G protein-coupled receptor 17 is regulated by WNT pathway during oligodendrocyte precursor cell differentiation.

G protein-coupled receptor 17 (GPR17) and the WNT pathway are critical players of oligodendrocyte (OL) differentiation acting as essential timers in developing brain to achieve fully-myelinating cells. However, whether and how these two systems are related to each other is still unknown. Of interest, both factors are dysregulated in developing and adult brain diseases, including white matter injury and cancer, making the understanding of their reciprocal interactions of potential importance for identifying new targets and strategies for myelin repair. Here, by a combined pharmacological and biotechnological approach, we examined regulatory mechanisms linking WNT signaling to GPR17 expression in OLs. We first analyzed the relative expression of mRNAs encoding for GPR17 and the T cell factor/Lymphoid enhancer-binding factor-1 (TCF/LEF) transcription factors of the canonical WNT/ß-CATENIN pathway, in PDGFRα+ and O4+ OLs during mouse post-natal development. In O4+ cells, Gpr17 mRNA level peaked at post-natal day 14 and then decreased concomitantly to the physiological uprise of WNT tone, as shown by increased Lef1 mRNA level. The link between WNT signaling and GPR17 expression was further reinforced in vitro in primary PDGFRα+ cells and in Oli-neu cells. High WNT tone impaired OL differentiation and drastically reduced GPR17 mRNA and protein levels. In Oli-neu cells, WNT/ß-CATENIN activation repressed Gpr17 promoter activity through both putative WNT response elements (WRE) and upregulation of the inhibitor of DNA-binding protein 2 (Id2). We conclude that the WNT pathway influences OL maturation by repressing GPR17, which could have implications in pathologies characterized by dysregulations of the OL lineage including multiple sclerosis and oligodendroglioma.

Quantitative Multiplexed Imaging Analysis Reveals a Strong Association between Immunogen-Specific B Cell Responses and Tonsillar Germinal Center Immune Dynamics in Children after Influenza Vaccination.

Generation of Ag-specific humoral responses requires the orchestrated development and function of highly specialized immune cells in secondary lymphoid organs. We used a multiparametric approach combining flow cytometry, confocal microscopy, and histocytometry to analyze, for the first time to our knowledge in children, tonsils from seasonal influenza-vaccinated children. We used these novel imaging assays to address the mucosal immune dynamics in tonsils investigating the spatial positioning, frequency, and phenotype of immune cells after vaccination. Vaccination was associated with a significantly higher frequency of follicular helper CD4 T cells compared with the unvaccinated control group. The imaging analysis revealed that potential suppressor (FOXP3hi) CD4 T cells are mainly located in extrafollicular areas. Furthermore, a significantly reduced frequency of both follicular and extrafollicular FOXP3hi CD4 T cells was found in the vaccine group compared with the control group. Levels of circulating CXCL13 were higher in those vaccinated compared with controls, mirroring an increased germinal center reactivity in the tonsils. Notably, a strong correlation was found between the frequency of tonsillar T follicular helper cells and tonsillar Ag-specific Ab-secreting cells. These data demonstrate that influenza vaccination promotes the prevalence of relevant immune cells in tonsillar follicles and support the use of tonsils as lymphoid sites for the study of germinal center reactions after vaccination in children.

T helper 9 cells induced by plasmacytoid dendritic cells regulate interleukin-17 in multiple sclerosis.

