Beneficial Effect of Phenytoin and Carbamazepine on GFAP Gene Expression and Mutant GFAP Folding in a Cellular Model of Alexander's Disease

Alexander's disease (AxD) is a rare, usually relentlessly progressive disorder of astroglial cells in the central nervous system related to mutations in the gene encoding the type III intermediate filament protein, glial fibrillary acidic protein (GFAP). The pathophysiology of AxD is only partially understood. Available data indicate that an excessive GFAP gene expression may play a role. In particular, a "threshold hypothesis" has been reported, suggesting that mutant GFAP representing about 20% of the total cellular GFAP should be sufficient to cause disease. Thus, strategies based on reducing cellular mutant GFAP protein levels and/or activating biological processes involved in the correct protein folding could be effective in counteracting the toxic effect of misfolded GFAP. Considering that clomipramine (CLM), which has been selected by a wide small molecules screening as the greatest inhibitory potential drug against GFAP expression, is contraindicated because of its proconvulsant activity in the infantile form of AxD, which is also characterized by the occurrence of epileptic seizures, two powerful antiepileptic agents, carbamazepine (CBZ) and phenytoin (PHT), which share specific stereochemical features in common with CLM, were taken into consideration in a reliable in vitro model of AxD. In the present work, we document for the first time that CBZ and PHT have a definite inhibitory effect on pathological GFAP cellular expression and folding. Moreover, we confirm previous results of a similar beneficial effect of CLM. In addition, we have demonstrated that CBZ and CLM play a refolding effect on mutant GFAP proteins, likely ascribed at the induction of CRYAB expression, resulting in the decrease of mutant GFAP aggregates formation. As CBZ and PHT are currently approved for use in humans, their documented effects on pathological GFAP cellular expression and folding may indicate a potential therapeutic role as disease-modifying agents of these drugs in the clinical management of AxD, particularly in AxD patients with focal epilepsy with and without secondary generalization

BoHV-4-based vector delivering Ebola virus surface glycoprotein

Background: Ebola virus (EBOV) is a Category A pathogen that is a member of Filoviridae family that causes hemorrhagic fever in humans and non-human primates. Unpredictable and devastating outbreaks of disease have recently occurred in Africa and current immunoprophylaxis and therapies are limited. The main limitation of working with pathogens like EBOV is the need for costly containment. To potentiate further and wider opportunity for EBOV prophylactics and therapies development, innovative approaches are necessary. Methods: In the present study, an antigen delivery platform based on a recombinant bovine herpesvirus 4 (BoHV-4), delivering a synthetic EBOV glycoprotein (GP) gene sequence, BoHV-4-syEBOVgD106TK, was generated. Results: EBOV GP was abundantly expressed by BoHV-4-syEBOVgD106TK transduced cells without decreasing viral replication. BoHV-4-syEBOVgD106TK immunized goats produced high titers of anti-EBOV GP antibodies and conferred a long lasting (up to 6 months), detectable antibody response. Furthermore, no evidence of BoHV-4-syEBOVgD106TK viremia and secondary localization was detected in any of the immunized animals. Conclusions: The BoHV-4-based vector approach described here, represents: an alternative antigen delivery system for vaccination and a proof of principle study for anti-EBOV antibodies generation in goats for potential immunotherapy applications

Study of susceptibility factors, modification and prognosis in the development of Hirschsprung’s disease-associated enterocolitis via omics analysis of data from whole-exome sequencing studies, genome-wide association studies and proteomics.

