Objective We hypothesized that autoaggressive immune responses observed in multiple sclerosis (MS) could be associated with an imbalance in proportion of immune cell subsets and in cytokine production in response to infection, including viruses. Methods We collected blood mononuclear cells (MNC) from 23 patients with MS and 23 sex- and age-matched healthy controls (HC) from the island of Sardinia, Italy, where the prevalence of MS is extraordinarily high. Using flow cytometry, we studied MNC for expression of blood dendritic cell antigens (BDCA)-2 and BDCA-4 surface markers reflecting the proportion of plasmacytoid dendritic cells (pDC) that produce type I interferons (IFNs) after virus challenge and promote Th2/anti-inflammtory cytokine production. In parallel, pro-inflammatory (interleukin [IL]-2, IL-12, IFN-γ), anti-inflammatory (IL-4, IL-10), and immuno-regulatory/pleiotropic cytokines (type I IFNs including IFN-α and β, IL-6) were measured before and after an in vitro exposure to herpes simplex virus type 1 (HSV-1). Results The subset of lineage negative (lin−), BDCA-2+ cells was lower in patients with MS compared with HC (0.08 ± 0.02% vs 0.24 ± 0.02%; P < 0.001). A similar pattern was observed for lin−BDCA-4+ cells (0.08 ± 0.02% vs 0.17% ± 0.03; P < 0.01). Spontaneous productions of IL-6 (45 ± 10 pg/mL vs 140 ± 26 pg/mL; P < 0.01) and IL-10 (17 ± 0.4 pg/mL vs 21 ± 1 pg/mL; P < 0.05) by MNC were lower in patients with MS compared with HC. Spontaneous production of IL-6 (6.5 ± 0.15 pg/mL vs 21 ± 5 pg/mL; P < 0.01 and IL-10 (11 ± 1 pg/mL vs 14 ± 3 pg/mL; P < 0.05) by pDC was also lower in patients with MS compared with HC. Exposure of MNC to HSV-1 showed, in both patients with MS and HC, increased production of IFN-α, IL-6, and IL-10 but decreased production of IL-4. In response to HSV-1 exposure, productions of IL-6 (165 ± 28 pg/mL vs 325 ± 35 pg/mL; P < 0.01) and IL-10 (27 ± 3 vs 33 ± 3 P < 0.05) by MNC as well as by pDC (IL-6: 28 ± 7 vs 39 ± 12 P < 0.05; IL-10: 14 ± 1 vs 16 ± 3 P < 0.05) were lower in patients with MS compared with HC. Conclusion The results implicate a new evidence for altered immune cells and reduced immune responses in response to viral challenge in MS.

Full text available Keywords: Chitotriosidase, Alzheimer's disease, macrophage

Dendritic cells (DC), as the most effective antigen presenting cells, are protagonists of the complex immune network involved in multiple sclerosis (MS) lesion formation. Glatiramer acetate (GA), a synthetic random copolymer, is thought to exert its therapeutical effect in MS by favouring both Th2 cell development and IL-10 production from peripheral lymphocytes as well as by systemically affecting the antigen presenting cells. In the present study we further analysed the mechanisms of action of GA by using an autologous DC-lymphocytes (Ly) coculture system from 11 MS patients and 12 matched healthy controls (HC). We found that, in MS patients, pretreatment with GA significantly decreases the in vitro proliferative effect of DC on lymphocytes as compared to HC and to unpulsed or myelin basic protein (MBP)-pulsed DC from MS patients (P < 0.05). In addition, GA-treated DC from both MS patients and HC significantly increase the lymphocyte production of IL-5 and IL-13 as compared to MBP-treated DC (P < 0.05). In conclusion our in vitro study may provide new therapeutical mechanisms of GA on lymphocytes, antiproliferative and Th2-favouring effects, which are mediated by monocyte-derived DC.