Multiple sclerosis (MS) is a chronic disease of the central nervous system (CNS) characterized by persistent inflammation orchestrated by cluster of differentiation (CD) 4 T helper (Th) cells. In particular, Th1 and Th17 cells amplify, whereas T regulatory (Treg) cells moderate inflammation. The role of other Th subsets in MS is not clear. In the present study, we investigated the generation of different Th responses by human dendritic cells (DCs) in MS. We compared the production of several Th cytokines by naive CD4+ T-cells polarized with myeloid and plasmacytoid DCs (mDCs and pDCs) in healthy donors (HD) and relapsing-remitting (RR)-MS patients. We found that resiquimod-stimulated mDCs were able to activate Th17 differentiation, whereas pDCs induced interleukin (IL)-10-producing Th cells. Surprisingly, resiquimod-stimulated pDCs from MS patients also significantly induced the differentiation of Th9 cells, which produce IL-9 and are known to be involved in allergic diseases. We investigated the potential role of IL-9 in MS. We found that IL-9 activated signal transducer and activator of transcription (STAT) 1 and STAT5 phosphorylation and interfered with IL-17 and interferon (IFN) regulatory transcription factor (IRF)-4 expression in Th17-polarized cells. Moreover, in the cerebrospinal fluid (CSF) of 107 RR-MS patients, IL-9 inversely correlated with indexes of inflammatory activity, neurodegeneration and disability progression of MS. High levels of IL-9 were associated with the absence of IL-17 in the CSF of RR-MS patients. Our results demonstrate a Th9-inducing potential of pDCs in MS, suggesting an immunoregulatory role leading to attenuation of the exaggerated Th17 inflammatory response.

Cerebrospinal fluid detection of interleukin-1ß in phase of remission predicts disease progression in multiple sclerosis.

BACKGROUND: Absence of clinical and radiological activity in relapsing-remitting multiple sclerosis (RRMS) is perceived as disease remission. We explored the role of persisting inflammation during remission in disease evolution. METHODS: Cerebrospinal fluid (CSF) levels of interleukin 1ß (IL-1ß), a major proinflammatory cytokine, were measured in 170 RRMS patients at the time of clinical and radiological remission. These patients were then followed up for at least 4 years, and clinical, magnetic resonance imaging (MRI) and optical coherence tomography (OCT) measures of disease progression were recorded. RESULTS: Median follow-up of RRMS patients was 5 years. Detection of CSF IL-1ß levels at the time of remission did not predict earlier relapse or new MRI lesion formation. Detection of IL-1ß in the CSF was instead associated with higher progression index (PI) and Multiple Sclerosis Severity Scale (MSSS) scores at follow-up, and the number of patients with sustained Expanded Disability Status Scale (EDSS) or Multiple Sclerosis Functional Composite worsening at follow-up was higher in individuals with detectable levels of IL-1ß. Patients with undetectable IL-1ß in the CSF had significantly lower PI and MSSS scores and a higher probability of having a benign MS phenotype. Furthermore, patients with undetectable CSF levels of IL-1ß had less retinal nerve fiber layer thickness and macular volume alterations visualized by OCT compared to patients with detectable IL-1ß. CONCLUSIONS: Our results suggest that persistence of a proinflammatory environment in RRMS patients during clinical and radiological remission influences midterm disease progression. Detection of IL-1ß in the CSF at the time of remission appears to be a potential negative prognostic factor in RRMS patients.

The impact of National Containment Measures on a Pediatric Italian regional Hub for COVID-19, an observational study.

BACKGROUND: Numerous studies described the epidemiological link and main clinical features of pediatric COVID-19, during the first pandemic period. Our study encompasses several different phases since the National Lockdown in Italy. The primary outcome is (I) to analyze the prevalence of positive NST (Nasopharyngeal Swab Test) among the largest Italian Pediatric cohort admitted to a single regional PED Hub for COVID-19 during an eight-month period. Secondary outcomes are: (II) the description of trend of admissions in our PED and (III) the categorization of the positive patients according to clinical manifestations and epidemiological link. METHODS: We described 316 patients with a positive NST for SARS-CoV2, on a total of 5001 nasopharyngeal swabs performed among 13,171 admissions at our PED, over a period starting from March 17th, 2020 to December 1st, 2020. Age, epidemiological link, clinical features and hospitalizations were analyzed according to different lockdown phases. Data were collected anonymously from electronic records and analyzed using SPSS 22.00 statistics software (Chicago, IL). RESULTS: Thirty-six percent of total admissions have been tested. During the post lockdown period, we performed the highest percentage of NST (Nasopharyngeal Swab Test) 49.7%, and among them 7.9% were positive. The prevalence of infection during a 10-month period was 2.3%. Mean age was 6.5 years old. Familial Link accounted for the 67.7% of infection, while Extrafamilial and Unknown link accounted for 17 and 14.9%, respectively. Familial link is predominant during all phases. Seventeen patients showed an intra-scholastic link, and the highest prevalence was observed in the 7-10 years age group, with a prevalence of 12.8% (5 patients). Fever was the most frequent symptom (66%), in particular among preschooler children aged 0-6 years (71.9%). Older children were more frequently symptomatic. Seven patients were admitted with MIS-C diagnosis. CONCLUSIONS: Different levels of containment measures caused important changes in number of positive NST for SARS-CoV2. Familial link was predominant in our cohort, during all phases of Lockdown. The risk of being infected at home is four time greater than the risk of being infected from an extra familial individual. Further studies are needed to evaluate the clear impact of intra-scholastic link. The constant improvement in knowledge on onset symptoms and risk factor for SARS-CoV2 infection and its complications (e.g. MIS-C), can impact on number of hospitalizations, ICU admissions and early management.

