Altered mucosal and plasma polyunsaturated fatty acids, oxylipins, and endocannabinoids profiles in Crohn's disease.

Selected mucosal and plasma polyunsaturated fatty acids (PUFAs) and related oxylipins and endocannabinoids were determined in 28 Crohn's disease (CD) patients and 39 controls. Fasting blood and colonic biopsies were collected in all participants, during a disease flare for the patients. Thirty-two lipid mediators including PUFAs, oxylipins, and endocannabinoids were assessed by LC-MS/MS. The pattern of lipid mediators in CD patients is characterized by an increase in arachidonic acid-derived oxylipins and endocannabinoids and a decrease in n-3 PUFAs and related endocannabinoids. A model combining increased 6-epi-lipoxin A4 and 2-arachidonyl glycerol with decreased docoasapentaenoic acid in plasma fairly discriminates patients from controls and may represent a lipidomic signature for CD flare. The study findings suggest that lipid mediators are involved in CD pathophysiology and may serve as biomarkers for disease flare. Further research is required to confirm the role of these bioactive lipids and test their therapeutic potential in CD.

Incidence and risk factors of SARS-CoV-2 infection among workers in a public health laboratory in Tunisia.

The aim of this study was to measure the extent of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection among workers at the Institut Pasteur de Tunis (IPT), a public health laboratory involved in the management of the COVID-19 pandemic in Tunisia, and to identify risk factors for infection in this occupational setting. A cross-sectional survey was conducted on IPT workers not vaccinated against coronavirus disease 2019 (COVID-19). Participants completed a questionnaire that included a history of reverse transcription-polymerase chain reaction (RT-PCR)-confirmed SARS-CoV-2 infection. Immunoglobulin G antibodies against the receptor-binding domain of the spike antigen (anti-S-RBD IgG) and the nucleocapsid protein (anti-N IgG) of the SARS-CoV-2 virus were detected by enzyme-linked immunoassay (ELISA). A multivariate analysis was used to identify factors significantly associated with SARS-CoV-2 infection. A total of 428 workers were enrolled in the study. The prevalence of anti-S-RBD and/or anti-N IgG antibodies was 32.9% [28.7-37.4]. The cumulative incidence of SARS-CoV-2 infection (positive serology and/or previous positive RT-PCR test) was 40.0% [35.5-44.9], while the proportion with asymptomatic infection was 32.9%. One-third of the participants with RT-PCR-confirmed infection tested seronegative more than 90 days postinfection. Participants aged over 40 and laborers were more susceptible to infection (adjusted OR [AOR] = 1.65 [1.08-2.51] and AOR = 2.67 [1.45-4.89], respectively), while tobacco smokers had a lower risk of infection (AOR = 0.54 [0.29-0.97]). The SARS-CoV-2 infection rate among IPT workers was not significantly different from that detected concurrently in the general population. Hence, the professional activities conducted in this public health laboratory did not generate additional risk to that incurred outside the institute in day-to-day activities.

RORC overexpression as a sign of Th17 lymphocytes accumulation in multiple myeloma bone marrow.

The role of the bone marrow microenvironment in supporting the proliferation and survival of the abnormal plasma cells in multiple myeloma (MM) is well established. Such microenvironment is rich of cytokines like IL-6, TGF-ß, IL-1 and IL-23 which are known to promote the differentiation of Th17 lymphocytes, a T helper subpopulation. Th17 cells secrete IL-17, a cytokine involved in the pathophysiology of several auto-immune diseases. Yet, its involvement in cancers remains unclear. Herein, we aimed to try to understand the role of Th17 lymphocytes in multiple myeloma. Bone marrow samples were prospectively collected from 29 MM patients and 23 healthy bone marrow donors for allograft. Mononuclear bone marrow cells were isolated by Ficoll-Hypaque gradient and CD138+ plasma cells were depleted using magnetic beads. The quantification of Th17 cells was performed by flow cytometry in the CD138 negative cells. The mRNA expression of IL17 and RORc was quantified using real time PCR in the same subset. The mRNA expression of IL17R was analyzed in plasma cells (CD138+ cells). Data obtained from patients and healthy donors were compared by both non-parametric Mann-Whitney U test and Spearman test. A significant increase of IL17 and RORC mRNA expression was found in the bone marrow microenvironment of MM patients compared to healthy donors. Th17 cells were also increased in the bone marrow of MM patients compared to healthy donors. Interestingly, the mRNA expression of IL17R was significantly decreased in MM patients. Yet, no correlation was found between the gene expression IL17, RORC and IL17R and the bone marrow infiltration or the stage of the disease. Collectively, our results suggest the involvement of Th17 cells in the pathophysiology of MM. Such data further support the use of anti-IL-17 antibodies as a therapeutic approach in MM.

