Antipsychotic drugs counteract autophagy and mitophagy in multiple sclerosis.

Multiple sclerosis (MS) is a neuroinflammatory and neurodegenerative disease characterized by myelin damage followed by axonal and ultimately neuronal loss. The etiology and physiopathology of MS are still elusive, and no fully effective therapy is yet available. We investigated the role in MS of autophagy (physiologically, a controlled intracellular pathway regulating the degradation of cellular components) and of mitophagy (a specific form of autophagy that removes dysfunctional mitochondria). We found that the levels of autophagy and mitophagy markers are significantly increased in the biofluids of MS patients during the active phase of the disease, indicating activation of these processes. In keeping with this idea, in vitro and in vivo MS models (induced by proinflammatory cytokines, lysolecithin, and cuprizone) are associated with strongly impaired mitochondrial activity, inducing a lactic acid metabolism and prompting an increase in the autophagic flux and in mitophagy. Multiple structurally and mechanistically unrelated inhibitors of autophagy improved myelin production and normalized axonal myelination, and two such inhibitors, the widely used antipsychotic drugs haloperidol and clozapine, also significantly improved cuprizone-induced motor impairment. These data suggest that autophagy has a causal role in MS; its inhibition strongly attenuates behavioral signs in an experimental model of the disease. Therefore, haloperidol and clozapine may represent additional therapeutic tools against MS.

Dimethyl Fumarate Treatment Reduces the Amount but Not the Avidity of the Epstein-Barr Virus Capsid-Antigen-Specific Antibody Response in Multiple Sclerosis: A Pilot Study.

(1) Multiple sclerosis (MS) is a chronic inflammatory disease of autoimmune origin. The Epstein−Barr virus (EBV) is associated with the onset of MS, as almost all patients have high levels of EBV-specific antibodies as a result of a previous infection. We evaluated longitudinally the effects of dimethyl fumarate (DMF), a first-line treatment of MS, on the quantity and quality of EBV-specific IgG in MS patients. (2) Serum samples from 17 MS patients receiving DMF were taken before therapy (T0) and after 1 week (T1) and 1 (T2), 3 (T3) and 6 (T4) months of treatment. Anti-EBV nuclear antigen (EBNA)-1 and capsid antigen (CA) IgG levels and anti-CA IgG avidity were measured in all samples. (3) Serum levels of anti-CA IgG were lower at T1 (p = 0.0341), T2 (p = 0.0034), T3 (p < 0.0001) and T4 (p = 0.0023) than T0. These differences were partially confirmed also in anti-EBNA-1 IgG levels (T3 vs. T0, p = 0.0034). All patients had high-avidity anti-CA IgG at T0, and no changes were observed during therapy. (4): DMF can reduce the amount but not the avidity of the anti-EBV humoral immune response in MS patients from the very early stages of treatment.

Gut Microbiota Changes during Dimethyl Fumarate Treatment in Patients with Multiple Sclerosis.

The gut microbiota is involved in the development of the immune system and can modulate the risk for immune-mediated disorders such as multiple sclerosis (MS). Dysbiosis has been demonstrated in MS patients and its restoration by disease-modifying treatments (DMTs) is hypothesized. We aimed to study the changes in gut microbiota composition during the first 6 months of treatment with dimethyl fumarate (DMF), an oral DMT, and to identify the microorganisms associated with DMF side effects. We collected and analyzed the gut microbiota of 19 MS patients at baseline and after 1, 3, and 6 months of DMF treatment. We then cross-sectionally compared gut microbiota composition according to the presence of gastrointestinal (GI) symptoms and flushing. Overall, the gut microbiota biodiversity showed no changes over the 6-month follow-up. At the genus level, DMF was associated with decreased Clostridium abundance after 6 months. In subjects reporting side effects, a higher abundance of Streptococcus, Haemophilus, Clostridium, Lachnospira, Blautia, Subdoligranulum, and Tenericutes and lower of Bacteroidetes, Barnesiella, Odoribacter, Akkermansia, and some Proteobacteria families were detected. Our results suggest that gut microbiota may be involved in therapeutic action and side effects of DMF, representing a potential target for improving disease course and DMT tolerability.