Hospitalization for acute cerebellitis in children affected by varicella: how much does it cost?

BACKGROUND: Chickenpox is a highly contagious airborne disease caused by the varicella zoster virus. It is generally benign and self-limiting, but it may be responsible of life-threatening complications. Acute cerebellitis (AC) is the most common neurological complication and is associated with prolonged hospitalization in the acute phase (HAP). AIM OF THE STUDY: To estimate the costs of AC HAP in children affected by varicella. MATERIALS AND METHODS: We retrospectively reviewed the medical records of a pediatric cohort hospitalized for chickenpox AC over a period of 15 years (from October 2003 to October 2018) and we analyzed acute care costs. For any patient the HAP has been calculated. The final value includes cost of hospital accommodation and management at the Pediatric and Infectious Diseases Unit. To this cost, the price of procedures (imaging, laboratory exams, medical and paramedical evaluations) and medical treatments was added. RESULTS: In the study period, 856 children had been hospitalized for varicella. Out of them, 65 met a diagnosis of AC and were included in the study. The hospitalization length was of 10 days (range 3-20 days). The median cost of HAP for each patient was of 5366 euro, with an average annual cost of 23,252 €. The most significant part of HAP is due to the cost of hospital accommodation and management at the Pediatric Infectious Diseases Unit, which was about € 537.78 for a single day. DISCUSSION: Although AC post-varicella is rare, its HAP cost is not negligible resulting in substantial economic burden. Vaccination would have probably prevented varicella and AC complication, avoiding hospitalization. CONCLUSIONS: Financial studies are important for evaluate the cost saving in order to influence public funding decisions. Further studies are necessary to investigate the economic burden of the disease.

Scurvy: still a threat in the well-fed first world?

We report three cases of scurvy in previously healthy children referred to us for leg pain and refusal to walk. All children had no significant medical history, symptoms had started months before and subtly advanced. Two of them presented with gingival hyperplasia and petechiae, another one reported night sweats and gingival bleeding in the past few weeks. Two had vitamin D deficiency, and all had microcytic anaemia (in one case requiring transfusional support). A nutritional screening revealed low or undetectable levels of ascorbic acid. This, along with the clinical and radiological findings, led to a diagnosis of scurvy. Vitamin C supplementation was started with rapid improvement of the children's clinical condition. Scurvy is a rare disease in the 'first world', but there are anecdotal reports of scurvy in children without any of the known risk factors for this condition. In our cases, a selective diet was the only risk factor.

Thymic Epithelium Abnormalities in DiGeorge and Down Syndrome Patients Contribute to Dysregulation in T Cell Development.