Muscle-Specific Kinase Autoimmune Myasthenia Gravis: Report of a Pediatric Case and Literature Review.

Myasthenia gravis (MG) with antibodies to the muscle-specific tyrosine kinase (MuSK-MG) receptor is a rare entity. It represents 5 to 8% of all MG patients. Few pediatric cases were reported. Clinical presentation is often atypical. It is characterized by predominant involvement of cranial, bulbar, and axial muscles and early respiratory crises. Myokymia and fasciculation are suggestive of MuSK-MG. The clinical course of patients with MuSK-MG is worse than other types of MG. Responses to standard therapies are variable. We report clinical, neurophysiological, serological, and outcome profile of a Tunisian child with MuSK-MG.

Impaired TGF-ß signaling in patients with active systemic lupus erythematosus is associated with an overexpression of IL-22.

The mechanisms leading to the disruption of self-tolerance in systemic lupus erythematosus (SLE) remain elusive. Herein, we aimed to decipher the molecular basis of the impaired response of mononuclear cells to TGF-ß1. The Smad3-pathway was explored on CD3+ lymphocytes in either active or non active SLE patients. An impaired transcription of TGF-ß1 target genes was demonstrated in the CD3+ lymphocytes of active SLE patients confirming that the defect involves T cells and pointing to its extrinsic nature. We further demonstrate that the defect did not result from an impaired TGF-ßRII expression or Smad2/3 phosphorylation suggesting that the mechanism lies downstream Smad2/3 translocation. Interestingly, the TGF-1 signaling defect did not correlate with an increased expression of soluble or membrane-bound IL-15. However, it was associated with an overexpression of IL-22. This suggests that an excessive activation of AhR pathway (through UV radiations, infections, etc.) could lead to the inhibition of immunosuppressive actions of TGF-ß thus disrupting immune homeostasis in SLE. Collectively, our data suggest that the impaired response to TGF-ß in SLE patients is associated with disease activity and provide new insights into the pathogenesis of SLE since it could establish the link between the environmental factors and the aberrancies of the immune system usually described in SLE.

Involvement of different CD4(+) T cell subsets producing granzyme B in the immune response to Leishmania major antigens.

The nature of effector cells and the potential immunogenicity of Leishmania major excreted/secreted proteins (LmES) were evaluated using peripheral blood mononuclear cells (PBMCs) from healed zoonotic cutaneous leishmaniasis individuals (HZCL) and healthy controls (HC). First, we found that PBMCs from HZCL individuals proliferate and produce high levels of IFN-γ and granzyme B (GrB), used as a marker of activated cytotoxic T cells, in response to the parasite antigens. IFN-γ is produced by CD4(+) T cells, but unexpectedly GrB is also produced by CD4(+) T cells in response to stimulation with LmES, which were found to be as effective as soluble Leishmania antigens to induce proliferation and cytokine production by PBMCs from immune individuals. To address the question of regulatory T cell (Tregs) involvement, the frequency of circulating Tregs was assessed and found to be higher in HZCL individuals compared to that of HC. Furthermore, both CD4(+)CD25(+) and CD4(+)CD25(-) T cells, purified from HZCL individuals, produced IFN-γ and GrB when stimulated with LmES. Additional experiments showed that CD4(+)CD25(+)CD127(dim/-) Tregs were involved in GrB production. Collectively, our data indicate that LmES are immunogenic in humans and emphasize the involvement of CD4(+) T cells including activated and regulatory T cells in the immune response against parasite antigens.

Dynamics of SARS-CoV-2 antibodies after natural infection: insights from a study on Pasteur Institute of Tunis employees.

