De novo transcriptome sequencing is a robust method for microRNA (miRNA) target gene prediction, especially for organisms without reference genomes. Following exposure of Megalobrama amblycephala to ammonia (0.1 or 20mgL−1 ), two cDNA libraries were constructed from the fish gills and sequenced using Illumina HiSeq 2000. Over 90 million reads were generated and de novo assembled into 46, 615 unigenes, which were then extensively annotated by comparing to different protein databases, followed by biochemical pathway prediction. The expression of 2666 unigenes significantly differed; 1961 were up-regulated, while 975 were down-regulated. Among these, 250 unigenes were identified as the targets for 10 conserved and 4 putative novel miRNA families by miRNA target computational prediction. We examined expression of ssa-miRNA-21 and its target genes by real-time quantitative PCR and found agreement with the sequencing data. This study demonstrates the feasibility of identifying miRNA targets by transcriptome analysis. The transcriptome assembly data represent a substantial increase in the genomic resources available for Megalobrama amblycephala and will be useful for gene expression profile analysis and miRNA functional annotation.
The thymus is the site of T cell maturation. Notch receptors (Notch1-4) and ligands (DLL1-3 and Jagged1-2) constitute one of several pathways involved in this process. Our data revealed differential constitutive expression of Notch genes and ligands in T lymphocytes and thymic dendritic cells (tDCs), suggesting their participation in human thymocyte maturation. nTreg analyses indicated that the Notch components function in parallel to promote maturation in the thymus.
Antibody-mediated depletion of neutrophils is commonly used to study neutropenia. However, the mechanisms by which antibodies deplete neutrophils have not been well defined. We noticed that mice deficient in complement and macrophages had blunted neutrophil depletion in response to anti-Ly6G monoclonal antibody (MAb) treatment. In vitro, exposure of murine neutrophils to anti-Ly6G MAb in the presence of plasma did not result in significant depletion of cells, either in the presence or absence of complement. In vivo, anti-Ly6G-mediated neutrophil depletion was abrogated following macrophage depletion, but not complement depletion, indicating a requirement for macrophages to induce neutropenia by this method. These results inform the use and limitations of anti-Ly6G antibody as an experimental tool for depleting neutrophils in various immunological settings.
OBJECTIVE:Patients with generalized autoimmune dysautonomia may also present with gastroparesis. Immune dysfunction in such patients can be evaluated using antibodies to glutamic acid decarboxylase (GAD) and full thickness biopsy of stomach. In this study, we utilize immunotherapy for treatment of drug and Gastric Electrical Stimulation (GES) resistant gastroparetic patients with evidence of neuroinflammation on full thickness gastric biopsy and had positive GAD65 autoantibodies.MATERIAL AND METHODS:We conducted a retrospective chart review of 11 female patients with drug and device resistant gastroparesis. Patients were treated for a total of 8-12 weeks with either intravenous immunoglobulin (IVIg), or combined mycophenolate mofetil (MM) and methylprednisolone, or only MM. Patients were excluded if they had previous side effects from steroid therapy, low scores on dual-energy X-ray absorptiometry (DEXA) scan results, immune-compromised conditions with infections like tuberculosis and zoster. Symptoms of nausea, vomiting, abdominal pain, early satiety/anorexia, bloating and total symptom score (TSS) as reported by the patients were recorded before and after the treatment at a follow up visit 2 to 16 weeks after initiation of therapy.RESULTS:Maximum symptom improvement was seen in patients treated with IVIg (67%). 6 patients (55%) had improvement in vomiting, whereas 5 patients (45%) had improvements in nausea, abdominal pain and bloating.CONCLUSIONS:Immunomodulatory therapy shows positive outcomes in improving vomiting symptom in some gastroparetic patients who have coexisting positive autoimmune profiles. This preliminary data suggests the need for further investigations in immunotherapy targeted to patients with gastroparetic symptoms refractory to approved drug and device therapies.
In this paper we demonstrate that patients treated with chemotherapy and/or hematopoietic stem cell transplantation (HSCT) have highly significant reduced serum ascorbic acid (AA) levels compared to healthy controls. We recently observed in in vitro experiments that growth of both T and NK cells from hematopoietic stem cells is positively influenced by AA. It might be of clinical relevance to study the function and recovery of immune cells after intensive treatment, its correlation to AA serum levels and the possible effect of AA supplementation.