The thymus plays a fundamental role in establishing and maintaining central and peripheral tolerance and defects in thymic architecture or AIRE expression result in the development of autoreactive lymphocytes. Patients with partial DiGeorge Syndrome (pDGS) and Down Syndrome (DS) present alterations in size and architecture of the thymus and higher risk to develop autoimmunity. We sought to evaluate thymic architecture and thymocyte development in DGS and DS patients and to determine the extent to which thymic defects result in immune dysregulation and T cell homeostasis perturbation in these patients. Thymi from pediatric patients and age-matched controls were obtained to evaluate cortex and medullary compartments, AIRE expression and thymocyte development. In the same patients we also characterized immunophenotype of peripheral T cells. Phenotypic and functional characterization of thymic and peripheral regulatory T (Treg) cells was finally assessed. Histologic analysis revealed peculiar alterations in thymic medulla size and maturation in DGS and DS patients. Perturbed distribution of thymocytes and altered thymic output was also observed. DGS patients showed lower mature CD4+ and CD8+ T cell frequency, associated with reduced proportion and function of Tregs both in thymus and peripheral blood. DS patients showed increased frequency of single positive (SP) thymocytes and thymic Treg cells. However, Tregs isolated both from thymus and peripheral blood of DS patients showed reduced suppressive ability. Our results provide novel insights on thymic defects associated with DGS and DS and their impact on peripheral immune dysregulation. Indeed, thymic abnormalities and defect in thymocyte development, in particular in Treg cell number and function could contribute in the pathogenesis of the immunodysregulation present in pDGS and in DS patients.

Laboratory Biomarkers to Facilitate Differential Diagnosis between Measles and Kawasaki Disease in a Pediatric Emergency Room: A Retrospective Study.

This retrospective study was conducted to analyze clinical and laboratoristic parameters to individuate specific differences and facilitate differential diagnosis between Measles and Kawasaki Disease (KD) at first evaluation in an emergency room. We found similar clinical features as duration of fever and number of KD criteria (p > 0.5) but significant differences in white blood cell count, neutrophils, CRP and LDH levels (p < 0.001). LDH value ≥ 800 mg/dl had sensibility of 89% and specificity of 90% for Measles while CRP ≥ 3 mg/dl had sensibility 89% and specificity of 85% for KD. The combined use of CRP, LDH and AST showed accuracy of 86.67%.

Neuroinflammation drives anxiety and depression in relapsing-remitting multiple sclerosis.

OBJECTIVE: To explore the inflammatory processes in the pathogenesis of psychiatric symptoms and the prognostic value of psychiatric comorbidities in multiple sclerosis (MS). METHODS: Four hundred five patients with relapsing-remitting (RR) MS underwent psychiatric evaluation by means of Beck Depression Inventory II (BDI-II) and State/Trait Anxiety Inventory (STAI-Y). The inflammatory activity level was assessed by MRI. In a subset of 111 treatment-naive patients, CSF levels of proinflammatory cytokines were determined. Correlation and regression analyses were performed to determine associations between variables. RESULTS: Relapsing patients demonstrated greater values of STAI-state and BDI-II compared with remitting patients but comparable trait-anxiety scores. There were no significant differences in psychometric parameters between relapsing and asymptomatic MRI-active patients, highlighting the effect of subclinical inflammation on mood disturbances. A significant reduction of STAI-state and BDI-II scores was recorded, along with the subsiding of neuroinflammation. Interleukin-2 CSF levels were found to correlate with STAI-state, while tumor necrosis factor-α and interleukin-1ß correlated with BDI-II. Because emotional disorders were associated with subclinical inflammation, variations of the psychometric profile were able to detect subclinical reactivation earlier. In line with this, high STAI-state values considerably predicted the possibility of disease reactivation. CONCLUSIONS: Mood alterations are induced by intrathecal inflammation, even though not clinically apparent, and are able to predict inflammatory reactivations in RRMS. Inflammation is therefore a biological event, not less important than the traditional psychosocial factors, involved in mood disorders.

Waning of vaccine-induced immunity to measles in kidney transplanted children.