This study aimed to assess the kinetics of antibodies against the SARS-CoV-2, following natural infection in a cohort of employees of the Institut Pasteur de Tunis (IPT) and to assess the risk of reinfection over a 12-months follow-up period. A prospective study was conducted among an open cohort of IPT employees with confirmed SARS-CoV-2 infection that were recruited between September 2020 and March 2021. Sera samples were taken at 1, 3, 6, 9 and 12 months after confirmation of COVID-19 infection and tested for SARS-CoV-2-specific immunoglobulin G (IgG) antibodies to the spike (S-RBD) protein (IgG anti-S-RBD) and for neutralizing antibodies. Participants who had an initial decline of IgG anti-S-RBD and neutralizing antibodies followed by a subsequent rise in antibody titers as well as those who tested positive for SARS-CoV-2 by RT-PCR after at least 60 days of follow up were considered as reinfected. In total, 137 individuals were included with a mean age of 44.7 ± 12.3 years and a sex-ratio (Male/Female) of 0.33. Nearly all participants (92.7%) were symptomatic, and 2.2% required hospitalization. Among the 70 participants with three or more prospective blood samples, 32.8% were reinfected among whom 11 (47.8%) reported COVID-19 like symptoms. Up to 12 months of follow up, 100% and 42.9% of participants had detectable IgG anti-S-RBD and neutralizing antibodies, respectively. This study showed that humoral immune response following COVID-19 infection may persist up to 12 months after infection despite the potential risk for reinfection that is mainly explained by the emergence of new variants.

A novel approach to designing viral precision vaccines applied to SARS-CoV-2.

Efficient precision vaccines against several highly pathogenic zoonotic viruses are currently lacking. Proteolytic activation is instrumental for a number of these viruses to gain host-cell entry and develop infectivity. For SARS-CoV-2, this process is enhanced by the insertion of a furin cleavage site at the junction of the spike protein S1/S2 subunits upstream of the metalloprotease TMPRSS2 common proteolytic site. Here, we describe a new approach based on specific epitopes selection from the region involved in proteolytic activation and infectivity for the engineering of precision candidate vaccinating antigens. This approach was developed through its application to the design of SARS-CoV-2 cross-variant candidates vaccinating antigens. It includes an in silico structural analysis of the viral region involved in infectivity, the identification of conserved immunogenic epitopes and the selection of those eliciting specific immune responses in infected people. The following step consists of engineering vaccinating antigens that carry the selected epitopes and mimic their 3D native structure. Using this approach, we demonstrated through a Covid-19 patient-centered study of a 500 patients' cohort, that the epitopes selected from SARS-CoV-2 protein S1/S2 junction elicited a neutralizing antibody response significantly associated with mild and asymptomatic COVID-19 (p<0.001), which strongly suggests protective immunity. Engineered antigens containing the SARS-CoV-2 selected epitopes and mimicking the native epitopes 3D structure generated neutralizing antibody response in mice. Our data show the potential of this combined computational and experimental approach for designing precision vaccines against viruses whose pathogenicity is contingent upon proteolytic activation.

A Longitudinal Study in Tunisia to Assess the Anti-RBD IgG and IgA Responses Induced by Three Different COVID-19 Vaccine Platforms.

BACKGROUND: Vaccination constitutes the best strategy against COVID-19. In Tunisia, seven vaccines standing for the three main platforms, namely RNA, viral vector, and inactivated vaccines, have been used to vaccinate the population at a large scale. This study aimed to assess, in our setting, the kinetics of vaccine-induced anti-RBD IgG and IgA antibody responses. METHODS: Using in-house developed and validated ELISA assays, we measured anti-RBD IgG and IgA serum antibodies in 186 vaccinated workers at the Institut Pasteur de Tunis over 12 months. RESULTS: We showed that RNA vaccines were the most immunogenic vaccines, as compared to alum-adjuvanted inactivated and viral-vector vaccines, either in SARS-CoV-2-naïve or in SARS-CoV-2-experienced individuals. In addition to the IgG antibodies, the vaccination elicited RBD-specific IgAs. Vaccinated individuals with prior SARS-CoV-2 infection exhibited more robust IgG and IgA antibody responses, as compared to SARS-CoV-2-naïve individuals. CONCLUSIONS: After following up for 12 months post-immunization, we concluded that the hierarchy between the platforms for anti-RBD antibody-titer dynamics was RNA vaccines, followed by viral-vector and alum-adjuvanted inactivated vaccines.