INTRODUCTION:Determination of antinuclear antibodies (ANA) by indirect immunofluorescence (IIF) is usually the initial test for the diagnosis of systemic rheumatic diseases (SRD). Assigning predictive values to positive and negative results of the test is vital because lack of knowledge about ANAs and their usefulness in classification criteria of SRD leads to inappropriate use.METHODS:Retrospective study, ANA tests requested by different specialties, correlation to patients' final diagnosis.RESULTS:The prevalence of autoimmune disease was relatively low in our population yielding a low PPV and a high NPV for the ANA test. 40% of the patients had no clinical criteria applied prior to test. Coexistence of two or more autoimmune disorders affects prevalence and predictive values.CONCLUSION:Application of the test after careful evaluation for clinical criteria remarkably improves the positive likelihood ratio for the diagnosis.
Schlafen (SLFN/Slfn) family members have been investigated for their involvement in fundamental cellular processes including growth regulation, differentiation and control of viral replication. However, most research has been focused on the characterization of Slfns within the murine system or in human cell lines. Since little is known about SLFNs in primary human immune cells, we set out to analyze the expression and regulation of the six human SLFN genes in monocytes, monocyte-derived dendritic cells (moDCs) and T cells. Comparison of SLFN gene expression across these three cell types showed high mRNA expression of SLFN11 in monocytes and moDCs and high SLFN5 expression in T cells, indicating functional importance within these cell types. Differentiation of monocytes to moDCs leads to the gradual upregulation of SLFN12L and SLFN13 while SLFN12 levels were decreased by differentiation stimuli. Stimulation of moDCs via human rhinovirus, lipopolysaccharide, or IFN-α lead to strong upregulation of SLFN gene expression, while peptidoglycan poorly stimulated regulation of both SLFNs and the classical interferon-stimulated gene MxA. T cell activation was found to downregulate the expression of SLFN5, SLFN12 and SLFN12L, which was reversible upon addition of exogenous IFN-α. In conclusion, we demonstrate, that SLFN gene upregulation is mainly dependent on autocrine type I interferon signaling in primary human immune cells. Rapid decrease of SLFN expression levels following T cell receptor stimulation indicates a role of SLFNs in the regulation of human T cell quiescence.
In this work were investigated the relationship between Hookworm/Schistosoma mansoni infections and allergy related risk factors in two endemic areas with distinct prevalence of infections and co-infection. The intensity of infections, eosinophilia, allergy risk factors, infections status and anti-Der p1 IgE levels before and 2 years (population 1) and 3 years (population 2) after anthelmintic treatment, were evaluated. It was observed that the population with lower prevalence and intensity of infection (population 2) had lower eosinophils counts (>600/mm3) and higher animal contact than the population with higher parasites intensity (population 1). After anthelmintic treatment the intensity of S. mansoni single infection decreased, but no changes were observed in Hookworm and co-infected individuals. The anthelmintic treatment also enhanced anti-Der p1 IgE optical density in ELISA on the subgroups that became negative for helminth infection regardless of their previous infection condition in population 1. Facing that, we evaluated the anti-Der p1 IgE reactivity index, and the ratio (after/before treatment) was significantly higher in patients co-infected before treatment. On the other hand, no association between anti-Der p1 IgE reactivity index and the intensity of infections were observed. In conclusion, effective anthelmintic therapy of subjects from endemic areas with high prevalence of Hookworm and S. mansoni infections enhances anti-Der p1 IgE levels.
Anti-Neutrophil Cytoplasmic Antibodies (ANCA)-associated vasculitides (AAV) are characterized by small vessel injury and in some cases granulomatous lesions and glomerular inflammation. The pathogenic bases of these clinical phenotypes are incompletely understood, but evidence from patients with AAV and other inflammatory diseases suggest a role for monocyte/macrophages in the perpetuation of tissue injury. Macrophage colony stimulating factor (M-CSF) is a promoter of monocyte recruitment and macrophage proliferation, involved in mesangial cell proliferation and experimental nephritis development. Serum concentrations of M-CSF mark and herald the onset of lupus nephritis. Plasma samples from 29 patients with AAV (18 granulomatosis with polyangiitis, GPA, 6 eosinophilic granulomatosis with polyangiitis, EGPA, and 5 microscopic polyangiitis, MPA) and from 10 healthy controls were collected together with clinical data. Patients with AAV had higher levels of M-CSF when compared to controls. M-CSF levels correlated positively with the BVAS, serum C-reactive protein and erythrocyte sedimentation rate, while haemoglobin correlated inversely with M-CSF. Patients with active renal disease had significantly higher levels of M-CSF when compared to the other subgroups. M-CSF levels did not differ between ANCA subserotypes and were not associated with the involvement of other organs. In conclusion, M-CSF is higher in patients with AAV and active nephritis and could contribute to the pathogenesis of these diseases. In addition, M-CSF could behave as a useful marker of renal involvement in AAV.
