Enrico Fainardi

Altered miRNA expression in T regulatory cells in course of multiple sclerosis.

OBJECTIVES Multiple sclerosis (MS) is a chronic inflammatory response against constituents of the central nervous system. It is known that regulatory T cells (Tregs) play a key role in the autoimmune balance and their improper function may facilitate the expansion of autoaggressive T cell clones. Recently, microRNAs (miRNAs) have been involved in autoimmune disorders and their loss-of-function in immune cells was shown to facilitate systemic autoimmune disorders. Here, we analyzed the miRNA expression profile in Tregs from MS-RR. METHODS We assessed miRNA genome-wide expression profile by microarray analysis on CD4(+)CD25(+high) T cells from 12 MS relapsing-remitting patients in stable condition and 14 healthy controls. Since CD4(+)CD25(+high) T cells comprise both T regulatory cells (CD4(+)CD25(+high)CD127(dim/-)) and T effector cells (CD4(+)CD25(+high)CD127(+)), we performed a quantitative RT-PCR on CD4(+)CD25(+high)CD127(dim/-) and CD4(+)CD25(+high)CD127(+) cells isolated from the same blood sample. RESULTS We found 23 human miRNAs differentially expressed between CD4(+)CD25(high)bona fide Treg cells from MS patients vs. healthy donors, but, conversely, among the deregulated miRNAs, members of the miR-106b-25 were found down-regulated in MS patients when compared to healthy donors in CD4(+)CD25(high)CD127(dim/-) T regulatory cells. More interesting, the ratio between Treg/Teff showed an enrichment of these microRNA in T regulatory cells derived from patients if compared to healthy controls. CONCLUSION miR-106b and miR-25 were previously shown to modulate the TGF-β signaling pathway through their action on CDKN1A/p21 and BCL2L11/Bim. TGF-β is involved in T regulatory cells differentiation and maturation. Therefore, the deregulation of this miRNA cluster may alter Treg cells activity in course of MS, by altering TGF-β biological functions.

Factors associated with neurological outcome and lesion progression in traumatic subarachnoid hemorrhage patients.

OBJECTIVE:Traumatic subarachnoid hemorrhage (tSAH) is a frequent finding after closed-head injuries, and its presence is a powerful factor associated with poor outcome. The exact mechanism linking tSAH and an adverse outcome is poorly understood. The aim of this study was to identify the factors that may predict outcomes and changes in the computed tomographic (CT) scans of lesions in a selected population of tSAH patients. METHODS:We evaluated 141 patients admitted consecutively from January 1, 1997, to January 31, 1999, with a CT diagnosis of tSAH. The admission and “worst” CT scans were recorded. CT scan changes were reported as “significant CT progression” (changes in the Marshall classification) or “any CT progression.” The amount of subarachnoid blood was recorded using a modified Fisher classification. Outcome was assessed at 6 months after injury with the Glasgow Outcome Scale. RESULTS:Twenty-eight patients (19.9%) had an unfavorable Glasgow Outcome Scale outcome. In the univariate analysis, prognosis was significantly related to age, admission Glasgow Coma Scale score, Marshall CT classification score at admission and on the worst CT scan, amount of tSAH, and volume of the associated brain contusions. From multivariate analysis, the only factors independently related to outcome were the Glasgow Coma Scale score (P < 0.01) and size of the tSAH at admission (P < 0.001). Thirty-four patients (24.1%) had significant CT lesion progression, and 66 patients (46.8%) had some lesion progression. Patients having significant progression of the lesion had a higher risk of an unfavorable outcome (32 versus 10%; P = 0.004). Unadjusted factors predicting CT progression were the Glasgow Coma Scale score at admission, the Marshall classification at admission, the amount of subarachnoid blood, and the presence or volume of associated brain contusions at admission. Independent factors associated with significant CT progression were the amount of tSAH (P < 0.001) and the presence or volume of brain contusions at admission (P < 0.001). CONCLUSION:The outcome of patients with tSAH at admission is related in a logistic regression analysis to the admission Glasgow Coma Scale score and to the amount of subarachnoid blood. These patients also have a significant risk of CT progression. The amount of subarachnoid blood and the presence of associated parenchymal damage are powerful independent factors associated with CT progression, thus linking poor outcomes and CT changes.

Cerebrospinal fluid and serum levels and intrathecal production of active matrix metalloproteinase-9 (MMP-9) as markers of disease activity in patients with multiple sclerosis.