Immunogenicity and Tolerance of BNT162b2 mRNA Vaccine in Allogeneic Hematopoietic Stem Cell Transplant Patients.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (ASCT) induces acquired immunodeficiency, potentially altering vaccine response. Herein, we aimed to explore the clinical tolerance and the humoral and cellular immune responses following anti-SARS-CoV-2 vaccination in ASCT recipients. METHODS: A prospective, non-randomized, controlled study that involved 43 ASCT subjects and 31 healthy controls. Humoral response was investigated using the Elecsys® test anti-SARS-CoV-2. Cellular response was assessed using the QFN® SARS-CoV-2 test. The lymphocyte cytokine profile was tested using the LEGENDplex™ HU Th Cytokine Panel Kit (12-plex). RESULTS: Adverse effects (AE) were observed in 69% of patients, encompassing pain at the injection site, fever, asthenia, or headaches. Controls presented more side effects like pain in the injection site and asthenia with no difference in the overall AE frequency. Both groups exhibited robust humoral and cellular responses. Only the vaccine transplant delay impacted the humoral response alongside a previous SARS-CoV-2 infection. Noteworthily, controls displayed a Th1 cytokine profile, while patients showed a mixed Th1/Th2 profile. CONCLUSIONS: Pfizer-BioNTech® anti-SARS-CoV-2 vaccination is well tolerated in ASCT patients, inducing robust humoral and cellular responses. Further exploration is warranted to understand the impact of a mixed cytokine profile in ASCT patients.

[Does multiple myeloma response to induction therapy depend on plasma cell IL6 receptor gene expression?]. / L'expression de l'IL6-r par les plasmocytes influe-t-elle la réponse au traitement d'induction au cours du myélome multiple ?

BACKGROUND: Interleukine 6 (IL-6) is the most important cytokine involved in malignant plasma cells growth and survival. AIM: To analyse bone marrow plasma cells IL6 receptor gene expression in both multiple myeloma patients at diagnosis and healthy bone marrow donors. METHODS: Clinical and biological patients' features and responses to Dexamethasone-Thalidomide induction therapy were gathered. 47 patients and 16 case controls were analyzed: Bone marrow plasma cells were isolated; and IL6 receptor gene expression was quantified using Taqman quantitative PCr technology and 2-ΔCT formula. RESULTS: Quantitative and qualitative IL6 receptor gene expression were negatively correlated with the degree of response to therapy (p= 0.02). In this study, plasma cells IL6 receptor gene expression seems to be decisive in predicting the response to treatment. CONCLUSION: Understanding the mechanisms involved in plasma cells IL6 receptor gene expression may offer a better appreciation of the physiopathologic and anti-oncogenic ways of drug resistance in multiple myeloma and consequently the discovery of new specific drugs.

Natriuretic-like Peptide Lebetin 2 Mediates M2 Macrophage Polarization in LPS-Activated RAW264.7 Cells in an IL-10-Dependent Manner.

Snake natriuretic peptide (NP) Lebetin 2 (L2) has been shown to improve cardiac function and reduce fibrosis as well as inflammation by promoting M2-type macrophages in a reperfused myocardial infarction (MI) model. However, the inflammatory mechanism of L2 remains unclear. Therefore, we investigated the effect of L2 on macrophage polarization in lipopolysaccharide (LPS)-activated RAW264.7 cells in vitro and explored the associated underlying mechanisms. TNF-α, IL-6 and IL-10 levels were assessed using an ELISA assay, and M2 macrophage polarization was determined by flow cytometry. L2 was used at non-cytotoxic concentrations determined by a preliminary MTT cell viability assay, and compared to B-type natriuretic peptide (BNP). In LPS-activated cells, both peptides reduced TNF-α and IL-6 release compared to controls. However, only L2 increased IL-10 release in a sustained manner and promoted downstream M2 macrophage polarization. Pretreatment of LPS-activated RAW264.7 cells with the selective NP receptor (NPR) antagonist isatin abolished both IL-10 and M2-like macrophage potentiation provided by L2. In addition, cell pretreatment with the IL-10 inhibitor suppressed L2-induced M2 macrophage polarization. We conclude that L2 exerts an anti-inflammatory response to LPS by regulating the release of inflammatory cytokines via stimulating of NP receptors and promoting M2 macrophage polarization through activation of IL-10 signaling.