The expression of the Adenovirus serotype 2 or serotype 5 (Ad2/5) E1A gene in tumor cells upregulates ligands that are recognized by the NKG2D activating receptor, which is expressed on NK cells and T cells, and reduces their tumorigenicity, a process dependent on NK cells and T cells. In some model systems, the forced overexpression of NKG2D ligands on tumor cells induced antigen-specific CD8+ T cells that mediated anti-tumor immunity. We wanted to determine if the interaction of NKG2D ligands on tumor cells that express E1A with NKG2D on immune cells contributed to the ability of E1A to induce a CD8+ T cell anti-tumor response or reduce tumorigenicity. To address these questions, we used the MCA-205 tumor cell line or MCA-205 cells that expressed Ad5 E1A (MCA-205-E1A cells), a fusion protein of E1A and ovalbumin (MCA-205-E1A-OVA) or OVA (MCA-205-OVA). We found that the expression of E1A or E1A-OVA, but not OVA, upregulated the expression of the NKG2D ligand RAE-1 on the surface of MCA-205 cells. Additionally, MCA-205-E1A cells and MCA-205-E1A-OVA cells were more sensitive to NK cell lysis than MCA-205 or MCA-205-OVA cells in WT B6 mice, but not NKG2D deficient B6 mice. Next, we adoptively transferred WT or NKG2D deficient OT-1 T cells (CD8 T cells that recognize OVA residues 257-264) into WT B6 mice or B6 mice that were deficient in NKG2D respectively and measured the expansion of OT-1 cells following immunization with MCA-205-E1A-OVA or MCA-205-OVA cells. We found that the expansion of OT-1 cells following immunization of either OVA-expressing MCA-205 cell lines was not affected by the presence or absence of NKG2D in B6 mice. Finally, we found that the capacity of E1A to reduce the tumorigenicity of MCA-205 cells was not impaired in B6-NKG2D deficient mice as compared to WT B6 mice. Our results suggest that the ability of E1A to reduce the tumorigenicity of MCA-205 cells, or induce an antigen-specific CD8+ T cell response, is independent of the interaction of NKG2D ligands with the NKG2D receptor.
Biomembranes undergo extensive shape changes as they perform vital cellular functions or become diseased. To understand the mechanisms by which lipids and proteins control membrane curvature during various processes, researchers have identified and engineered many curvature-inducing and curvature-sensing proteins and peptides. In this paper, a simple experiment was performed to show qualitatively how membrane remodeling by N-terminal amphipathic helix of Amphiphysin affects the spatial distribution of the transmembrane Fc receptor protein (FcεRI) in mast cells. Results indicate that an elevated concentration of amphipathic helices can interfere with the formation of a typical mast cell synapse.
Glucocorticoid-Induced Leucine Zipper (GILZ) is a glucocorticoid-inducible gene that mediates glucocorticoid anti-inflammatory effects. GILZ and the isoform L-GILZ are expressed in a variety of cell types, especially of hematopoietic origin, including macrophages, lymphocytes and epithelial cells, and strongly upregulated upon glucocorticoid treatment. A quantitative analysis of GILZ expression in mouse tissues is technically difficult to perform because of the presence of a pseudogene and the high homology of GILZ gene with other genes of TSC22 family. We here propose specific primer pairs to be used in Real Time PCR to avoid unwanted amplification of GILZ pseudogene and TSC-22 family member d1iso3. These primer pairs were used to determine GILZ and L-GILZ expression, in either untreated or in vivo and in vitro dexamethasone-treated tissues. Results indicate that GILZ and L-GILZ are upregulated by glucocorticoids, being GILZ more sensitive to glucocorticoid induction than L-GILZ, but they are differently expressed in all examined tissues, confirming a different role in specific cells. An inappropriate primer pair amplified also GILZ pseudogene and TSC22d1iso3, thus producing misleading results. This quantitative evaluation may be used to better characterize the role of GILZ and L-GILZ in mice and may be translated to humans.