Specific antibodies reacting to JC polyomavirus capsid protein mimotopes in sera from multiple sclerosis and other neurological diseases-affected patients.

Published data support the hypothesis that viruses could be trigger agents of multiple sclerosis onset. This link is based on evidence of early exposure to viral agents in patients affected by this neurologic disease. JC (JC polyomavirus [JCPyV]), BK (BKPyV), and simian virus 40 (SV40) neurotropic polyomavirus footprints have been detected in brain tissue specimens and samples from patients affected by different neurological diseases. In this investigation, serum samples from patients affected by multiple sclerosis and other inflammatory and noninflammatory neurologic diseases, as well as healthy subjects representing the control, were investigated for immunoglobulin G (IgG) antibodies against JCPyV. To this end, an immunologic approach was employed, which consists of employing indirect enzyme-linked immunosorbent assay testing with synthetic peptides mimicking viral capsid protein 1 antigens. A significantly lower prevalence of IgG antibodies against JCPyV VP1 epitopes, with a low titer, was detected in serum samples from patients with multiple sclerosis (MS) and other neurologic diseases than in healthy subjects. Our study indicates that the prevalence of JCPyV antibodies from patients with multiple sclerosis is 50% lower than in healthy subjects, suggesting specific immune impairments. These results indicate that patients affected by neurological diseases, including MS, respond poorly to JCPyV VP1 antigens, suggesting specific immunologic dysfunctions.

Sexual dimorphism in the cerebrospinal fluid total protein content.

Objectives Cerebrospinal fluid (CSF) is a clear, colorless body fluid filling the central nervous system. The determination of the CSF total protein (TP) content represents an important screening test of various pathologies. We aimed to address the effect of sex and age on CSF-TP content and the use of the current upper reference limits (URLs). Methods CSF-TP content was analysed in a selected population of 1,252 patients (648 women and 604 men; age 18-89 years) who underwent lumbar puncture as a part of the diagnostic work-up. Samples presenting (i) more than 5 white blood cells (WBC)/µL, (ii) discolorations and (iii) reduced glucose were not included. Results The CSF-TP content median values were significantly higher in men than in women (46 vs. 37 mg/dL) even after adjusting for age and different hospital inpatients. CSF-TP content positively correlated with age both in men and in women with a constant difference between sexes of 8.5 mg/dL. Applying the most used URLs (mainly 45 and 50 mg/dL, but also 60 mg/dL), men received a laboratory report suggestive of altered CSF-TP content more frequently than women. The use of age- and sex-calibrated CSF-TP URLs reduced, but not eliminated, this sex-gap. Conclusions Using the current URLs, a condition of "elevated CSF-TP content" may be overestimated in men or, conversely, underestimated in women, regardless of the age and of the diagnosis. These results highlighted the need to apply CSF-TP URLs values ​​normalized for both sex and age.

Correlation between auto/mitophagic processes and magnetic resonance imaging activity in multiple sclerosis patients.

BACKGROUND: An alteration of autophagy and mitophagy, two highly conserved lysosome-dependent degradation pathways involved in the maintenance of cellular homeostasis, has been associated with multiple sclerosis (MS). OBJECTIVE: To search the level of autophagy-related 5 (ATG5) and Parkin proteins, as markers of autophagy and mitophagy respectively, and lactate in a cohort of MS patients. METHODS: Cerebrospinal fluid (CSF) and serum samples from 60 MS patients were analyzed: 30 with magnetic resonance imaging (MRI) evidence of disease activity, gadolinium (Gd)-based contrast agent positive (Gd+), and 30 without MRI evidence of disease activity (Gd-). ATG5, Parkin, and lactate were measured using commercially available products. RESULTS AND CONCLUSIONS: Serum levels of ATG5, Parkin, and lactate were more elevated in Gd+ than in Gd- MS patients (p < 0.0001), and CSF concentrations of ATG5 and Parkin were greater in Gd+ than in Gd- MS (p < 0.0001). Our results demonstrated that molecular markers of autophagy and mitophagy are increased in CSF of MS patients during the active phases of the disease and that these catabolic markers, together with lactate, are also remarkably augmented in blood suggesting a role of these processes in MS pathogenesis and the possible use of these molecules as biomarkers of disease activity.

