TLR2 stimulation drives human naive and effector regulatory T cells into a Th17-like phenotype with reduced suppressive function.

Naturally occurring CD4(+)CD25(+)FOXP3(+) regulatory T cells suppress the activity of pathogenic T cells and prevent development of autoimmune responses. There is growing evidence that TLRs are involved in modulating regulatory T cell (Treg) functions both directly and indirectly. Specifically, TLR2 stimulation has been shown to reduce the suppressive function of Tregs by mechanisms that are incompletely understood. The developmental pathways of Tregs and Th17 cells are considered divergent and mutually inhibitory, and IL-17 secretion has been reported to be associated with reduced Treg function. We hypothesized that TLR2 stimulation may reduce the suppressive function of Tregs by regulating the balance between Treg and Th17 phenotype and function. We examined the effect of different TLR2 ligands on the suppressive functions of Tregs and found that activation of TLR1/2 heterodimers reduces the suppressive activity of CD4(+)CD25(hi)FOXP3(low)CD45RA(+) (naive) and CD4(+)CD25(hi)FOXP3(hi)CD45RA(-) (memory or effector) Treg subpopulations on CD4(+)CD25(-)FOXP3(-)CD45RA(+) responder T cell proliferation while at the same time enhancing the secretion of IL-6 and IL-17, increasing RORC, and decreasing FOXP3 expression. Neutralization of IL-6 or IL-17 abrogated Pam3Cys-mediated reduction of Treg suppressive function. We also found that, in agreement with recent observations in mouse T cells, TLR2 stimulation can promote Th17 differentiation of human T helper precursors. We conclude that TLR2 stimulation, in combination with TCR activation and costimulation, promotes the differentiation of distinct subsets of human naive and memory/effector Tregs into a Th17-like phenotype and their expansion. Such TLR-induced mechanism of regulation of Treg function could enhance microbial clearance and increase the risk of autoimmune reactions.

Optic neuropathy associated with CANOMAD: description of 2 cases.

CANOMAD is a chronic ataxic neuropathy associated with IgM paraproteinemia and reactivity against disialosyl gangliosides. Ophthalmoplegia is a typical feature, but optic pathway involvement has not been reported previously. We describe 2 cases of CANOMAD associated with optic neuropathy. Severe visual loss was present in 1 case. We postulate that optic nerve damage may be related to antibody reactivity against gangliosides. Our report broadens the spectrum of cranial nerve involvement in this rare entity.

Lack of evidence for oxidative stress in sporadic amyotrophic lateral sclerosis fibroblasts.

BACKGROUND: It is conceivable that an early therapeutic intervention in amyotrophic lateral sclerosis (ALS) would lead to better results in terms of disease progression for these patients. One possible strategy to increase the sensitivity of the diagnosis is represented by the use of biological parameters reflecting, for example, oxidative stress alterations associated with ALS. Such biomarkers would be valuable tools both for a better diagnostic evaluation and for studying the impact of therapeutic interventions on the disease course. A special category of experimental models is represented by peripheral cells obtained directly from patients (ex vivo). OBJECTIVE: In this study, primary fibroblasts obtained from 10 sporadic ALS (SALS) patients and 10 healthy matched controls were used to investigate a panel of parameters related to the oxidative status. METHODS: Reactive oxygen species production, protein carbonylation and nitration, susceptibility to hydrogen peroxide exposure, p38-mitogen-activated protein kinase activation and adenosine triphosphate intracellular content were evaluated. RESULTS: No significant difference was observed in all investigated parameters between patient and control cells, and no correlation with the disease severity was found. CONCLUSION: Collectively, our data show no major alterations of the oxidative and bioenergetic status in SALS cultured fibroblasts, suggesting that these cells do not represent a useful model to study the oxidative dysfunction associated with SALS.

Novel therapeutic approaches to autoimmune demyelinating disorders.

Multiple Sclerosis (MS) is the most common autoimmune demyelinating disorder in Western countries and can lead to permanent disability. Over the past decades remarkable progress has been made in providing new therapeutic strategies to tackle the burden of the disease. Oral drugs and monoclonal antibodies are the main innovative approaches that have been tested in advanced stage clinical trials. Several new drugs have been shown to be superior to traditional disease modifying treatments (DMTs), in terms of both clinical and imaging outcome measures. Oral drugs have the advantage of offering a convenient route of administration. Recently fingolimod has received approval for the treatment of relapsing remitting (RR)-MS in several countries, becoming the first oral drug available to patients. Whilst the majority of the current studies focus on RR-MS, some trials investigate the primary or secondary progressive subtypes as well as the early forms of the disease aiming at delaying the conversion to clinically definite MS. Overall the future of the treatment options looks promising, although the occurrence of significant adverse events in some instances points to cautious evaluation of risks and benefits. Extension studies for most of the new drugs are under way and will provide evidence on the efficacy and long term effects of the new treatment strategies.

Increase of Ki-67+ natural killer cells in multiple sclerosis patients treated with interferon-ß and interferon-ß combined with low-dose oral steroids.

Interferon-ß (IFN-ß) is known to expand regulatory CD56(bright) natural killer (NK) cells in multiple sclerosis (MS). In this cross-sectional study we show that MS patients treated with IFN-ß alone or in combination with low-dose prednisolone displayed increased proportion of all NK cell subsets in the active phase of the cell cycle (Ki-67+). There was no difference in NK cell apoptosis markers. In vitro experiments showed that both IFN-ß and IFN-ß in combination with corticosteroids increased the proportion of Ki-67(+) NK cells. This study, although limited, shows that treatment with IFN-ß affects NK cell cycle without altering NK cell apoptosis in MS patients.

Circulating subsets and CD4(+)CD25(+) regulatory T cell function in chronic inflammatory demyelinating polyradiculoneuropathy.

Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is an inflammatory disease of the peripheral nervous system that is probably autoimmune in origin. Different components of the adaptive and innate immunity may be responsible for the aberrant response towards nerve antigens. To investigate this, we examined lymphocyte subsets and regulatory T cell (Treg) function in the blood of CIDP patients, healthy controls (HC) and subjects with non-immune mediated neuropathies (other neuropathies, ON). We used flow cytometry to determine the frequency of monocytes, B cells, natural killer (NK) and NK-T cells, total and activated CD4(+) and CD8(+) T cells, effector memory and central memory CD4(+) and CD8(+) T cells, and CD4(+)CD25(high)Foxp3(+) Tregs. Treg function was studied after polyclonal stimulation and antigen specific stimulation with myelin protein peptides in CIDP and HC. There was an increased frequency of monocytes (p = 0.02) and decreased frequency of NK cells (p = 0.02) in CIDP compared with HC but not ON. There were no significant differences in other populations. Treg function was impaired in CIDP compared to HC (p = 0.02), whilst T cell proliferation to myelin protein peptides before and after depletion of Tregs was not different between patients and controls. This study shows increased circulating monocytes and reduced NK cells in CIDP. Although Treg frequency was not altered, we confirm that Tregs display a defect of suppressive function. Myelin protein peptides were not the target of the altered peripheral regulation of the immune response. The mechanisms of peripheral immune tolerance in CIDP and their relevance to the pathogenesis deserve further exploration.

Modulation of regulatory T cells in health and disease: role of toll-like receptors.

Naturally arising regulatory T cells (Tregs) originate from the thymus and are characterised by the expression of Foxp3 as a key control gene for their development and function. Their pivotal role is maintaining immunological self tolerance. Recently, Tregs have been shown to express Toll-like receptors (TLRs), which are essential components of the innate immune system for the detection of microbial infections and the activation of dendritic cells (DC) maturation programs to induce adaptive immune responses. TLRs are type 1 transmembrane receptors characterised by a highly variable extracellular region containing a leucine rich repeat domain (LRR) involved in ligand binding and an intracellular tail containing a highly conserved region, the TIR homology domain, which mediates interaction between TLRs and downstream signalling molecules. Recent data suggest that the activation of TLRs on Tregs can increase or decrease their suppressive activity, thus providing an important link between innate and adaptive immune responses. Treg modulation by TLRs might influence such processes as the response to infections, immune surveillance to cancer, transplant rejection, and the induction of autoimmunity. Understanding the link between Tregs and TLR could be beneficial to the discovery of new therapeutic targets and strategies.

Chronic sensorimotor polyradiculopathy with antibodies to P2: an electrophysiological and immunoproteomic analysis.

In this study we report a patient with chronic progressive sensory ataxia, proximal weakness, immunoglobulin M (IgM) monoclonal gammopathy, and elevated protein levels in the cerebrospinal fluid, who showed a good response to prednisone. Electrophysiological study disclosed abnormalities predominantly of late responses (F waves and H reflexes), with no evidence of demyelination in the peripheral nerves, suggesting motor and preganglionic sensory nerve roots as the site of the lesion. An immune-mediated pathogenesis was considered and, to identify possible target antigens, we performed bidimensional electrophoresis and a Western blot study. Based on the suspected lesion site, we used human anterior and posterior root extracts. We identified IgM reactivity against peripheral nerve myelin protein P2. Enzyme-linked immunosorbent assay confirmed IgM reactivity toward one synthetic peptide from P2. To our knowledge, reactivity against P2 has not been reported previously in a paraproteinemic neuropathy. Furthermore, we demonstrated that bidimensional electrophoresis and Western blot of the tissue involved, as determined by clinical and electrophysiological studies, may be useful to establish clinical-immunological correlations in paraproteinemic neuropathies.

Lymphocyte subset patterns and cytokine production in Alzheimer's disease patients.

To investigate the signs of inflammatory processes in Alzheimer's disease (AD), we examined peripheral blood mononuclear cells (PBMC) from 51 AD patients (29 with mild and 22 with moderately severe dementia) and 51 age-matched healthy controls (HC), using flow cytometry to analyse the absolute number and the percentage of T, B and NK cells. We also studied the surface expression of CD25, CD28, CD57, CD71, CD45RA and CD45RO markers on cells CD4+ and CD8+. In 30 AD patients and 20 HC the production of IL-2, IFN-gamma, IL-10 and TNF-alpha by PBMC after stimulation with [25-35], [1-40] and [1-16] beta-amyloid (betaA) fragments was also evaluated. A significant decrease in circulating B and CD8+CD28- cells, as well as an increase in CD8+ cells expressing CD71+ and CD28+, was observed in AD patients. A significant decrease in IL-10 production was also found after stimulation of PMBC with betaA [1-40]. The decreased IL-10 production was not related to disease severity. The observed imbalance of immune peripheral cell subpopulations and decreased IL-10 production point to a reduction of suppressor cell function in AD patients.

Characterization of a monoclonal antibody specific for human peripheral myelin protein 22 and its use in immunohistochemical studies of the fetal and adult nervous system.

We produced a mouse monoclonal antibody using cDNA and peptide immunization against the putative second extra-cellular domain of human peripheral myelin protein 22 (PMP22). It reacted specifically with human PMP22 and not with other human myelin proteins and did not react with bovine, rat, or mouse PMP22. The antibody stained the compact myelin of human peripheral nerve motor and sensory axons and did not stain central nervous system tissue. PMP22 reactivity was detected in the spinal roots of the human fetus at 19-20 weeks of gestation. The staining pattern of the PMP22 antibody resembled that of a monoclonal antibody directed against the myelin protein zero.