Vaccine-preventable diseases are a significant cause of morbidity and mortality in solid organ transplant recipients who undergo immunosuppression after transplantation. Data on immune responses and long-term maintenance after vaccinations in such population are still limited.We cross-sectionally evaluated the maintenance of immune response to measles vaccine in kidney transplanted children on immunosuppressive therapy. Measles-specific enzyme-linked immunosorbent assay and B-cell enzyme-linked immunosorbent spot were performed in 74 kidney transplant patients (Tps) and in 23 healthy controls (HCs) previously vaccinated and tested for humoral protection against measles. The quality of measles antibody response was measured by avidity test. B-cell phenotype, investigated via flow cytometry, was further correlated to the ability of Tps to maintain protective humoral responses to measles over time.We observed the loss of vaccine-induced immunity against measles in 19% of Tps. Nonseroprotected children showed signs of impaired B-cell distribution as well as immune senescence and lower antibody avidity. We further reported as time elapsed between vaccination and transplantation, as well as the vaccine administration during dialysis are clinical factors affecting the maintenance of the immune memory response against measles.Tps present both quantitative and qualitative alterations in the maintenance of protective immunity to measles vaccine. Prospective studies are needed to optimize the vaccination schedules in kidney transplant recipients in order to increase the immunization coverage over time in this population.

Interleukin-1ß causes excitotoxic neurodegeneration and multiple sclerosis disease progression by activating the apoptotic protein p53.

BACKGROUND: Understanding how inflammation causes neuronal damage is of paramount importance in multiple sclerosis (MS) and in other neurodegenerative diseases. Here we addressed the role of the apoptotic cascade in the synaptic abnormalities and neuronal loss caused by the proinflammatory cytokines interleukin-1ß (IL-1ß) and tumor necrosis factor (TNF-α) in brain tissues, and disease progression caused by inflammation in relapsing-remitting MS (RRMS) patients. RESULTS: The effect of IL-1ß, but not of TNF-α, on glutamate-mediated excitatory postsynaptic currents was blocked by pifithrin-α (PFT), inhibitor of p53. The protein kinase C (PKC)/transient receptor potential vanilloid 1 (TRPV1) pathway was involved in IL-1ß-p53 interaction at glutamatergic synapses, as pharmacological modulation of this inflammation-relevant molecular pathway affected PFT effects on the synaptic action of IL-1ß. IL-1ß-induced neuronal swelling was also blocked by PFT, and IL-1ß increased the expression of p21, a canonical downstream target of activated p53.Consistent with these in vitro results, the Pro/Pro genotype of p53, associated with low efficiency of transcription of p53-regulated genes, abrogated the association between IL-1ß cerebrospinal fluid (CSF) levels and disability progression in RRMS patients. The interaction between p53 and CSF IL-1ß was also evaluated at the optical coherence tomography (OCT), showing that IL-1ß-driven neurodegenerative damage, causing alterations of macular volume and of retinal nerve fibre layer thickness, was modulated by the p53 genotype. CONCLUSIONS: Inflammatory synaptopathy and neurodegeneration caused by IL-1ß in RRMS patients involve the apoptotic cascade. Targeting IL-1ß-p53 interaction might result in significant neuroprotection in MS.

Treatment options to reduce disease activity after natalizumab: paradoxical effects of corticosteroids.

AIM: Natalizumab (NTZ) discontinuation leads to multiple sclerosis (MS) recurrence, but represents the only known strategy to limit the risk of progressive multifocal leukoencephalopathy (PML) in JCV seropositive patients. Here, we compared the clinical and imaging features of three groups of patients who discontinued NTZ treatment. METHODS: We treated 25 patients with subcutaneous INFß-1b (INF group), 40 patients with glatiramer acetate (GA group), and 40 patients with GA plus pulse steroid (GA+CS group). RESULTS: Six of 25 patients (24%) of the INF group were relapse-free 6 months after NTZ suspension. In GA group, a significant higher proportion of patients (26 of 40 patients, 65%) were relapse-free (P<0.05). Far from improving the clinical effects of GA in post-NTZ setting, combination of GA+CS was associated with lower relapse-free rate than GA alone (40% vs. 65%, P=0.04). Also on MRI parameters, combination of GA+CS was associated with worse outcome than GA alone, as 22 of 26 subjects (84.6%) had MRI evidence of disease activity 6 months after NTZ discontinuation. CONCLUSION: Corticosteroids should not be used in combination with GA to prevent post-NTZ disease recurrence.