Incidence study of Guillain-Barré syndrome in the province of Ferrara, Northern Italy, between 2003 and 2017. A 40-year follow-up.

BACKGROUND: Guillain-Barré syndrome (GBS) is an acute/subacute autoimmune inflammatory polyradiculoneuropathy. Previous epidemiological studies carried out in the province of Ferrara, Italy, from 1981 to 2002 indicated that GBS incidence had tendency of increase in the period considered. OBJECTIVES: We aimed at updating the epidemiology of GBS in the years 2003-2017 and carrying on the work started in the 1980s. METHODS: We conducted an incidence study, by adopting a complete enumeration approach. Cases were identified from administrative, medical records, and database of the Ferrara Hospital and other provincial structures of the study area. Case ascertainment and definition are analogous to those adopted in previous surveys. RESULTS: In the period 1 January 2003 to 31 December 2017, 73 patients living in the province of Ferrara (mean population 353,142) were found to be new cases of GBS fulfilling the NINCDS criteria. Male/female ratio 1.15. The mean incidence rate was 1.38 per 100,000 (95% CI 1.08-1.74), 1.54 per 100,000 for men and 1.23 per 100,000 for women, a nonsignificant difference. During the period considered, the rates had slow increase or mild decrease, without nonsignificant difference. The highest rates were observed for the age groups 70-79 years for both sexes. A half of patients reported infectious events in the weeks before the onset of symptoms. CONCLUSION: In line with many epidemiological data, in the whole period 2003-2017, we observed a trend towards increase or decrease in incidence and periods of relative stability. Similar temporal heterogeneity with the comparison to our previous works was found.

Multiple Sclerosis in Italy: A 40-Year Follow-Up of the Prevalence in Ferrara.

BACKGROUND: To assess a longitudinal follow-up of the prevalence of multiple sclerosis (MS) through 4 decades in the province of Ferrara, northern Italy, and reappraise the current rates on December 31, 2016. METHODS: We conducted a community-based intensive prevalence study, by adopting a complete enumeration approach. MS cases were identified from administrative health data and medical records from the Units of Neurology and Motor Rehabilitation, Ferrara University Hospital, from other provincial neurological structures and from archives of the National Pension Institute and National Health Insurance scheme of the study area. Case ascertainment method and case definition are analogous to those adopted in previous surveys in the same area of study. RESULTS: On December 31, 2016, 685 patients (478 women and 207 men) affected by definite or probable MS (Poser's criteria) were living in the province of Ferrara (population 386,896), yielding a crude prevalence ratio of 194.91 (95% CI 180.4-209.6) per 100,000, 260.8 (95% CI 238.10-285.82) for women and 123.1 (95% CI 106.98-141.21) for men The prevalence ratio was 26.9 per 100,000 in 1978, increased to a value of 46.1 per 100,000 in 1981, 69.4 per 100,000 in 1993, 120.9 per 100,000 in 2004. Female to male ratio was 2.31 (1.2 on December 31, 1978). The mean duration of the disease at prevalence day was 17.5 ± 11.9 years (13.9 ± 10.8 years in 1978). The mean age at prevalence day was 52.04 ± 10.8 years (13.8 ± 10.8 years in 1978). CONCLUSION: Our study has confirmed the province of Ferrara is an area at high risk for MS, in line with epidemiological data from the regions of continental and insular Italy. The sharp increase in MS prevalence over time in this population can be imputed in part to a greater exposition to risk factors in genetically susceptible subjects but also to an increased survival and improved ascertainment. So, the results suggest that both methodologic and environmental factors are essential in determining the real distribution of MS. The need to get reliable estimates of MS prevalence must be highlighted as a public health and research priority, essential to support planning and prioritization of care services and to reduce the overall burden of chronic disease.

Cerebrospinal fluid analysis and the determination of oligoclonal bands.

