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Background: Cerebrospinal fluid (CSF) free light chains (FLCs) can be an alternative assay to oligoclonal bands (OCBs) in inflammatory neurological disorders, but threshold has no consensus. Objective: To assess the diagnostic accuracy of CSF FLCs in multiple sclerosis (MS) and other neurological diseases. Methods: A total of 406 patients from five Italian centers. FLCs were measured in CSF and serum using Freelite MX assays on Optilite. Results: A total of 171 patients were diagnosed as MS, 154 non-inflammatory neurological diseases, 48 inflammatory central nervous system (CNS) diseases, and 33 peripheral neurological diseases. Both kFLC and λFLC indices were significantly higher in patients with MS compared to other groups ( p Conclusion: Our findings support the diagnostic utility of FLC indices in MS and other CNS inflammatory disorders, suggesting a combined use of FLC and OCB to help clinicians with complementary information.

Background:There is emerging evidence that intrathecal IgM synthesis (ITMS) is a risk factor for conversion to clinically defined multiple sclerosis (CDMS) in clinically isolated syndrome (CIS) patients.Objectives:The objective of this study is to verify the prognostic role of ITMS as a risk factor for the second clinical attack in patients after the first demyelinating event.Methods:Monocentric observational study performed on prospectively acquired clinical data and retrospective evaluation of magnetic resonance imaging (MRI) data. ITMS was assessed according to Reiber’s non-linear function. We compared time to the second attack by using Kaplan–Meier curves and performed adjustment by Cox regression analysis.Results:Demographics and clinical data were collected prospectively in a cohort of 68 patients. ITMS occurred in 40% (27/68) of patients who had a higher T1-hypointense lesion load at brain MRI ( p = 0.041). In multivariate Cox regression analysis (adjusted for age, sex, baseline Expanded Disability Status Scale, IgG oligoclonal bands and disease-modifying treatment exposure), relapsing-remitting multiple sclerosis (MS) patients with ITMS were at higher risk to experience a second clinical attack (adjusted hazard ratio (aHR) = 6.3, 95% confidence interval (CI) = 2.1–18.4, p = 0.001).Conclusion:Together with previous studies, our findings support the role of ITMS as a prognostic biomarker in MS.

The presence of intrathecal IgM synthesis (ITMS) has been associated with an aggressive multiple sclerosis (MS) clinical course. In the present systematic review, we aimed at assessing the prevalence of ITMS among different MS phenotypes. Moreover, we aimed at quantifying the risk of a second relapse in ITMS positive and oligoclonal IgG bands (OCGBs)-positive patients. We selected clinical studies reporting the ITMS prevalence assessed as oligoclonal IgM Bands (OCMBs), lipid-specific OCMBs (LS-OCMBs), and/or as an intrathecal IgM production > 0% (IgMLoc, Reiber formula). The overall prevalence of ITMS was higher in relapsing-remitting (RR) than clinically isolated syndrome (CIS) patients (40.1% versus 23.8%, p < 0.00001), while was in line with that detected in primary progressive MS (PPMS, 26.7%). Almost all patients (98%) with ITMS had also OCGBs. The risk of having a second relapse was higher in OCGBs positive patients (HR = 2.18, p = 0.007) but much higher in ITMS positive patients (HR = 3.62, p = 0.0005). This study revealed that the prevalence of ITMS is higher in RRMS patients. It suggests that the risk of having a second relapse, previously ascribed to OCGBs, may, to a certain extent, be related to the presence of intrathecal IgM.

Background Anti-ganglioside antibodies are currently used in the differential diagnosis of suspected immune-mediated neuropathies. In-house and increasingly used commercial assays seem to perform suboptimally, and comparative information on their analytical performance are essentially lacking. Born within the frame of guidelines and standardization activities by the Italian Association of Neuroimmunology, this external quality assessment scheme (EQAS) is a real-life snapshot of the laboratory diagnostics in this field. Methods The EQAS consisted of five surplus, anonymized serum samples from patients with clinically-defined neuropathies and two serum samples from healthy blood donors. Eight laboratories used commercial line-/dot-blots, seven in-house/commercial ELISAs (in addition, 13 laboratories tested a recently released ELISA by Bühlmann). Only high anti-ganglioside antibody reactivities were considered, in accordance with consolidated recommendations. Results Large variations in anti-ganglioside antibody profiles were observed, even, although to a lesser extent, within homogeneous classes of assays. Concordance between the profiles and clinical phenotypes was also partial. Conclusions Although conducted on a relatively small, but representative number of Italian laboratories, this EQAS shows a critical between-laboratory disagreement in the test results of anti-ganglioside antibodies. Also considering the trend for using certified assays in generalist laboratories, strong efforts toward standardization and the identification of the best method(s) for their determinations are compellingly needed.