Inflammatory Bowel Disease Increases the Severity of Myocardial Infarction after Acute Ischemia-Reperfusion Injury in Mice.

(1) Background: Increased risk of myocardial infarction (MI) has been linked to several inflammatory conditions, including inflammatory bowel disease (IBD). However, the relationship between IBD and MI remains unclear. Here, we implemented an original mouse model combining IBD and MI to determine IBD's impact on MI severity and the link between the two diseases. (2) Methods: An IBD model was established by dextran sulfate sodium (DSS) administration in drinking water, alone or with oral C. albicans (Ca) gavage. IBD severity was assessed by clinical/histological scores and intestinal/systemic inflammatory biomarker measurement. Mice were subjected to myocardial ischemia-reperfusion (IR), and MI severity was assessed by quantifying infarct size (IS) and serum cardiac troponin I (cTnI) levels. (3) Results: IBD mice exhibited elevated fecal lipocalin 2 (Lcn2) and IL-6 levels. DSS mice exhibited almost two-fold increase in IS compared to controls, with serum cTnI levels strongly correlated with IS. Ca inoculation tended to worsen DSS-induced systemic inflammation and IR injury, an observation which is not statistically significant. (4) Conclusions: This is the first proof-of-concept study demonstrating the impact of IBD on MI severity and suggesting mechanistic aspects involved in the IBD-MI connection. Our findings could pave the way for MI therapeutic approaches based on identified IBD-induced inflammatory mediators.

PpSP32, the Phlebotomus papatasi immunodominant salivary protein, exerts immunomodulatory effects on human monocytes, macrophages, and lymphocytes.

BACKGROUND: The saliva of sand flies, vectors of Leishmania parasites, contains several components that exert pharmacological activity facilitating the acquisition of blood by the insect and contributing to the establishment of infection. Previously, we demonstrated that PpSP32 is the immunodominant salivary antigen in humans exposed to Phlebotomus papatasi bites and validated its usefulness as a predictive biomarker of disease. PpSP32, whose functions are little known to date, is an intriguing protein due to its involvement in the etiopathogenesis of pemphigus, an auto-immune disease. Herein, we aimed to better decipher its role through the screening of several immunomodulatory activity either on lymphocytes or on monocytes/macrophages. METHODS: Peripheral mononuclear cells from healthy volunteers were stimulated with anti-CD3/anti-CD28 antibodies, phytohemagglutinin, phorbol 12-myristate 13-acetate/ionomycin, or lipopolysaccharide in the presence of increasing doses of PpSP32. Cell proliferation was measured after the addition of tritiated thymidine. Monocyte activation was tested by analyzing the expression of CD86 and HLA-DR molecules by flow cytometry. Cytokine production was analyzed in culture supernatants by ELISA. THP-1-derived macrophages were stimulated with LPS in the presence of increasing doses of PpSP32, and cytokine production was analyzed in culture supernatants by ELISA and multiplex technique. The effect of PpSP32 on NF-kB signaling was tested by Western blot. The anti-inflammatory activity of PpSP32 was assessed in vivo in an experimental inflammatory model of carrageenan-induced paw edema in rats. RESULTS: Our data showed that PpSP32 down-modulated the expression of activation markers in LPS-stimulated monocytes and THP1-derived macrophages. This protein negatively modulated the secretion of Th1 and Th2 cytokines by human lymphocytes as well as pro-inflammatory cytokines by monocytes, and THP1-derived macrophages. PpSP32 treatment led to a dose-dependent reduction of IκB phosphorylation. When PpSP32 was injected into the paw of carrageenan-injected rats, edema was significantly reduced. CONCLUSIONS: Our data indicates that PpSP32 induces a potent immunomodulatory effect on monocytes and THP-1-derived macrophages. This inhibition could be mediated, among others, by the modulation of the NF-kB signaling pathway. The anti-inflammatory activity of PpSP32 was confirmed in vivo in the carrageenan-induced paw edema model in rats.

VISTA+/CD8+ status correlates with favorable prognosis in Epithelial ovarian cancer.