The efficacy of pEGFP (plasmid expressing enhanced green fluorescent protein)-encapsulated PEGylated (meaning polyethylene glycol coated) magnesium phosphate nanoparticles (referred to as MgPi-pEGFP nanoparticles) for the induction of immune responses was investigated in a mouse model. MgPi-pEGFP nanoparticles induced enhanced serum antibody and antigen-specific T-lymphocyte responses, as well as increased IFN-γ and IL-12 levels compared to naked pEGFP when administered via intravenous, intraperitoneal or intramuscular routes. A significant macrophage response, both in size and activity, was also observed when mice were immunized with the nanoparticle formulation. The response was highly specific for the antigen, as the increase in interaction between macrophages and lymphocytes as well as lymphocyte proliferation took place only when they were re-stimulated with recombinant green fluorescence protein (rGFP). Thus the nanoparticle formulation elicited both humoral as well as cellular responses. Cytokine profiling revealed the induction of Th-1 type responses. The results suggest DNA-encapsulated magnesium phosphate (MgPi) nanoparticles may constitute a safer, more stable and cost-efficient DNA vaccine formulation.
Type 1 diabetes (T1D) is a multigenic disease caused by T-cell mediated destruction of the insulin producing pancreatic islet β-cells. The earliest sign of islet autoimmunity in NOD mice, islet leukocytic infiltration or insulitis, is obvious at around 5 weeks of age. The molecular alterations that occur in T cells prior to insulitis and that may contribute to T1D development are poorly understood. Since CD4 T-cells are essential to T1D development, we tested the hypothesis that multiple genes/molecular pathways are altered in these cells prior to insulitis. We performed a genome-wide transcriptome and pathway analysis of whole, untreated CD4 T-cells from 2, 3, and 4 week-old NOD mice in comparison to two control strains (NOR and C57BL/6). We identified many differentially expressed genes in the NOD mice at each time point. Many of these genes (herein referred to as NOD altered genes) lie within known diabetes susceptibility (insulin-dependent diabetes, Idd) regions, e.g. two diabetes resistant loci, Idd27 (tripartite motif-containing family genes) and Idd13 (several genes), and the CD4 T-cell diabetogenic activity locus, Idd9/11 (2 genes, KH domain containing, RNA binding, signal transduction associated 1 and protein tyrosine phosphatase 4a2). The biological processes associated with these altered genes included, apoptosis/cell proliferation and metabolic pathways (predominant at 2 weeks); inflammation and cell signaling/activation (predominant at 3 weeks); and innate and adaptive immune responses (predominant at 4 weeks). Pathway analysis identified several factors that may regulate these abnormalities: eight, common to all 3 ages (interferon regulatory factor 1, hepatic nuclear factor 4, alpha, transformation related protein 53, BCL2-like 1 (lies within Idd13), interferon gamma, interleukin 4, interleukin 15, and prostaglandin E2); and two each, common to 2 and 4 weeks (androgen receptor and interleukin 6); and to 3 and 4 weeks (interferon alpha and interferon regulatory factor 7). Others were unique to the various ages, e.g. myelocytomatosis oncogene, jun oncogene, and amyloid beta (A4) to 2 weeks; tumor necrosis factor, transforming growth factor, beta 1, NFκB, ERK, and p38MAPK to 3 weeks; and interleukin 12 and signal transducer and activator of transcription 4 to 4 weeks. Thus, our study demonstrated that expression of many genes that lie within several Idds (e.g. Idd27, Idd13 and Idd9/11) was altered in CD4 T-cells in the early induction phase of autoimmune diabetes and identified their associated molecular pathways. These data offer the opportunity to test hypotheses on the roles played by the altered genes/molecular pathways, to understand better the mechanisms of CD4 T-cell diabetogenesis, and to develop new therapeutic strategies for T1D.