Study results from differentgeographical areas providesome circumstantial evidence that,when compared with the generalpopulation, people who later in lifedevelop multiple sclerosis (MS)have a pattern of birth excess numbersin spring and late summer,which may disclose an associationwith MS–predisposing environmentalagents. To identify the presenceof season–related cluster ofMS birth in Sardinia we have designeda case–control study in theprovince of Sassari, Northern Sardinia,insular Italy, an area at veryhighand increasing risk for MS.Mean birth incidence rate of peoplewith MS (810 cases) on a threeandsix–months basis were comparedwith that of two control populations:the MS unaffected siblings(1069), sharing genetic materialwith patients, and a representativenumber of births (247,612) of thegeneral population of the studyarea. We found that the birth inmonths peaking in spring significantlyrepresents one risk factor forfuture MS development. This seasonaldeviation of MS births revealsan intriguing epidemiologicaloverlap with common environmentalagents, which may open anew scenario of hypothetical explanationsfor environmental factorsperhaps affecting the CNS at thecrucial time of myelination orshaping the newborn immune system.

The 1,2-fucosyl-oligosaccharides, and among these the 2'-fucosyl-lactose (2'-FL) and lacto-N-fucopentaose (LNFP)-I, are quantitatively the most represented oligosaccharides of human milk. They are also seen to represent an important immune device to prevent nursing infants from severe infectious diarrhoea. Recent evidences show that the appearance of 2'-FL and LNFP-I in human colostrums is synchronised with the macrophage inhibition and that LNFP-III induces a Th2 response from the mouse peripheral immune system. Since mannosyl-fucosyl receptors are described on the macrophage surface, all these evidences allow us to investigate on the possible immune function of human 2'-FL and LNFP-I in vitro on LPS-activated mononuclear cells (MNC) from 12 patients with multiple sclerosis (MS) and 20 matched health controls (HC). We found that 2'-FL and LNFP-I significantly decrease, to a different extent, the MNC proliferation from both HC and MS patients, in a linear and dose-dependent manner. 2'-FL and LNFP-I also reduce the production of IL-12 and IFN-γ, particularly in MS patients as compared to HC (p=0.01 and p<0.001, respectively), while increasing that of IL-10. The overall immunomodulatory effect of 2'-FL and LNFP I here presented may represent a future therapeutic option for the abnormal immune response found in some monocyte-mediated diseases.

Activated macrophages are major effectors at all stages of lesion formation in multiple sclerosis (MS) brain. Here, we report that the macrophage enzyme chitotriosidase (Chit) is significantly elevated both in plasma and cerebrospinal fluid (CSF) of patients with MS as compared to healthy controls and other neurological patients (P< 0.001). Furthermore, the Chit activity in blood significantly associates with the MS clinical course (higher in secondary progressive relative to relapsing remitting, P = 0.01) and the clinical severity as measured by Kurtkze’s Expanded Disability Status Scale (P<0.001). Also, we found that Chit activity is compartmentalized in the central nervous system of early MS patients and that its CSF/plasma quotient, in the presence of a preserved albumin quotient, correlates with the extent of future clinical deterioration (r = 0.91; P<0.001). These findings confirm that innate immunity, here represented by Chit, is clinically relevant in MS and allows, if confirmed, reconsidering novel MS therapeutic strategies specifically aimed at this branch of the immune response.

The human genome has been subjected to selective pressures to resist to infectious agents in spite of a heavy segregational load. With this regard, thalassaemia and glucose-6-phosphate dehydrogenase deficiency have been considered an efficient genetic protection against P. falciparum malaria in Sardinia, insular Italy. In this island, some multiple sclerosis (MS)-associated HLA haplotypes have the highest odds ratios in the same highestrate malarious areas of the island. Moreover, tumor necrosis factor (TNF) polymorphisms epidemiologically associated with both MS and malaria are ten-fold more frequent amongst Sardinians compared to other populations worldwide4. A possible association between MS and malaria in this island was never analysed experimentally. We studied the immunological response of mononuclear cells to P. falciparum and the killing effect of macrophages on parasites in Sardinian MS patients and in matched healthy controls (HC).