This document presents the guidelines for the cerebrospinal fluid (CSF) analysis and the determination of oligoclonal bands (OCBs) as pivotal tests in neuroinflammatory 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.

Serum IgG against Simian Virus 40 antigens are hampered by high levels of sHLA-G in patients affected by inflammatory neurological diseases, as multiple sclerosis.

BACKGROUND: Many investigators detected the simian polyomavirus SV40 footprints in human brain tumors and neurologic diseases and recently it has been indicated that SV40 seems to be associated with multiple sclerosis (MS) disease. Interestingly, SV40 interacts with human leukocyte antigen (HLA) class I molecules for cell entry. HLA class I antigens, in particular non-classical HLA-G molecules, characterized by an immune-regulatory function, are involved in MS disease, and the levels of these molecules are modified according with the disease status. OBJECTIVE: We investigated in serum samples, from Italian patients affected by MS, other inflammatory diseases (OIND), non-inflammatory neurological diseases (NIND) and healthy subjects (HS), SV40-antibody and soluble sHLA-G and the association between SV40-prevalence and sHLA-G levels. METHODS: ELISA tests were used for SV40-antibodies detection and sHLA-G quantitation in serum samples. RESULTS: The presence of SV40 antibodies was observed in 6 % of patients affected by MS (N = 4/63), 10 % of OIND (N = 8/77) and 15 % of NIND (N = 9/59), which is suggestive of a lower prevalence in respect to HS (22 %, N = 18/83). MS patients are characterized by higher sHLA-G serum levels (13.9 ± 0.9 ng/ml; mean ± St. Error) in comparison with OIND (6.7 ± 0.8 ng/ml), NIND (2.9 ± 0.4 ng/ml) and HS (2.6 ± 0.7 ng/ml) subjects. Interestingly, we observed an inverse correlation between SV40 antibody prevalence and sHLA-G serum levels in MS patients. CONCLUSION: The data obtained showed a low prevalence of SV40 antibodies in MS patients. These results seems to be due to a generalized status of inability to counteract SV40 infection via antibody production. In particular, we hypothesize that SV40 immune-inhibitory direct effect and the presence of high levels of the immune-inhibitory HLA-G molecules could co-operate in impairing B lymphocyte activation towards SV40 specific peptides.

Cerebrospinal fluid amounts of HLA-G in dimeric form are strongly associated to patients with MRI inactive multiple sclerosis.

BACKGROUND: The relevance of human leukocyte antigen (HLA)-G in dimeric form in multiple sclerosis (MS) is still unknown. OBJECTIVE: To investigate the contribution of cerebrospinal fluid (CSF) HLA-G dimers in MS pathogenesis. METHODS: CSF amounts of 78-kDa HLA-G dimers were measured by western blot analysis in 80 MS relapsing-remitting MS (RRMS) patients and in 81 inflammatory and 70 non-inflammatory controls. RESULTS: CSF amounts of 78 kDa HLA-G dimers were more frequent in RRMS than in inflammatory (p<0.01) and non-inflammatory controls (p<0.001) and in magnetic resonance imaging (MRI) inactive than in MRI active RRMS (p<0.00001). CONCLUSION: Our findings suggest that HLA-G dimers may be implicated in termination of inflammatory response occurring in MS.

TIMP-1 resistant matrix metalloproteinase-9 is the predominant serum active isoform associated with MRI activity in patients with multiple sclerosis.