In this study, we employed a sensitive activity assay system to measure cerebrospinal fluid (CSF) and serum levels of active matrix metalloproteinase-9 (MMP-9) in 37 relapsing-remitting (RR), 15 secondary progressive (SP) and nine primary progressive (PP) multiple sclerosis (MS) patients, grouped according to clinical and magnetic resonance imaging (MRI) evidence of disease activity. We also studied, as neurological controls, 48 patients with other inflammatory neurological disorders (OIND) and 48 with non-inflammatory neurological disorders (NIND). To assess active MMP-9/TIMP-1 circuit, CSF and serum levels of MMP-9 tissue inhibitor TIMP-1 were quantified by ELISA in the same patient population. CSF mean levels of active MMP-9, CSF active MMP-9/TIMP-1 ratios and intrathecal active MMP-9 synthesis, as indicated by specific index, were more elevated in MS than in NIND (P <0.05, <0.02 and <0.02, respectively), serum active MMP-9/TIMP-1 ratio was higher in MS (P<0.01) and OIND (P<0.02) than in NIND, and serum TIMP-1 concentrations were lower in MS than in NIND (P<0.05). More importantly, serum active MMP-9 mean levels, serum active MMP-9/TIMP-1 ratio and intrathecal production of active MMP-9 were increased in MS patients with clinical (P<0.001, <0.001 and <0.05, respectively) and MRI (P<0.001, <0.001 and <0.02, respectively) disease activity, whereas CSF mean concentrations of active MMP-9 and CSF active MMP-9/TIMP-1 ratio were enhanced only in MS patients with MRI evidence of disease activity (P<0.02 and <0.01, respectively). Altogether, these findings suggest that a shift in MMP-9/TIMP-1 balance towards proteolytic activity of MMP-9 could be relevant in MS immune dysregulation. In addition, our results indicate that CSF and serum levels of active MMP-9 may represent a potential surrogate biomarker for monitoring MS disease activity. In particular, serum active MMP-9/TIMP-1 ratio seems to be a very appropriate indicator of ongoing MS inflammation, since it is easily measurable.

Presence of detectable levels of soluble HLA-G molecules in CSF of relapsing–remitting multiple sclerosis: relationship with CSF soluble HLA-I and IL-10 concentrations and MRI findings

We have investigated the presence of non-classical soluble HLA-G molecules (sHLA-G) in cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients and the possible relationships between CSF levels of sHLA-G, classical soluble HLA-I (sHLA-I) molecules, IL-10 amounts and Magnetic Resonance Imaging (MRI) findings were evaluated. We studied by ELISA technique the sHLA-I, sHLA-G and IL-10 levels in CSF of 50 relapsing-remitting (RR) MS patients stratified according to clinical and MRI evidence of disease activity. Thirty-six patients with other inflammatory neurological disorders (OIND) and 41 with non-inflammatory neurological disorders (NIND) were used as controls. CSF mean levels were significantly higher in MS and OIND than in NIND for sHLA-I (p<0.001) and in MS than in controls for sHLA-G (p<0.001), with no differences among the various groups for IL-10 mean concentrations. An increase in CSF sHLA-I was found in MS patients with Gd-enhancing lesions (p<0.01), while sHLA-G and IL-10 were more represented in MS patients without lesional activity on MRI scans (p<0.02). In MRI-inactive MS, CSF IL-10 mean concentrations were significantly greater in patients with CSF-detectable levels of sHLA-G than in those without any evidence of CSF sHLA-G expression (p<0.05). Our findings suggest that CSF classical sHLA-I and non-classical sHLA-G levels may modulate MS activity as assessed by MRI acting in opposite directions. The association observed between sHLA-G and IL-10 when Gd-enhancing lesion resolved indicates a potential immunoregulatory role for IL-10 in the control of MS disease activity by shifting the sHLA-I/sHLA-G balance towards sHLA-G response.

Environmental Risk Factors and Multiple Sclerosis: A Community-Based, Case-Control Study in the Province of Ferrara, Italy

The frequency of multiple sclerosis (MS) in Italy and in other areas of the world seems to have increased over time, suggesting that some environmental factors operate in its etiology. We performed a retrospective, community-based case-control study on MS in order to verify the etiologic role of selected environmental factors. We found an association between MS and higher educational level, employment in public administration, past history of allergies, and infection at an early age with measles, rubella and whooping cough. Our data seem to confirm that exogenous factors play a role in the etiology of MS although some confounding variables could have accounted for the associations.

25-Hydroxyvitamin D in cerebrospinal fluid during relapse and remission of multiple sclerosis

Hypovitaminosis D may play a role in multiple sclerosis (MS), but little is known about intrathecal vitamin D. 25-Hydroxyvitamin D was measured in cerebrospinal fluid and sera from 36 patients with relapsing-remitting MS, 20 patients with other inflammatory neurological diseases and 18 patients with non-inflammatory neurological diseases with liquid chromatography-mass spectrometry. There were no significant differences in cerebrospinal fluid concentrations of 25-hydroxyvitamin D, but the cerebrospinal fluid:serum ratio was significantly lower in MS compared with other inflammatory neurological diseases (p=0.0012) and non-inflammatory neurological diseases (p=0.041) patients. The concentrations of 25-hydroxyvitamin D in cerebrospinal fluid and serum were positively correlated and their ratio was similar to that of albumin. Neither the concentrations of 25-hydroxyvitamin D in cerebrospinal fluid or serum nor their ratio were associated with the presence of relapses or gadolinium-enhanced lesions. These results do not support that 25-hydroxyvitamin D is actively transported to the cerebrospinal fluid, or that the cerebrospinal fluid or serum levels or their ratio exert a major impact on MS activity.