Although many failed surrogate markers are provided in the literature, inflammation may contribute to the outcome of ischemic stroke. In 50 consecutive patients with acute ischemic stroke, in the absence of symptoms and signs of concomitant infection, we evaluated a panel of biomarkers reported to be variably associated with brain ischemia, and correlate their serum level with the brain lesion volume and clinical outcome. Infarct size was calculated on computed tomography (CT) scans by means of the Cavalieri's method. Neurological impairment was scored by using the Glasgow Coma Scale, Glasgow Outcome Scale and National Institutes of Health (NIH) scales at stroke onset and 3-month follow-up. Some markers showed a direct significant correlation with both initial and final NIH scale and with infarct size, particularly tumor necrosis factor alpha (TNF-alpha) (P=0.002), intercellular adhesion molecule-1 (P<0.01) and matrix metalloproteinase-2/9 (P=0.001). In contrast to previous reports, interleukin-6 (IL-6) serum level showed a significant inverse correlation with both final neurological impairment and infarct size (P<0.001). This novel finding allows us suggesting that IL-6, in the context of a complex pro-inflammatory network occurring during stroke, is associated with neuroprotection rather than neurotoxicity in patients with ischemic brain injury.

Subjects from Sardinia, Italy, are relatively homogeneous compared to Swedes. Although ethnically distant, both populations have similarly high multiple sclerosis (MS) incidence rates. Pro- and anti-inflammatory cytokines and their receptors, signalling molecules and other immune response-associated factors might influence MS pathogenesis, though definite proof is missing. The study of populations with similar MS incidence but different genetic and environmental background could make possible the definition of factors that relate to such background differences. We selected untreated female MS patients from Sassari, Sardinia, and Stockholm, Sweden, and corresponding sexand age-matched healthy controls (HC), to study blood mononuclear cells (MNC) for mRNA expression of 20 immune response-related genes considered relevant in MS, employing real-time PCR. Higher expression of IL-12p40 mRNA was confined to MS from both Sassari and Stockholm, compared to corresponding HC. MS patients from Sassari, but not Stockholm, expressed higher TNF-a compared to corresponding HC. MS patients from Stockholm, but not Sassari, expressed higher IL-6. Indoleamine 2,3 dioxygenase (IDO), a molecule necessary in tolerance induction, was lower in MS from Stockholm compared to corresponding HC. This was not observed in Sassari. No differences were detected for other members of the IL-12 family, other Th1 and Th2 cytokines, and the signalling molecules Stat 4 and 6. The results corroborate a pro-inflammatory state in MS as reflected by high expression of IL-12, TNF-a and IL-6, although the extent of expression of TNF-a, IL-6 and IDO differs between strictly matched MS patients from different high-incidence areas. This might result from genetic and/or environmental differences. They may account for some of the discrepancies regarding immune response-related molecules previously reported in MS. In conclusion, a pro-inflammatory state exists in MS patients from Sassari as well as Stockholm. The changes of pro-inflammatory and other immune response-related variables differ however between the two MS populations. This may be attributed to the genetic and/or environmental background.

The establishment of new hygienic conditions plays a role in the appearance of autoimmunity in "westernalised" countries. Consistently, but still unconvincingly, several epidemiological and immunogenetic evidences link the disappearance of malaria with the increase of multiple sclerosis (MS) in Sardinia, insular Italy. To this purpose, we have made an attempt to build a relationship between malaria disappearance and MS under the light of the hygiene hypothesis. This relationship has taken into account the MS frequency increase soon after malaria eradication in Sardinia, the present malaria endemism in Africa, the innate immune system activity here represented by Chitotriosidase (Chit), an hydrolytic enzyme produced by macrophages, and an unproductive polymorphism of Chit gene (CHIT1) as a measure of the genetic weight of Plasmodium-related immunity in these populations. Data were derived from both experimental results specifically designed for this study and other data obtained from the available literature. The experimental and the hystorical-epidemiological findings concur to indicate that whilst in Africa CHIT1 mutation is rare and MS incidence is very low due to unmodified parasitic influence and hygienic conditions, in Sardinia a relationships between CHIT1 mutation, plasma Chit activity and MS prevalence rate is detected, even to a higher extent compared to Sicily, area at former lower rate of malaria endemy. Upon such a basis, we have found convincing argumentations that, at least in part, MS has increased over the last four decades in Sardinia also because of the eradication of malaria, 50 years ago. This infectious disease that run for centuries in Sardinia, besides well documented enzyme deficiencies and red cell pathologies, have left an abnormal macrophage reactivity against Plasmodium falciparum. As a result, some Sardinian individuals secrete abnormally high levels of mediators of the innate immunity, relics of former protective anti-malaria infection, in response to new environmental factors. Therefore, MS, an immune-conditioned pathology of the central nervous system has been subject to an unexplained epidemiological increase in the last few decades in Sardinia because cells of the innate immune system, immuno-genetically selected over the centuries in response to widespread P. falciparum malaria, have kept the tendency to over-respond to triggering factors even after the disappearance of malaria. This hypothesis may have an influence in re-directing clinicians toward a innate immunity-based rather than an antigen specific-based new MS therapies.

