor. Another crucial finding is that substance P does not regulate the production of SDF-1alpha in stroma. The studies indicate that SDF-1alpha levels above baseline production in bone marrow stroma induce the production of substance P to stimulate hemopoiesis. Substance P, however, does not act as autocrine stimulator to induce the production of SDF-1alpha. This study adds SDF-1alpha as a mediator within the neural-immune-hemopoietic axis.

Methods for sample preparation for direct immunoassay measurement of analytes in tissue homogenates: ELISA assay of amyloid beta-peptides.

Use of low abundance analytes in whole tissue homogenates has been realized with the development of assays in which a specific analyte is captured and detected using immunological reagents. One of the many advantages of analyte immunoassay in crude homogenates is its relative simplicity, allowing high throughput analysis of samples. In this unit, some major key determinants in sample and standard preparation and handling are described that have been shown to improve the performance and reliability of these assay systems. The ELISA assay of amyloid peptides from brain tissue is described as an example, since the protocols for this analysis exemplify many of the techniques and problems that are encountered in the development of new assays.

The IA-2 gene family: homologs in Caenorhabditis elegans, Drosophila and zebrafish.

AIMS/HYPOTHESIS: IA-2 and IA-2beta are major autoantigens in Type I (insulin-dependent) diabetes mellitus and are expressed in neuroendocrine tissues including the brain and pancreatic islets of Langerhans. Based on sequence analysis, IA-2 and IA-2beta are transmembrane protein tyrosine phosphatases but lack phosphatase activity because of critical amino acid substitutions in the catalytic domain. We studied the evolutionary conservation of IA-2 and IA-2beta genes and searched for homologs in non-mammalian vertebrates and invertebrates. METHODS: IA-2 from various species was identified from EST sequences or cloned from cDNA libraries or both. Expression in tissues was determined by transfection and in situ hybridization. RESULTS: We identified homologs of IA-2 in C. elegans, Drosophila, and zebrafish which showed 46, 58 and 82 % identity and 60, 65 and 87 % similarity, respectively, to the amino acids of the intracellular domain of human IA-2. Further studies showed that IA-2 was expressed in the neural tissues of the three species. Comparison of the genomic structure of the intracellular domain of human IA-2 with that of human IA-2beta showed that they were nearly identical and comparison of the intron-exon boundaries of Drosophila IA-2 with human IA-2 and IA-2beta showed a high degree of relatedness. CONCLUSION/INTERPRETATION: Based on these findings and sequence analysis of IA-2 homologs in mammals, we conclude that there is an IA-2 gene family which is a part of the larger protein tyrosine phosphatase superfamily. The IA-2 and IA-2beta genes represent two distinct subgroups within the IA-2 family which originated over 500 million years ago, long before the development of the pancreatic islets of Langerhans.