In neuro Behçet disease with multiple sclerosis‐like features, diagnosis could be challenging. Here, we studied the cerebrospinal fluid and serum inflammatory profile of 11 neuro Behçet and 21 relapsing‐remitting multiple sclerosis patients. Between the soluble factors analyzed (MMP9, TNF α, IL6, CXCL13, CXCL10, CXCL8, IFN γ, IL10, IL17, IL23, and others) we found MMP9 increased in neuro Behçet serum compared to multiple sclerosis and decreased in cerebrospinal fluid. Furthermore, neuro Behçet analysis of circulating natural killer CD56DIM subset suggests their potential involvement in increased MMP9 production. We believe that these findings may have a translational utility in clinical practice.

This document presents the guidelines for the cerebrospinal fluid (CSF) analysis and the determination of oligoclonal bands (OCBs) as pivotal tests in neuroin flammatory pathologies of the central nervous system. The guidelines have been developed following a consensus process built on questionnaire-based surveys, internet contacts, and discussions at workshops of the sponsoring Italian Association of Neuroimmunology (AINI) congresses. Essential clinical information on the pathologies in which the CSF analysis is indicated, and, particularly, on those characterized by the presence of OCBs in the intrathecal compartment, indications and limits of CSF analysis and OCB determination, instructions for result interpretation, and agreed laboratory protocols (Appendix) are reported for the communicative community of neurologists and clinical pathologists.

This document presents the guidelines for onconeural antibody testing that have been developed following a consensus process built on questionnaire-based surveys, internet contacts, and discussions at workshops of the sponsoring Italian Association of Neuroimmunology (AINI) congresses. Essential clinical information on paraneoplastic neurological syndromes, indications and limits of onconeural antibody testing, instructions for result interpretation, and an agreed laboratory protocol (Appendix) are reported for the communicative community of neurologists and clinical pathologists.

Introduction Cellular analysis in cerebrospinal fluid (CSF) provides important diagnostic information in many pathological settings. The aim of this two-site study was to evaluate the Sysmex XN Body Fluid mode (XN-BF) for cell analysis of CSF compared to light microscopy (LM). Methods Two hundred and seven consecutive CSF samples were analyzed in parallel with XN-BF and LM. The study also included the estimation of the limit of blank (LoB), limit of detection (LoD), limit of quantitation (LoQ), carry-over and linearity of XN-BF module. Results LoQ of white blood cells (WBC) was 3×106 cells/L; linearity was good and carry-over negligible. XN-BF parameters were compared to LM for the following cell classes: total cells, WBC, polymorphonuclear (PMN), and mononuclear (MN) cells. The bias ranged from 1.3 to 15.2×106 cells/L. The receiver operating characteristics curve analysis for WBC showed an area under the curve of 0.98, and the global diagnostic agreement was 95% at a cutoff of 5×106 cells/L. Conclusions XN-BF provides rapid and accurate counts in clinically relevant ranges of CSF values, thus providing a valuable alternative to conventional LM analysis. However, microscopic review remains advisable in samples with abnormal cell counts or high fluorescent (HF-BF) cell parameter exceeding 5×106 cells/L.

Anti-myelin-associated glycoprotein (MAG) antibody is associated with clinically heterogeneous polyneuropathies. Our purpose was to compare neuropathy phenotypes identified by different anti-MAG tests’ results. Cohort study: Sera from 40 neuropathy anti-MAG EIA positive patients were tested for anti-MAG by Western blot (WB), for anti-peripheral nerve myelin (PNM) on monkey nerve by immunofluorescence assay (IFA), and for anti-HNK1 on rat CNS slices by IFA. Anti-sulfatide antibodies, for comparison, were also tested by EIA. Among 40 anti-MAG EIA positive sera, 85% also had anti-PNM IFA reactivity and 67.5% bind HNK1 on rat CNS. Anti-HNK1 positive patients had the classical predominantly distal acquired demyelinating symmetric (DADS) neuropathy with a benign course, while anti-PNM positive but anti-HNK1 negative patients had predominantly axonal neuropathy with a high frequency of anti-sulfatide reactivity and the worst long-term prognosis. Anti-MAG EIA positive patients without anti-PNM or anti-HNK1 IFA reactivity had a CIDP-like polyneuropathy. Different methods to test for anti-MAG antibodies identify different clinical and electrophysiological findings, as well as long-term outcome. HNK1 reactivity is the strongest marker of DADS.