Cytokines of the IL-6 family have been related to infarct size and prognosis in patients with myocardial infarction. The aims of the present study were to elucidate possible associations between myocardial necrosis and left ventricular impairment and members of the IL-6 transsignalling system including soluble (s) IL-6R and (s) glycoprotein 130 (sgp130) in patients with ST-elevation myocardial infarction (STEMI) treated with primary PCI. In blood samples from 1028 STEMI patients, collected in-hosptial, we found significant correlations between peak TnT and IL-6 and CRP (p < 0.001, all) and between IL-6 and CRP and LV ejection fraction and NT-proBNP (p < 0.001, all). On the contrary, no significant associations were found between peak TnT and sgp130 or sIL-6R. Furthermore sgp130 was significantly elevated in diabetic patients and also associated with the glucometabolic state. In conclusion, circulating levels of IL-6 and CRP, but not the soluble forms of the receptor (sIL-6R) or the receptor signalling subunit (sgp130) were associated with the extent of myocardial necrosis. The biological importance of the IL-6/gp130-mediated signalling pathways in patients with acute myocardial infarction and dysglycemia should be further elucidated.
The P2X7 receptor (P2X7R) is an ATP-gated cation channel that is abundantly expressed in monocytes/macrophages. P2X7R activation by ATP results in various cellular responses including Ca(2+) influx, membrane pore formation, and cytokine secretion. Since P2X7R has low affinity for ATP, high concentrations of ATP (in the mM range) are generally required to activate this receptor in vitro. Functional expression of P2X7R has been detected in monocytes/macrophages obtained from different animal species including humans, rodents, dogs, and bovines, but so far it has not been detected in swine (Sus scrofa). In this study, we investigated the expression and functions of P2X7R in swine macrophages, which were isolated from mixed primary cultures of swine kidney or liver tissue. The P2X7R mRNA and protein expression observed in the swine macrophages was comparable to that seen in a c-myc-immortalized mouse kidney-derived clonal macrophage cell line (KM-1). However, extracellular ATP did not induce P2X7R-dependent sustained Ca(2+) influx, membrane pore formation, or the secretion of the bioactive cytokine interleukin-1β in the swine macrophages, whereas these responses were clearly observed in the mouse KM-1 cells after stimulation with millimolar concentrations of ATP as a positive control. These findings suggest that the ATP/P2X7R pathway is impaired in swine macrophages at least in the culture conditions used in the present study.
Simvastatin (SMV) has been shown to exhibit promising anti-inflammatory properties alongside its classic cholesterol lowering action. We tested these emerging effects in a major thermal injury mouse model (3rd degree scald, ~20% TBSA) with previously documented, inflammation-mediated intestinal defects. Neutrophil extracellular traps (NETs) inflammation measurement methods were used alongside classic gut mucosa inflammation and leakiness measurements with exogenous melatonin treatment as a positive control. Our hypothesis is that simvastatin has protective therapeutic effects against early postburn gut mucosa inflammation and leakiness. To test this hypothesis, we compared untreated thermal injury (TI) adult male mice with TI littermates treated with simvastatin (0.2 mg/kg i.p., TI + SMV) immediately following burn injury and two hours before being sacrificed the day after; melatonin-treated (Mel) (1.86 mg/kg i.p., TI + Mel) mice were compared as a positive control. Mice were assessed for the following: (1) tissue oxidation and neutrophil infiltration in terminal ileum mucosa using classic carbonyl, Gr-1, and myeloperoxidase immunohistochemical or biochemical assays, (2) NETosis in terminal ileum and colon mucosa homogenates and peritoneal and fluid blood samples utilizing flow cytometric analyses of the surrogate NETosis biomarkers, picogreen and Gr-1, and (3) transepithelial gut leakiness as measured in terminal ileum and colon with FITC-dextran and transepithelial electrical resistance (TEER). Our results reveal that simvastatin and melatonin exhibit consistently comparable therapeutic protective effects against the following: (1) gut mucosa oxidative stress as revealed in the terminal ileum by markers of protein carbonylation as well as myeloperoxidase (MPO) and Gr-1 infiltration, (2) NETosis as revealed in the gut milieu, peritoneal lavage and plasma utilizing picogreen and Gr-1 flow cytometry and microscopy, and (3) transepithelial gut leakiness as assessed in the ileum and colon by FITC-dextran leakiness and TEER. Thus, simvastatin exhibits strong acute anti-inflammatory actions associated with marked decreases in gut tissue and systemic NETosis and decreased gut mucosa leakiness.