Emerging topics and new perspectives on HLA-G

Following the Fifth International Conference on non-classical HLA-G antigens (HLA-G), held in Paris in July 2009, we selected some topics which focus on emerging aspects in the setting of HLA-G functions. In particular, HLA-G molecules could play a role in: (1) various inflammatory disorders, such as multiple sclerosis, intracerebral hemorrhage, gastrointestinal, skin and rheumatic diseases, and asthma, where they may act as immunoregulatory factors; (2) the mechanisms to escape immune surveillance utilized by several viruses, such as human cytomegalovirus, herpes simplex virus type 1, rabies virus, hepatitis C virus, influenza virus type A and human immunodeficiency virus 1 (HIV-1); and (3) cytokine/chemokine network and stem cell transplantation, since they seem to modulate cell migration by the downregulation of chemokine receptor expression and mesenchymal stem cell activity blocking of effector cell functions and the generation of regulatory T cells. However, the immunomodulatory circuits mediated by HLA-G proteins still remain to be clarified.

Detection of JC virus DNA in cerebrospinal fluid from multiple sclerosis patients

JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), has been proposed as a possible aetiopathogenic factor in multiple sclerosis (MS). We performed a study to search the LT region of JCV genome by nested PCR in cerebrospinal fluid (CSF), peripheral blood mononuclear cell (PBMC) and urine samples collected from 121 MS patients, 24 patients with other neurological disorders (OND), 30 non neurological patients (NND) and in PBMCs and urine of 40 healthy subjects. JCV DNA has been found in the CSF of 11 MS patients (9%) while all the CSFs from the 24 OND and the 30 NND cases were negative. No significant differences have been observed as regard to the frequency of JCV DNA detection in PBMCs and urine between the MS patients and the control groups. Nucleotide sequences analysis of seven JCV CSF isolates showed that five strains were identical the prototypal strain, while the other two had a base mutation (T→C) in 4286 nucleotide (nt). The finding of JCV DNA in the CSF of MS patients suggest that JCV could play a role in the triggering and/or in the maintenance of MS aetiopathogenic process, and therefore it should be taken in consideration when monitoring this disease.

Inhibition of multiple sclerosis-associated retrovirus as biomarker of interferon therapy

The authors performed a longitudinal evaluation of multiple sclerosis (MS) patients, during 1 year of therapy with interferon-β (IFN-β), by clinical examination and detection of presence in the blood and viral load of MS-associated retrovirus (MSRV), by MSRVenv-specific, fully quantitative, real time reverse transcriptase-polymerase chain reaction (RT-PCR). MSRV load in the blood was directly related to MS duration and fell below detection limits within 3 months of IFN therapy; one patient had strong progression, accompanied by total MSRV rescue. These findings suggest that evaluation of plasmatic MSRV could be considered the first prognostic marker for the individual patient, to monitor disease progression and therapy outcome.

Intrathecal synthesis of soluble HLA-G and HLA-I molecules are reciprocally associated to clinical and MRI activity in patients with multiple sclerosis

The aim of this study was to provide further insight into the effective contribution of classical soluble HLA-A, B and C class Ia (sHLA-I) and non-classical soluble HLA-G class Ib (sHLA-G) molecules in immune dysregulation occurring in multiple sclerosis (MS). We evaluated by enzyme-linked immunosorbent assay (ELISA) technique intrathecal synthesis and cerebrospinal fluid (CSF) and serum levels of sHLA-I and sHLA-G in 69 relapsing-remitting (RR), 21 secondary progressive (SP) and 13 primary progressive (PP) MS patients stratified according to clinical and magnetic resonance imaging (MRI) evidence of disease activity. We also tested, as neurological controls, 91 patients with other inflammatory neurological disorders (OIND) and 92 with non-inflammatory neurological disorders (NIND). Eighty-two healthy volunteers served as further controls for sHLA-I and sHLA-G determinations. An intrathecal production of sHLA-I and sHLA-G detected by specific indexes was significantly more frequent in MS patients than in controls (p<0.01). An intrathecal synthesis of sHLA-I was prevalent in clinically (p<0.02) and MRI active (p<0.001) MS, whereas a CSF-restricted release of sHLA-G predominated in clinically (p<0.01) and MRI stable (p<0.001) MS. sHLA-I levels were low in the serum of clinically active (p<0.001) and high in the CSF of MRI active (p<0.01) MS. Conversely, sHLA-G concentrations were decreased in the serum of clinically stable MS (p<0.01) and increased in the CSF of MRI inactive MS (p<0.001). The trends towards a negative correlation observed between CSF and serum concentrations and intrathecal synthesis of sHLA-I and sHLA-G in patients without evidence of clinical and MRI activity confirmed that intrathecal production and fluctuations in CSF and serum concentrations of sHLA-I and sHLA-G were reciprocal in MS. Our results suggest that, in MS, a balance between classical sHLA-I and non-classical sHLA-G products modulating both MRI and clinical disease activity in opposite directions may exist.