BACKGROUND:To facilitate the clinical diagnosis of ventilator-associated pneumonia (VAP) in the ICU, the Clinical Pulmonary Infection Score (CPIS) has been proposed but has shown a low diagnostic performance in subsequent studies. We propose a new score based on procalcitonin level and chest echography with the aim of improving VAP diagnosis: the Chest Echography and Procalcitonin Pulmonary Infection Score (CEPPIS). METHODS:This retrospective pilot study recruited patients admitted to the Intensive Care Unit of the Emergency Department, Careggi University Hospital (Florence, Italy), from January 2009 to December 2011. Patients were retrospectively divided into a microbiologically confirmed VAP group or a control group based on diagnosis of VAP and positive tracheal aspirate culture. RESULTS:A total of 221 patients were included, with 113 in the microbiologically confirmed VAP group and 108 in the control group. A CEPPIS > 5 retrospectively fixed was significantly better in predicting VAP (OR, 23.78; sensitivity, 80.5%; specificity, 85.2%) than a CPIS > 6 (OR, 3.309; sensitivity, 39.8%; specificity, 83.3%). The receiver operating characteristic area under the curve analysis also showed a significantly higher diagnostic value for CEPPIS > 5 than CPIS > 6 (0.829 vs 0.616, respectively;P< .0001). CONCLUSIONS:In this pilot, exploratory analysis, CEPPIS is effective in predicting VAP. Prospective validation is needed to confirm the potential value of this score to facilitate VAP diagnosis.

This document presents the guidelines for testing antibodies against neuronal surface antigens that have been developed following a consensus process built on questionnaire-based surveys, internet contacts, and discussions at workshops of the sponsoring Italian Association of Neuroimmunology (AINI) congresses. Essential clinical information on autoimmune encephalitis associated with antibodies against neuronal surface antigens, indications and limits of testing for such antibodies, instructions for result interpretation, and an agreed laboratory protocol (Appendix A) are reported for the communicative community of neurologists and clinical pathologists.

This document presents the guidelines for anti-myelin-associated glycoprotein (MAG) antibody testing that have been developed following a consensus process built on questionnaire-based surveys, internet contacts, and discussions at workshops of sponsoring Italian Association of Neuroimmunology (AINI) congresses. The main clinical information on anti-MAG antibody polyneuropathy, indications and limits of anti-MAG antibody testing, instructions for result interpretation, and an agreed laboratory protocol (Appendix) are reported for the communicative community of neurologists and clinical pathologists.

This document presents the guidelines for anti-aquaporin-4 (AQP4) antibody testing that has been developed following a consensus process built on questionnaire-based surveys, internet contacts, and discussions at workshops of the sponsoring Italian Association of Neuroimmunology (AINI) congresses. Essential clinical information on neuromyelitis optica spectrum disorders, indications and limits of anti-AQP4 antibody testing, instructions for result interpretation, and an agreed laboratory protocol (Appendix) are reported for the communicative community of neurologists and clinical pathologists.

This document presents the guidelines for anti-ganglioside antibody testing that have been developed following a consensus process built on questionnaire-based surveys, internet contacts, and discussions at workshops of the sponsoring Italian Association of Neuroimmunology (AINI) congresses. Main clinical information on dysimmune peripheral neuropathies, indications and limits of anti-ganglioside antibody testing, instructions for result interpretation, and an agreed laboratory protocol (Appendix) are reported for the communicative community of neurologists and clinical pathologists.

Microbiological tests on cerebrospinal fluid (CSF) utilize a common urgent-care procedure that does not take into account the chemical and cytological characteristics of the CSF, resulting sometimes in an unnecessary use of human and diagnostic resources. The aim of this study was to retrospectively validate a simple scoring system (bacterial meningitis-Careggi score [BM-CASCO]) based on blood and CSF sample chemical/cytological parameters for evaluating the probability of acute bacterial meningitis (ABM) in adults. BM-CASCO (range, 0 to 6) was defined by the following parameters: CSF cell count, CSF protein levels, CSF lactate levels, CSF glucose-to-serum glucose ratio, and peripheral neutrophil count. BM-CASCO was retrospectively calculated for 784 cases of suspected ABM in adult subjects observed during a four-and-a-half-year-period (2010 to 2014) at the emergency department (ED) of a large tertiary-care teaching hospital in Italy. Among the 28 confirmed ABM cases (3.5%), Streptococcus pneumoniae was the most frequent cause (16 cases). All ABM cases showed a BM-CASCO value of ≥3. Most negative cases (591/756) exhibited a BM-CASCO value of ≤1, which was adopted in our laboratory as a cutoff to not proceed with urgent microbiological analysis of CSF in cases of suspected ABM in adults. During a subsequent 1-year follow-up, the introduction of the BM-CASCO in the diagnostic workflow of ABM in adults resulted in a significant decrease in unnecessary microbiological analysis, with no false negatives. In conclusion, BM-CASCO appears to be an accurate and simple scoring system for optimization of the microbiological diagnostic workflow of ABM in adults.