Immunotherapy by blocking immune checkpoint regulators has emerged as a new targeted therapy for some cancers. Among them V-domain Ig suppressor of Tcell activation (VISTA) which is identified as a novel checkpoint regulator in ovarian cancer. This study aimed to investigate the VISTA role in Epithelial ovarian cancer (EOC), and its relationship with tumor-infiltrating lymphocytes (TILs) markers and its prognostic value. The expression of VISTA, CD3, CD8, CD4, FOXP3, and CD56 was assessed in 168 EOC tissue microarrays (TMA) by immunohistochemistry (IHC). In addition, associations between VISTA, TILs, clinicopathological variables, and overall survival (OS) were analyzed. VISTA expression in IGRov1 cells, as well as in PBMC of EOC patient, was evaluated by western blot. VISTA expression was detected in 64,28% of tissues, among which 42.3% were positive for tumor cells (TCs), and 47,9% were positive for immune cells (ICs). In univariate analysis, VISTA expression was significantly associated with a high density of TILs:CD3+ (p = 0,001), CD4+ (p = 0,002) and CD8+ (p≤0,001), in ICs but not in TCs. In terms of OS, multivariate analysis showed a significant association between the high density of CD8+ TILs and VISTA positive staining in ICs (p = 0,044), but not in TCs (p = 0,108). Kaplan-Meier curves demonstrated no correlation between VISTA expression and prolonged OS in both ICs (p = 0,841) and TCs (p = 0,090). Classification of EOC tumor microenvironment based on VISTA and CD8+TILs expression, demonstrated four immune subtypes: VISTA+/CD8+, VISTA+/CD8-, VISTA-/CD8+ and VISTA-/CD8-. The dual positive VISTA+/CD8+ subtype was significantly associated with prolonged OS in both TCs and ICs (p = 0,012 and p≤0,01, respectively), whereas patients with VISTA+/CD8- had the worst OS. Our results showed that VISTA is highly expressed in the IGRov1 cell line and LT-CD8 from a patient with EOC. Our results highlighted the association of VISTA expression and CD8+ TILs in EOC, with prolonged OS in patients with VISTA+/CD8+ and proposed VISTA as a potential immunotherapeutic target in EOC.

Development of an Optimized Process for Functional Recombinant SARS-CoV-2 Spike S1 Receptor-Binding Domain Protein Produced in the Baculovirus Expression Vector System.

To map the spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and evaluate immune response variations against this virus, it is essential to set up efficient serological tests locally. The SARS-CoV-2 immunogenic proteins were very expensive and not affordable for lower- middle-income countries (LMICs). For this purpose, the commonly used antigen, receptor-binding domain (RBD) of spike S1 protein (S1RBD), was produced using the baculovirus expression vector system (BEVS). In the current study, the expression of S1RBD was monitored using Western blot under different culture conditions. Different parameters were studied: the multiplicity of infection (MOI), cell density at infection, and harvest time. Hence, optimal conditions for efficient S1RBD production were identified: MOI 3; cell density at infection 2-3 × 106 cells/mL; and time post-infection (tPI or harvest time) of 72 h and 72-96 h, successively, for expression in shake flasks and a 7L bioreactor. A high production yield of S1RBD varying between 4 mg and 70 mg per liter of crude cell culture supernatant was achieved, respectively, in the shake flasks and 7L bioreactor. Moreover, the produced S1RBD showed an excellent antigenicity potential against COVID-19 (Wuhan strain) patient sera evaluated by Western blot. Thus, additional serological assays, such as in-house ELISA and seroprevalence studies based on the purified S1RDB, were developed.

Whole-exome sequencing reveals novel variants of monogenic diabetes in Tunisia: impact on diagnosis and healthcare management.