Hirschsprung’s disease (HSCR) is a congenital gut malformation caused by a lack of innervation. One of the most serious is HSCR associated enterocolitis (HAEC), a potentially lethal condition with 30% of incidence. However, the causes of HAEC are still unknown and the onset is difficult to predict. This thesis work focuses on the study of susceptibility factors, modifications, and prognosis in the development of Hirschsprung’s (HSCR) disease-associated enterocolitis (HAEC) using different omics technologies. Genetic Investigation of HAEC by Whole-Exome Sequencing (WES) on 24 HSCR patients affected (HAEC) or not affected (HSCR-only) by enterocolitis and replication of results on a larger panel of patients allowed the identification of the HAEC susceptibility variant p.H187Q in the Oncostatin-M receptor (OSMR) gene (14.6% in HAEC and 5.1% in HSCR-only,p= 0.0024). Proteomic analysis on the lymphoblastoid cell lines from one HAEC patient homozygote for this variant and one HAEC wild-type patient revealed two well distinct clusters of proteins significantly up or downregulated upon OSM stimulation. I have then carried out a transcriptome analysis on Intraepithelial lymphocytes (IEL) derived from gut biopsies and on Peripheral Blood Monocyte Cells (PBMCs) from HAEC, HSCR-only and pediatric patients affected by neither Hirschsprung, nor inflammatory related diseases. The analysis on the IELs showed a clear clustering between the groups of patients and an enrichment in immune and inflammatory pathways in the HAEC group. The results showed an interesting role for the gene Onco- statin M (OSMR) as a potential modifier from HSCR to HAEC. Additionally, a potential genetic connection between Inflammatory Bowel Disease and HSCR using the UKBioBank was explored. The finding of a shared genetic background with other inflammatory disorders affecting the gut, and the evaluation of gene networks and pathways involved in inflammation provides further knowledge on the mechanisms of gut inflammation

Flu and Pneumococcal Vaccine Coverage in Scleroderma Patients Still Need to Be Prompted: A Systematic Review

Systemic sclerosis (scleroderma, SSc) is an autoimmune connective tissue disease characterized by excessive production of collagen and multiorgan involvement. Scleroderma patients are at increased risk of influenza complications and pneumonia; thus, vaccinations are recommended. This systematic review evaluated the influenza and pneumococcus vaccination coverage for SSc patients. We included all studies from Pubmed reporting on influenza and pneumococcal vaccination rate in Scleroderma patients up to May 2021. The 14 studies thus selected identified a suboptimal vaccination rate in autoimmune and SSc patients, ranging from 28 to 59% for the flu vaccine, and from 11 to 58% for the pneumo vaccine in absence of specific vaccination campaigns, variously considering also other variables such as age, gender, vaccination settings, and possible vaccination campaigns. We also considered the reasons for low coverage and the approaches that might increase the vaccination rates. A lack of knowledge about the importance of vaccination in these patients and their doctors underlined the need to increase the awareness for vaccination in this patients’ category. Current guidelines recommend vaccination in elderly people and people affected by particular conditions that widely overlap with SSc, yet autoimmune diseases are not always clearly mentioned. Improving this suboptimal vaccination rate with clear guidelines is crucial for SSc patients and for clinicians to immunize these categories based principally on the pathology, prior to the age. Recommendations by the immunologist and the direct link to the vaccine providers can highly improve the vaccine coverage

Flu and Pneumococcal Vaccine Coverage in Scleroderma Patients Still Need to Be Prompted: A Systematic Review

Systemic sclerosis (scleroderma, SSc) is an autoimmune connective tissue disease characterized by excessive production of collagen and multiorgan involvement. Scleroderma patients are at increased risk of influenza complications and pneumonia; thus, vaccinations are recommended. This systematic review evaluated the influenza and pneumococcus vaccination coverage for SSc patients. We included all studies from Pubmed reporting on influenza and pneumococcal vaccination rate in Scleroderma patients up to May 2021. The 14 studies thus selected identified a suboptimal vaccination rate in autoimmune and SSc patients, ranging from 28 to 59% for the flu vaccine, and from 11 to 58% for the pneumo vaccine in absence of specific vaccination campaigns, variously considering also other variables such as age, gender, vaccination settings, and possible vaccination campaigns. We also considered the reasons for low coverage and the approaches that might increase the vaccination rates. A lack of knowledge about the importance of vaccination in these patients and their doctors underlined the need to increase the awareness for vaccination in this patients’ category. Current guidelines recommend vaccination in elderly people and people affected by particular conditions that widely overlap with SSc, yet autoimmune diseases are not always clearly mentioned. Improving this suboptimal vaccination rate with clear guidelines is crucial for SSc patients and for clinicians to immunize these categories based principally on the pathology, prior to the age. Recommendations by the immunologist and the direct link to the vaccine providers can highly improve the vaccine coverage