Background: Pharmacological inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1) are currently evaluated in clinical trials for various malignancies but, interestingly, also proved of remarkable efficacy in preclinical models of autoimmune disorders including experimental autoimmune encephalomyelitis (EAE). Objectives: The objectives of the study were to determine molecular mechanisms underlying suppression of the encephalitogenic response by these drugs; likewise, whether clinically-relevant post-treatment paradigms with PARP-1 inhibitors could prevent EAE relapses. Methods: Adopted both in vitro techniques (bone marrow-derived cultured DC) as well as in vivo models of chronic or relapsing–remitting (RR) EAE. Results: We report that two structurally unrelated PARP-1 inhibitors negatively regulated NFκB activation, as well as maturation, cytokine production and APC function of cultured mouse bone marrow-derived dendritic cells (DCs). PARP-1 inhibitors also reduced the number and APC function of DCs migrating in the draining lymph nodes of ovalbumin-immunized mice. In C57Bl mice with chronic EAE or SJL mice with RR EAE, pharmacological inhibition of PARP-1 reduced CNS DC migration and demyelination as well as neurological impairment to an extent similar to that achieved with the potent immunosuppressant cyclosporine A. Remarkably, PARP-1 inhibitors injected after the first phase of disease reduced relapse incidence and severity, as well as the spinal cord number of autoreactive Th17 cells. Under this clinically-relevant treatment paradigm, PARP inhibitors also suppressed epitope spreading of the encephalitogenic response. Conclusions: Overall, data underscore the potential relevance of PARP-1 inhibitors to MS therapy and suppression of autoimmunity.

Azathioprine (Aza), 6-Mercaptopurine (6-MP) and 6-Thioguanine (6-TG) are thiopurine drugs widely used as immunosuppressants/anti-inflammatory agents in organ transplantation and chemotherapy. Aza is well tolerated and effective in modifying the course of MS. Here we investigated the action of 6-MP on human dendritic cells (DCs). We described for the first time that 6-MP impairs in vitro differentiation of DCs, has an inhibitory effect during DC activation processes inducing a functionally less immunogenic phenotype. Moreover, 6-MP significantly reduces DC IL-23 production and CCR7 expression, at the same time induces IL-10 augmentation. All these findings add a novel action mechanism in Aza immune modulation.

Multiple sclerosis (MS) is the most common inflammatory demyelinating disorder of the central nervous system (CNS). An approach to improve MS treatment is to identify a rational combination of new medications or existing therapies that impact different aspects of the disease process. Statins are effective in the treatment of MS animal models and are promising candidates for future treatment. Minocycline ameliorates clinical severity of experimental autoimmune encephalomyelitis (EAE) and exhibits several anti-inflammatory and neuroprotective activities. In this study, we tested whether the combination of these two drugs could produce beneficial effects in EAE mice immunized with myelin oligodendrocyte protein (MOG). Our findings show that combined treatment, compared to using the medications alone, resulted in a significant reduction in disease severity, in both the acute and chronic phases of the disease, along with attenuation of inflammation, demyelination and axonal loss. Stereological analysis revealed that the combined treatment significantly guarded against neuroinflammation and neurodegeneration. Moreover, a significant suppression of anti-MOG antibody production in animals treated with the two medications was found. In conclusion, our findings prove that this combination of drugs is neuroprotective and suppresses the severity of EAE. Furthermore, this pharmacological approach appears to be promising as a future therapeutic strategy to control MS.