BACKGROUND: The activity of matrix metalloproteinase-9 (MMP-9) depends on two isoforms, an 82 kDa active MMP-9 modulated by its specific tissue inhibitor (TIMP-1), and a 65 kDa TIMP-1 resistant active MMP-9. The relevance of these two enzymatic isoforms in multiple sclerosis (MS) is still unknown. OBJECTIVE: To investigate the contribution of the TIMP-1 modulated and resistant active MMP-9 isoforms to MS pathogenesis. METHODS: We measured the serum levels of the 82 kDa and TIMP-1 resistant active MMP-9 isoforms by activity assay systems in 86 relapsing-remitting MS (RRMS) patients, categorized according to clinical and magnetic resonance imaging (MRI) evidence of disease activity, and in 70 inflammatory (OIND) and 69 non-inflammatory (NIND) controls. RESULTS: Serum levels of TIMP-1 resistant MMP-9 were more elevated in MS patients than in OIND and NIND (p < 0.05, p < 0.02, respectively). Conversely, 82 kDa active MMP-9 was higher in NIND than in the OIND and MS patients (p < 0.01 and p < 0.00001, respectively). MRI-active patients had higher levels of TIMP-1 resistant MMP-9 and 82 kDa active MMP-9, than did those with MRI inactive MS (p < 0.01 and p < 0.05, respectively). CONCLUSION: Our findings suggested that the TIMP-1 resistant MMP-9 seem to be the predominantly active isoform contributing to MS disease activity.

Epstein-Barr virus-specific intrathecal oligoclonal IgG production in relapsing-remitting multiple sclerosis is limited to a subset of patients and is composed of low-affinity antibodies.

BACKGROUND: The purpose of this study was to investigate intrathecal production and affinity distributions of Epstein-Barr virus (EBV)-specific antibodies in multiple sclerosis (MS) and controls. METHODS: Cerebrospinal fluid (CSF) and serum concentrations, quantitative intrathecal synthesis, oligoclonal bands (OCB) patterns and affinity distributions of anti-Epstein Barr virus (EBV) antibodies were evaluated in 100 relapsing-remitting MS (RRMS) patients and 200 age- and sex-matched controls with other inflammatory neurological disorders (OIND) and other noninflammatory neurological disorders (NIND). RESULTS: Levels of anti-EBNA-1 and anti-viral capsid antigen (VCA) IgG were different in both the CSF (P <0.0001 and P <0.01, respectively) and serum (P <0.001 and P <0.05, respectively) among the RRMS, OIND and NIND. An intrathecal synthesis of anti-EBNA-1 IgG and anti-VCA IgG, as indicated by the antibody index, was underrepresented in the RRMS, OIND and NIND (range 1 to 7%). EBV-specific OCB were detected in 24% of the RRMS patients and absent in the controls. High-affinity antibodies were more elevated in the RRMS and in the OIND than in the NIND for CSF anti-EBNA-1 IgG (P <0.0001) and anti-VCA IgG (P <0.0001). After treatment with increasing concentrations of sodium thiocyanate, the EBV-specific IgG OCB had low affinity in all 24 RRMS patients analyzed. CONCLUSIONS: Our findings do not support the potential role of an EBV persistent brain chronic infection in MS and suggest that an EBV-specific intrathecal oligoclonal IgG production can occur in a subset of MS patients as part of humoral polyreactivity driven by chronic brain inflammation.

Cerebrospinal Fluid Analysis in Rheumatological Diseases with Neuropsychiatric Complications and Manifestations: A Narrative Review.

The analysis of cerebrospinal fluid (CSF) remains a valuable diagnostic tool in the evaluation of inflammatory and infectious conditions involving the brain, spinal cord, and meninges. Since many rheumatic inflammatory diseases can involve the central and peripheral nervous system, the aims of this narrative review were to summarize the latest evidence on the use of CSF analysis in the field of neuropsychiatric manifestations of rheumatic diseases. Routine CSF parameters were taken into consideration for this review: appearance; total protein and cellular content (pleocytosis); lactate and/or glucose; CSF/serum albumin quotient; intrathecal synthesis of IgG. Data regarding the role of CSF analysis in the clinical management of neuropsychiatric systemic lupus erythematosus, primary Sjogren's syndrome, rheumatoid arthritis, and Behçet's syndrome are presented. Although no disease-specific picture has been identified, CSF analysis remains a useful diagnostic tool to confirm the presence of a neuro-inflammatory state or, conversely, to exclude the concomitant presence of other inflammatory/infectious diseases affecting the CNS in the context of systemic rheumatologic conditions.

Breaking Barriers: Unveiling Sex-Related Differences in Cerebrospinal Fluid Analysis-A Narrative Review.