BoHV-4-based vector delivering Ebola virus surface glycoprotein

Abstract Background Ebola virus (EBOV) is a Category A pathogen that is a member of Filoviridae family that causes hemorrhagic fever in humans and non-human primates. Unpredictable and devastating outbreaks of disease have recently occurred in Africa and current immunoprophylaxis and therapies are limited. The main limitation of working with pathogens like EBOV is the need for costly containment. To potentiate further and wider opportunity for EBOV prophylactics and therapies development, innovative approaches are necessary. Methods In the present study, an antigen delivery platform based on a recombinant bovine herpesvirus 4 (BoHV-4), delivering a synthetic EBOV glycoprotein (GP) gene sequence, BoHV-4-syEBOVgD106ΔTK, was generated. Results EBOV GP was abundantly expressed by BoHV-4-syEBOVgD106ΔTK transduced cells without decreasing viral replication. BoHV-4-syEBOVgD106ΔTK immunized goats produced high titers of anti-EBOV GP antibodies and conferred a long lasting (up to 6 months), detectable antibody response. Furthermore, no evidence of BoHV-4-syEBOVgD106ΔTK viremia and secondary localization was detected in any of the immunized animals. Conclusions The BoHV-4-based vector approach described here, represents: an alternative antigen delivery system for vaccination and a proof of principle study for anti-EBOV antibodies generation in goats for potential immunotherapy applications

Alexander Disease Modeling in Zebrafish: An In Vivo System Suitable to Perform Drug Screening

Alexander disease (AxD) is a rare astrogliopathy caused by heterozygous mutations, either inherited or arising de novo, on the glial fibrillary acid protein (GFAP) gene (17q21). Mutations in the GFAP gene make the protein prone to forming aggregates which, together with heat-shock protein 27 (HSP27), \u3b1B-crystallin, ubiquitin, and proteasome, contribute to form Rosenthal fibers causing a toxic effect on the cell. Unfortunately, no pharmacological treatment is available yet, except for symptom reduction therapies, and patients undergo a progressive worsening of the disease. The aim of this study was the production of a zebrafish model for AxD, to have a system suitable for drug screening more complex than cell cultures. To this aim, embryos expressing the human GFAP gene carrying the most severe p.R239C under the control of the zebrafish gfap gene promoter underwent functional validation to assess several features already observed in in vitro and other in vivo models of AxD, such as the localization of mutant GFAP inclusions, the ultrastructural analysis of cells expressing mutant GFAP, the effects of treatments with ceftriaxone, and the heat shock response. Our results confirm that zebrafish is a suitable model both to study the molecular pathogenesis of GFAP mutations and to perform pharmacological screenings, likely useful for the search of therapies for AxD

The OSMR Gene Is Involved in Hirschsprung Associated Enterocolitis Susceptibility through an Altered Downstream Signaling

Hirschsprung (HSCR) Associated Enterocolitis (HAEC) is a common life-threatening complication in HSCR. HAEC is suggested to be due to a loss of gut homeostasis caused by impairment of immune system, barrier defense, and microbiome, likely related to genetic causes. No gene has been claimed to contribute to HAEC occurrence, yet. Genetic investigation of HAEC by Whole-Exome Sequencing (WES) on 24 HSCR patients affected (HAEC) or not affected (HSCR-only) by enterocolitis and replication of results on a larger panel of patients allowed the identification of the HAEC susceptibility variant p.H187Q in the Oncostatin-M receptor (OSMR) gene (14.6% in HAEC and 5.1% in HSCR-only, p = 0.0024). Proteomic analysis on the lymphoblastoid cell lines from one HAEC patient homozygote for this variant and one HAEC patient not carrying the variant revealed two well distinct clusters of proteins significantly up or downregulated upon OSM stimulation. A marked enrichment in immune response pathways (q < 0.0001) was shown in the HAEC H187 cell line, while proteins upregulated in the HAEC Q187 lymphoblasts sustained pathways likely involved in pathogen infection and inflammation. In conclusion, OSMR p.H187Q is an HAEC susceptibility variant and perturbates the downstream signaling cascade necessary for the gut immune response and homeostasis maintenance