Multiple myeloma (MM) is characterized, in about 80% of cases, by the production of monoclonal intact immunoglobulin and more than 95% of them have elevated concentrations of involved (i.e. of the same class of intact immunoglobulin) free light chain (FLC). The introduction of novel therapeutic strategies has changed the natural history of the disease, leading to new manifestations of relapse. Light chain escape (LCE) is a pattern of relapse in which the FLC increase is not accompanied by a concomitant raise of the original monoclonal component (MC). Here we present a case of a 55-year-old man with an IgG kappa MM stage III diagnosed in September 2007. At presentation an IgG kappa MC and urine Bence Jones protein (BJP) kappa were present. Bone marrow biopsy (BMB) showed the presence of 80% monotypic kappa plasma cells (PCs). The patient received bortezomib, thalidomide, dexamethasone before undergoing a double autologous stem cell transplantation (ASCT) in October 2008 and April 2009. In May 2011 he relapsed showing the same pattern of presentation and treatment with lenalidomide and dexamethasone was started. ln May 2013 serum and urine immunofixation and FLC became negative. In September 2014, an increase of kappa FLC was observed, while serum and urine immunofixations remained negative until January 2015, when urine immunofixation became positive. Eventually, in February 2015, serum immunofixation revealed the presence of a free kappa MC. After a new BMB showing 80% of monotypic kappa PCs, a LCE relapse was diagnosed and the patient started the treatment with bendamustine, bortezomib and dexamethasone. In the present case, the increase of kappa FLC has indicated relapse 4 and 5 months earlier than urine and serum IFE, respectively. Our observation confirms that it is advisable to routinely perform FLC or BJP during follow up of MM patients undergoing ASCT and/or treatment with biological drugs to ensure that LCE is not missed.

Nanomaterials interact with cells and modify their function and biology. Manufacturing this ability can provide tissue-engineering scaffolds with nanostructures able to influence tissue growth and performance. Carbon nanotube compatibility with biomolecules motivated ongoing interest in the development of biosensors and devices including such materials. More recently, carbon nanotubes have been applied in several areas of nerve tissue engineering to study cell behavior or to instruct the growth and organization of neural networks. To gather further knowledge on the true potential of future constructs, in particular to assess their immune-modulatory action, we evaluate carbon nanotubes interactions with human dendritic cells (DCs). DCs are professional antigen-presenting cells and their behavior can predict immune responses triggered by adhesion-dependent signaling. Here, we incorporate DC cultures to carbon nanotubes and we show by phenotype, microscopy, and transcriptional analysis that in vitro differentiated and activated DCs show when interfaced to carbon nanotubes a lower immunogenic profile. journal article research support, non-u.s. gov't 2013 2013 11 15 imported

Pixantrone is an immunesuppressor similar to mitoxantrone but with lower cardiotoxicity. We evaluated the effect of pixantrone on B cells and lymphomononuclear cells in the course of acute EAE. Pixantrone reduced the number of B cells and suppressed myelin basic protein (MBP) specific IgG production. In vitro, pixantrone induced apoptosis of rat B lymphocytes in a way similar to mitoxantrone. In addition, pixantrone inhibited antigen specific and mitogen induced lymphomononuclear cell proliferation, as well as IFN-γ production, during EAE. These findings suggest a similar mechanism of action for pixantrone and mitoxantrone on the effector function of lymphomonocyte B and T cells.

In this study, we investigated the in vitro proliferative response of peripheral blood T lymphocytes from MS patients and controls to MBP and MOG either in the absence or in the presence of the conditioning factor IL-7. In the absence of IL-7, T-cell reactivity to MOG and MBP was similar in MS patients and controls even if an increased MBP response was found in a subgroup of patients with active disease. In the presence of IL-7, increased T-cell reactivity to MBP was observed in MS patients suggesting that their MBP-specific T cells are in a different functional state.

T cells reactive to self-antigens are present in the peripheral blood of patients with autoimmune diseases as well as in healthy subjects. Although T cell-response to the self-myelin antigen myelin basic protein (MBP) has been widely investigated in multiple sclerosis (MS) patients, very little is known about the evolution over time of this response and its correlation with the disease activity. In recent years magnetic resonance imaging (MRI) techniques have provided new tools for following the inflammatory activity in the central nervous system (CNS) of MS patients. In the present study the T cell response to MBP was longitudinally investigated in terms of frequency, epitope specificity, and cytokine production profile in four patients with relapsing-remitting MS enrolled in a gadolinium-enhanced MRI serial study. In spite of different profiles of inflammatory activity within the CNS, all the patients examined showed major changes in their reactivity to MBP during the follow-up period in terms of both frequency and epitope specificity. Episodic expansions of MBP-specific T cell populations were observed in each patient, and overall they did not correlate with disease activity. In these patients the expansions: 1) occurred in the context of a steady level of disease activity, 2) correlated with a burst of CNS inflammation, 3) followed the appearance of a new active lesion, and 4) were observed even in the absence of detectable signs of CNS inflammation during the entire follow-up period. These results suggest that the evolution over time of the T cell response to a self-antigen such as MBP is more complex than previously expected. The short-term repertoire dynamics of autoreactive T cells in MS underscore the importance of longitudinal studies for evaluating autoreactivity to myelin antigens and probably to any self-antigen in other autoimmune diseases. J. Neurosci. Res. 66:517–524, 2001. © 2001 Wiley-Liss, Inc.