Background: Following an acute brain ischemia, local endothelia allow monocyte chemoattraction into the lesion site which contributes to brain damage through a group of neurotoxic factors. A relationship exists between the extent of brain damage and the plasma level of monocyte products, including chitotriosidase, though usually strictly related to preexisting infectious-inflammatory diseases. Purpose: Since chitotriosidase activity is also elevated in pathogen-free conditions, we tested whether chitotriosidase upregulation might be specifically related to stroke and unrelated to clinically relevant infectious diseases. Methods: We studied the plasma level of chitotriosidase activity, TNF-alpha and IL-6 in 44 consecutive patients with acute brain ischemia without concomitant symptoms or signs of inflammatory-infectious diseases. Results were compared with stroke severity and outcome as detected by brain CT and NIH scale. Blood samples were collected, on average, 11 h after stroke onset. Results: Chitotriosidase activity positively correlates with stroke severity, as measured by NIH scale ( r = 0.69, p < 0.01), to the extent of brain damage as documented by CT ( r = 0.75, p <= 0.001) and the TNF-alpha level ( r = 0.76, p < 0.001); it also inversely correlates with the IL-6 level ( r = - 0.43, p <= 0.05). Conclusion: Our results indicate that chitotriosidase is a specific marker of macrophage activation occurring in stroke which directly correlates with stroke severity independently of preexisting inflammatory or infectious conditions. Copyright (C) 2005 S. Karger AG, Basel.

Several nuclear hormone receptors have been associated with inflammatory reactions. Particularly, liver X receptors (LXRs) have recently been identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. LXRs are negative regulators of macrophage inflammatory gene expression. Multiple sclerosis (MS), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. Sweden belongs to the countries with a high MS incidence. In Italy, the MS incidence is lower, except on the island of Sardinia where the incidence is even higher than in Sweden. Subjects from Sardinia are ethnically more homogeneous, and differ from Swedes also regarding genetic background and environment. We studied mRNA expression of several nuclear hormone receptors in blood mononuclear cells (MNC) from female patients with untreated relapsing-remitting MS from Sassari, Sardinia, and Stockholm, Sweden. Sex- and age-matched healthy controls (HC) were from both areas. mRNA expression was evaluated by quantitative real-time PCR. We found altered mRNA expression of LXRs, estrogen receptors (ERs), and androgen receptor (AR) in MS. mRNA expression of both LXRα and LXRβ is lower in MS from Stockholm but not from Sassari. In particular, LXRα mRNA expression was significantly lower in MS from Stockholm as compared with all groups in the study including MS from Sassari. Low levels of ERα mRNA are seen in MS from both Stockholm and Sassari. The splice variant ERβcx showed significantly higher mRNA expression in MS from Sassari and Stockholm as compared with corresponding HC. In particular, ERβcx mRNA in MS from Sassari was remarkably higher as compared with all other groups in the study. Higher levels of AR mRNA are present in HC from Sassari. The findings indicate that the expression levels of anti-inflammatory nuclear receptor superfamily genes in MS appear to reflect both ethnic and environmental influences.