We recently demonstrated in several mammalian species, a novel procedure to obtain liver-macrophages (Kupffer cells) in sufficient numbers and purity using a mixed primary culture of hepatocytes. In this study, we applied this method to the C57BL/6 mouse liver and established an immortalized Kupffer cell line from this mouse strain. The hepatocytes from the C57BL/6 adult mouse liver were isolated by a two-step collagenase perfusion method and cultured in T25 culture flasks. Similar to our previous studies, the mouse hepatocytes progressively changed their morphology into a fibroblastic appearance after a few days of culture. After 7–10 days of culture, Kupffer-like cells, which were contaminants in the hepatocyte fraction at the start of the culture, actively proliferated on the mixed fibroblastic cell sheet. At this stage, a retroviral vector containing the human c-myc oncogene and neomycin resistance gene was introduced into the mixed culture. Gentle shaking of the culture flask, followed by the transfer and brief incubation of the culture supernatant, resulted in a quick and selective adhesion of Kupffer cells to a plastic dish surface. After selection with G418 and cloning by limiting dilutions, a clonal cell line (KUP5) was established. KUP5 cells displayed typical macrophage morphology and were stably passaged at 4–5 days intervals for more than 5 months, with a population doubling time of 19h. KUP5 cells are immunocytochemically positive for mouse macrophage markers, such as Mac-1, F4/80. KUP5 cells exhibited substantial phagocytosis of polystyrene microbeads and the release of inflammatory cytokines upon lipopolysaccharide stimulation. Taken together, KUP5 cells provide a useful means to study the function of Kupffer cells in vitro.
Fungal colonization with Pneumocystis is associated with increased airway mucus in infants during their primary Pneumocystis infection, and to severity of COPD in adults. The pathogenic mechanisms are under investigation. Interestingly, increased levels of hCLCA1 – a member of the calcium-sensitive chloride conductance family of proteins that drives mucus hypersecretion – have been associated with increased mucus production in patients diagnosed with COPD and in immunocompetent rodents with Pneumocystis infection. Pneumocystis is highly prevalent in infants; therefore, the contribution of Pneumocystis to hCLCA1 expression was examined in autopsied infant lungs. Respiratory viruses that may potentially increase mucus, were also examined. hCLCA1 expression was measured using actin-normalized Western-blot, and the burden of Pneumocystis organisms was quantified by qPCR in 55 autopsied lungs from apparently healthy infants who died in the community. Respiratory viruses were diagnosed using RT-PCR for RSV, metapneumovirus, influenza, and parainfluenza viruses; and by PCR for adenovirus. hCLCA1 levels in virus positive samples were comparable to those in virus-negative samples. An association between Pneumocystis and increased hCLCA1 expression was documented (P=0.028). Additionally, increasing Pneumocystis burden correlated with increasing hCLCA1 protein expression levels (P=0.017). Results strengthen the evidence of Pneumocystis-associated up-regulation of mucus-related airway responses in infant lungs. Further characterization of this immunocompetent host-Pneumocystis-interaction, including assessment of potential clinical significance, is warranted.
OBJECTIVE:The pathophysiology of the postpolio syndrome is not fully understood. Increased cytokine levels in cerebrospinal fluid and peripheral blood indicate a systemic inflammatory process. Decreased cytokine levels and the clinical effect of intravenous immunoglobulin treatment further indicate an inflammatory/immunological pathogenesis. The aim of the present study was to evaluate whether an autoimmune process follows the initial infection, by means of analyzing immune complexes.PATIENTS AND METHODS:Circulating immune complexes were analyzed from blood samples of 20 postpolio patients and 95 healthy controls. To compensate for differences in age between patients and controls, a sub-analysis was performed using only the 30 oldest controls. Tumor necrosis factor-inducing properties of polyethylene glycol-precipitated immune complexes were compared between the postpolio patients and 10 healthy controls.RESULTS:When comparing levels in postpolio patients to the whole control group, including the 30 oldest investigated, there were no statistically significant differences. No difference was found in tumor necrosis factor levels induced by immune complexes when comparing patients and controls.CONCLUSIONS:There was no increase in circulating immune complex or in tumor necrosis factor-inducing effects of circulating immune complex between postpolio patients and healthy controls, indicating that the postpolio syndrome is not due to an autoimmune reaction.