The NapA protein of B. burgdorferi is essential for the persistence of spirochetes in ticks. One of the most intriguing aspects of NapA is its potential to interfere with the host immune system. Here, we investigated the role of the acquired immune responses induced by NapA in the cerebrospinal fluids (CSF) of patients with chronic Lyme borreliosis. We evaluated the cytokine profile induced in microglia cells and CSF T cells following NapA stimulation. We report here that NapA induced a regulatory T (Treg) response in the CSF of patients with chronic Lyme borreliosis and it is able to expand this suppressive response by promoting the production of TGF-β and IL-10 by microglia cells. Collectively, these data strongly support a central role of NapA in promoting both Treg response and immune suppression in the CSF of patients with chronic Lyme borreliosis and suggest that NapA and the Treg pathway may represent novel therapeutic targets for the prevention and treatment of the disease.

In this study, we investigate the role of the C→G mutation in position 77 of exon 4 of the protein tyrosine phosphatase receptor-type C (PTPRC) gene, coding for the CD45 molecule, for the development of multiple sclerosis (MS) in an Italian continental population. The PTPRC mutated genotype has been recently described as associated with MS in three different case–control studies carried out in German MS patients, whereas similar studies performed in the US and Swedish populations failed to demonstrate such an association. The C→G transition in position 77 was found in a small number of Italian MS patients and in none of the matched group of healthy controls (Fisher exact test, P value=0.02). This finding suggests a role, in at least a group of patients, for the PTPRC mutation in genetic susceptibility to MS.

Immune synapse formation between dendritic cells (DCs) and T cells is one of the key events in immune reaction. In immunogenic synapses, the presence of fully mature DCs is mandatory; consequently, the modulation of DC maturation may promote tolerance and represents a valuable therapeutic approach in autoimmune diseases. In the field of cell therapy, bone marrow mesenchymal stem cells (MSCs) have been extensively studied for their immunoregulatory properties, such as inhibiting DC immunogenicity during in vitro differentiation and ameliorating in vivo models of autoimmune diseases (e.g., experimental allergic encephalomyelitis). MSCs seem to play different roles with regard to DCs, depending on cell concentration, mechanism of stimulation, and accompanying immune cells. The aim of this work was to elucidate the immunogenic effects of MSC/DC interactions during DC activation (LPS stimulation or Ag loading). Human monocyte-derived DCs, bone marrow-derived MSCs, and circulating lymphocytes obtained from healthy donors, as well as the laboratory-generated influenza virus hemagglutinin-derived peptide, aa 306–318 peptide-specific T cell line were used for this study. We demonstrate that MSCs mediate inhibition of DC function only upon cell–cell contact. Despite no modification observed in cell phenotype or cytokine production, MSC-treated DCs were unable to form active immune synapses; they retained endocytic activity and podosome-like structures, typical of immature DCs. The transcriptional program induced by MSC–DC direct interaction supports at the molecular pathway level the phenotypical features observed, indicating the genes involved into contact-induced rearrangement of DC cytoskeleton.

Neuromyelitis optica spectrum disorder (NMOsd) is a group of demyelinating disorders recently redefined and associated with NMO-IgG/anti-aquaporin 4 antibodies. Because NMOsd is of unknown prevalence worldwide, we conducted a retrospective, cross-sectional study of 850 patients with demyelinating disorders hospitalized in North East Tuscany from 1998 to 2006 to examine the prevalence of NMO and related disorders among unselected consecutive neurological patients with inflammatory CNS diseases and to evaluate the clinical phenotype spectrum of identified cases. Clinical data were updated after at least 2 years of follow-up. An immunofluorescence technique was used to detect NMO-IgG on rat brain tissue. Sera from other 828 neurological patients, 65 non-neurological patients and 50 healthy donors served as controls. The prevalence of NMOsd was 1.5%, with a MS:NMOsd ratio of 42.7. Among 13 NMOsd patients, 77% had long spinal cord lesions, 38% had severe optic neuritis and 23% had brain or brainstem lesions. Only 56% had clinically definite NMO at follow-up. The final EDSS score ranged from 1 to 10, mainly depending on brainstem involvement occurrence. Our findings confirm a low prevalence of NMO and related disorders among demyelinating inflammatory diseases in a Caucasian population. Moreover, this study demonstrates an unexpectedly high prevalence of limited and atypical variants of this disease, not previously documented.