(1) Background: The recent emphasis on sexual and gender diversity's impact on human health underscores the need for tailored diagnostic and therapeutic approaches in neurology. The aim of this article is to conduct a narrative review of the available scientific literature on sex differences in cerebrospinal fluid analysis. (2) Methods: The literature search encompassed PubMed databases, focusing on cerebrospinal fluid analysis and sex differences, considering parameters like cerebrospinal fluid protein content, cell count, albumin quotient (QAlb) and intrathecal IgG synthesis. (3) Results: Nine articles from the past two decades were identified, revealing limited research in this area. Males consistently exhibited higher cerebrospinal fluid protein content and albumin quotient values across various pathologies and age groups. Consequently, males more frequently manifested blood-cerebrospinal fluid barrier dysfunction than females. No significant sex differences were observed in cerebrospinal fluid leukocyte count or intrathecal IgG synthesis. (4) Conclusions: This review highlights the dearth of research on sex differences in cerebrospinal fluid analysis, despite consistent findings of higher protein content and albumin quotient values in males. Revisiting current diagnostic thresholds based on sex is crucial for accurate prognosis and personalised treatment strategies in neurological disorders. Moving towards sex-specific approaches in clinical practice is imperative for advancing personalised medicine.

Sex Differences in Albumin Quotient and Cerebrospinal Fluid Total Protein Content Do Not Depend on Anthropometric Factors.

(1) Background: Cerebrospinal fluid (CSF)/serum albumin quotient (QAlb) and CSF total protein (TP) are more elevated in males than females, and this has been hypothesised to be due to anthropometric differences between the sexes. This study aimed to investigate QAlb and CSF TP as a function of body height, weight, and body mass index (BMI). (2) Methods: A total of 207 patients were included in the study and analysed blinded to clinical diagnosis. (3) Results: Multivariable linear regressions were run to predict log-transformed Qalb and log-transformed CSF TP value from age, sex, weight, and height (first model) or from age, sex, and BMI (second model). In both models, age (ß = 0.004, 95% CI = 0.002 to 0.006) and sex (ß = -0.095, 95% CI = -0.169 to -0.021, and ß = -0.135, 95% CI = -0.191 to -0.079) were significant predictors for QAlb, but weight, height, and BMI were not. Similarly, age (ß = 0.004, 95% CI = 0.003 to 0.006) and sex (ß = -0.077, 95% CI = -0.142 to -0.013, and ß = -0.109, 95% CI = -0.157 to -0.060) were significant predictors for CSF TP, while anthropometric characteristics were not. No differences in QAlb and CSF TP were found when grouping males and females by BMI status. (4) Conclusions: Our data suggest that anthropometric characteristics could not explain the sex-related differences in QAlb and CSF TP.

Matrix metalloproteinase-2 (MMP-2) generates soluble HLA-G1 by cell surface proteolytic shedding.

Human leukocyte antigen-G (HLA-G) molecules are non-classical HLA class I antigens with an important role in pregnancy immune regulation and inflammation control. Soluble HLA-G proteins can be generated through two mechanisms: alternative splicing and proteolytic release, which is known to be metalloprotease mediated. Among this class of enzymes, matrix metalloproteinases (MMPs) might be involved in the HLA-G1 membrane cleavage. Of particular interest are MMP-2 and MMP-9, which regulate the inflammatory process by cytokine and chemokine modulation. We evaluated the effect of MMP-9 and MMP-2 on HLA-G1 membrane shedding. In particular, we analyzed the in vitro effect of these two gelatinases on the secretion of HLA-G1 via proteolytic cleavage in 221-G1-transfected cell line, in JEG3 cell line, and in peripheral blood mononuclear cells. The results obtained by both cell lines showed the role of MMP-2 in HLA-G1 shedding. On the contrary, MMP-9 was not involved in this process. In addition, we identified three possible highly specific cleavage sites for MMP-2, whereas none were detected for MMP-9. This study suggests an effective link between MMP-2 and HLA-G1 shedding, increasing our knowledge on the regulatory machinery beyond HLA-G regulation in physiological and pathological conditions.

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.