MSCs have been proposed as possible treatment in MS: In this study MSCs obtained from 10 MS patients and 6 healthy donors (HD) were compared in terms of phenotypical and functional characteristics. We show that MSCs isolated from MS and HD differ significantly for IP10 production. Therefore, although MSCs isolated from MS patients exhibit the same properties of HD MSCs in terms of proliferation, phenotype, in vitro differentiation, TLR expression, immunosuppressive ability, inhibition of DC differentiation and activation, the use of autologous MSCs in cell therapy of autoimmune diseases should be submitted to attentive evaluation and treatment.

Aims: Depression and other psychiatric disorders are frequent in HCV-infected patients, especially during interferon treatment. The molecular mechanism(s) maderlying this finding is still unknown but it has been suggested that HCV and/or interferon administration may increase indoleamine 2,3-dioxygenase (IDO) activity, and reduce plasma tryptophan (TRP) levels and brain serotonin synthesis thus leading to psychopathological disorders. Methods: We studied 89 subjects: (a) 39 patients with chronic hepatitis C virus (HCV) infection and mild liver damage; (b) 39 healthy controls; and (c) 10 patients with chronic hepatitis B virus (HBV) infection. 15 of the patients with HCV infection were re-evaluated after antiviral treatment with pegylated interferon α-2a plus ribavirin leading to viral eradication. We measured serum TRP and kynurenine levels and IDO activity in macrophages. Furthermore, each patient had an accurate psychopathological evaluation. Results: HCV-infected patients had lower (28%) serum TRP and kynuxenine levels than healthy volunteers or HBV-infected patients with comparable liver damage. Depression and anxiety symptoms were particularly common in HCV patients. After viral clearance, macrophage IDO activity, plasma TRP and kynuxenine levels returned toward normal values and psychopathology improved. Conclusion: Our study shows that HCV patients have reduced serum TRP levels and confirms that they frequently suffer from anxiety and depression-related symptoms. The reduced IDO activity found in the macrophages of these patients suggests that HCV infection may hamper macrophage fmactions. After successful antiviral treatment, in spite of the expected increase of IDO activity in macrophages, we noticed that TRP and kynurenine plasma levels returned toward physiological levels and psychopathology decreased significantly.

A cyclic analogue, [cyclo(87-99)MBP(87)(-)(99)], of the human immunodominant MBP(87)(-)(99) epitope, was designed based on ROESY/NMR distance information and modeling data for linear epitope 87-99, taking into account T-cell (Phe(89), Lys(91), Pro(96)) and HLA (His(88), Phe(90), Ile(93)) contact side-chain information. The cyclic analogue was found to induce experimental allergic encephalomyelitis (EAE), to bind HLA-DR4, and to increase CD4 T-cell line proliferation, like that of the conformationally related linear MBP(87)(-)(99) epitope peptide. The mutant cyclic peptides, the cyclo(91-99)[Ala(96)]MBP(87)(-)(99) and the cyclo(87-99)[Arg(91)Ala(96)]MBP(87)(-)(99), reported previously for suppressing, to a varying degree, autoimmune encephalomyelitis in a rat animal model, were found in this study to possess the following immunomodulatory properties: (i) they suppressed the proliferation of a CD4 T-cell line raised from a multiple sclerosis patient, (ii) they scored the best in vitro TH2/TH1 cytokine ratio in peripheral blood mononuclear cell cultures derived from 13 multiple sclerosis patients, inducing IL-10 selectively, and (iii) they bound to HLA-DR4, first to be reported for cyclic MBP peptides. In addition, cyclic peptides were found to be more stable to lysosomal enzymes and Cathepsin B, D, and H, compared to their linear counterparts. Taken together, these data render cyclic mimics as putative drugs for treating multiple sclerosis and potentially other Th1-mediated autoimmune diseases.

Pixantrone is less cardiotoxic and is similarly effective to mitoxantrone (MTX) as an antineoplastic drug. In our study, pixantrone reduced the severity of acute and decreased the relapse rate of chronic relapsing experimental allergic encephalomyelitis (EAE) in rats. A marked and long-lasting decrease in CD3+, CD4+, CD8+ and CD45RA+ blood cells and reduced anti-MBP titers were observed with both pixantrone and MTX. In vitro mitogen- and antigen-induced T-cell proliferation tests of human and rodents cells evidenced that pixantrone was effective at concentrations which can be effectively obtained after i.v. administration in humans. Cardiotoxicity was present only in MTX-treated rats. The effectiveness and the favorable safety profile makes pixantrone a most promising immunosuppressant agent for clinical use in multiple sclerosis (MS).

We have systematically screened the genome for evidence of linkage disequilibrium (LD) with multiple sclerosis (MS) by typing 6000 microsatellite markers in case-control and family based (AFBAC) cohorts from the Italian population. DNA pooling was used to reduce the genotyping effort involved. Four DNA pools were considered: cases (224 Italian MS patients), controls (231 healthy Italians), index (185 index cases from trio families) and parents (the 370 parents of the patient included in the Index pool), respectively. After refining analysis of the most promising 14 markers to emerge from this screening process, only marker D2S367 retained evidence for association. © 2003 Elsevier B.V. All rights reserved.

We report for the first time the immunoadjuvant effects of lipoconjugation of peptide antigens in an in vitro system by using CD4+ T-cells. The lipopeptides obtained by conjugating a palmitoyl moiety at the N(alpha)-terminal of Gln(74) or at the epsilon-NH(2) of Lys(75) of GpMBP(74-85) induced increased T-cell responsiveness compared to the native nonlipidated peptide. On the other hand, lipoderivatives of GpMBP(82-98) did not increase the T-cell response, demonstrating that the superagonist inducing effect of lipoconjugation is epitope-specific. Digestion of the two native peptides with cathepsin D and L, both implicated in antigen processing, and with a complete lysosomal fraction of a EBV-transformed B cell line shows that GpMBP(74-85) is resistant to cellular proteases, while GpMBP(82-98) is easily digested by these enzymes. These results suggest that the first prerequisite for increasing the T-cell response by lipoconjugation is the stability of the native peptide to peptidases, providing an important insight into the understanding of the immunoadjuvant effect of lipoderivative antigens.

Background The mechanisms by which human dendritic cells (DCs) activate a T H 1-polarizing or T H 2-polarizing program are still partially unclear. Objective Study of the mechanisms responsible for the T H 1/T H 2-polarizing activity of human circulating myeloid DCs before and after ligation of their Toll-like receptors (TLRs). Methods IL-4 and IFN-γ production by CD4 + T cells was assessed in cocultures with myeloid DCs before or after TLR triggering. Expression of Jagged-1 and Delta-4 Notch ligands and of GATA-3 and T-box expressed in T cells transcription factors was evaluated by real-time quantitative PCR. Signal transducer and activator of transcription 4 and 6 phosphorylation was assessed by flow cytometry. Knockdown of Jagged-1 or Delta-4 was performed by transfection of DCs with appropriate silencing mRNAs. Results Myeloid immature DCs constitutively expressed Jagged-1, which induces in CD4 + T cells a T H 2 polarization, as shown by Jagged-1 gene silencing. The T H 2 polarization associated with high GATA-3/T-box expressed in T cells ratio and was at least partially dependent on the early induction of IL-4. Maturation of DCs by TLR ligation resulted in the reduction of Jagged-1 and upregulation of Delta-4, which was at least in part responsible for the polarization of CD4 + T cells to the T H 1 phenotype. Conclusion CD4 + T-cell responses are usually characterized by a prevalent T H 2 phenotype unless TLRs are triggered on DCs by microbial components.

Poly(ADP-ribose) (PAR) polymerase (PARP)-1 is a nuclear enzyme regulating protein that functions by targeting PAR chains. Besides its classic role in DNA repair, PARP-1 is emerging as a key transcriptional regulator in different cell types including the immune ones. In this study, we investigated the role of PARP-1 in human dendritic cell (DC) function. We report that both PARP-1 mRNA and protein levels significantly increased during in vitro DC differentiation from monocytes. Of note, inhibitors of PARP-1 such as phenanthridinone and thieno[2,3-c]isoquinolin-5-one reduced expression of CD86 and CD83 in a concentration-dependent manner, having no effects on expression of CD80 and HLA-DR in mature DCs. In the same cultures, PARP-1 inhibitors also reduced production of IL-12 and IL-10. Addition of exogenous IL-12 to the culture medium partially restored CD86 expression in DCs exposed to PARP-1 inhibitors. In line with the role of PAR formation in NF-κB-dependent transactivation, we also report that phenanthridinone and thieno[2,3-c]isoquinolin-5-one impaired NF-κB and AP-1 subunit DNA binding activity in cellular extract of activated DCs. Finally, we show that PARP-1 inhibitors reduced the T cell allostimulatory activity of mature DCs, and that this reduction was prevented when DCs matured in the presence of PARP-1 inhibitors plus IL-12. Of note, nonproliferating T cells exposed to PARP-1 inhibitor-challenged DCs could undergo efficient proliferation when exposed to a subsequent activation stimulus such as anti-CD3 plus anti-CD-28. Together, data provide evidence for a key role of PARP-1 and poly ADP-ribosylation in DC immunocompetence and underscore the relevance of PARP-1 inhibitors to treatment of immune disorders.