Introduction: Monogenic diabetes (MD) accounts for 3%-6% of all cases of diabetes. This prevalence is underestimated due to its overlapping clinical features with type 1 and type 2 diabetes. Hence, genetic testing is the most appropriate tool for obtaining an accurate diagnosis. In Tunisia, few cohorts of MD have been investigated until now. The aim of this study is to search for pathogenic variants among 11 patients suspected of having MD in Tunisia using whole-exome sequencing (WES). Materials and methods: WES was performed in 11 diabetic patients recruited from a collaborating medical center. The pathogenicity of genetic variation was assessed using combined filtering and bioinformatics prediction tools. The online ORVAL tool was used to predict the likelihood of combinations of pathogenic variations. Then, Sanger sequencing was carried out to confirm likely pathogenic predicted variants among patients and to check for familial segregation. Finally, for some variants, we performed structural modeling to study their impact on protein function. Results: We identified novel variants related to MD in Tunisia. Pathogenic variants are located in several MODY and non-MODY genes. We highlighted the presence of syndromic forms of diabetes, including the Bardet-Biedl syndrome, Alström syndrome, and severe insulin resistance, as well as the presence of isolated diabetes with significantly reduced penetrance for Wolfram syndrome-related features. Idiopathic type 1 diabetes was also identified in one patient. Conclusion: In this study, we emphasized the importance of genetic screening for MD in patients with a familial history of diabetes, mainly among admixed and under-represented populations living in low- and middle-income countries. An accurate diagnosis with molecular investigation of MD may improve the therapeutic choice for better management of patients and their families. Additional research and rigorous investigations are required to better understand the physiopathological mechanisms of MD and implement efficient therapies that take into account genomic context and other related factors.

Immunogenicity of Mix-and-Match CoronaVac/BNT162b2 Regimen versus Homologous CoronaVac/CoronaVac Vaccination: A Single-Blinded, Randomized, Parallel Group Superiority Trial.

(1) Background: This study aimed to compare the immunogenicity of the mix-and-match CoronaVac/BNT162b2 vaccination to the homologous CoronaVac/CoronaVac regimen. (2) Methods: We conducted a simple-blinded randomized superiority trial to measure SARS-CoV-2 neutralization antibodies and anti-spike receptor binding domain (RBD) IgG concentrations in blood samples of participants who had received the first dose of CoronaVac vaccine followed by a dose of BNT162b2 or CoronaVac vaccine. The primary endpoint for immunogenicity was the serum-neutralizing antibody level with a percentage of inhibition at 90% at 21-35 days after the boost. A difference of 25% between groups was considered clinically relevant. (3) Results: Among the 240 eligible participants, the primary endpoint data were available for 100 participants randomly allocated to the mix-and-match group versus 99 participants randomly allocated to the homologous dose group. The mix-and-match regimen elicited significantly higher levels of neutralizing antibodies (median level of 96%, interquartile range (IQR) (95-97) versus median level of 94%, IQR (81-96) and anti-spike IgG antibodies (median level of 13,460, IQR (2557-29,930) versus median level of 1190, IQR (347-4964) compared to the homologous group. Accordingly, the percentage of subjects with a percentage of neutralizing antibodies > 90% was significantly higher in the mix-and-match group (90.0%) versus the homologous (60.6%). Interestingly, no severe events were reported within 30 days after the second dose of vaccination in both groups. (4) Conclusions: Our data showed the superiority of the mix-and-match CoronaVac/BNT162b2 vaccination compared to the CoronaVac/CoronaVac regimen in terms of immunogenicity, thus constituting a proof-of-concept study supporting the use of inactivated vaccines in a mix-and-match strategy while ensuring good immunogenicity and safety.

Impaired control of effector T cells by regulatory T cells: a clue to loss of oral tolerance and autoimmunity in celiac disease?

OBJECTIVES: Regulatory T cells (Tregs) are instrumental for tolerance to self-antigens and dietary proteins. We have previously shown that interleukin (IL)-15, a cytokine overexpressed in the intestine of patients with celiac disease (CD), does not impair the generation of functional Tregs but renders human T cells resistant to Treg suppression. Treg numbers and responses of intestinal and peripheral T lymphocytes to suppression by Tregs were therefore compared in CD patients and controls. METHODS: Intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) were isolated from duodenal biopsy specimens of CD patients and controls. Concomitantly, CD4+CD25+ T lymphocytes (Tregs) were purified from blood. Responses of IELs and of LPLs, and peripheral lymphocytes (PBLs) to suppression by Tregs were tested by analyzing anti-CD3-induced proliferation and interferon (IFN)-γ production in the presence or absence of peripheral Tregs. Lamina propria and peripheral CD4+CD25+FOXP3+ T cells were assessed by flow cytometry. RESULTS: Although percentages of CD4+CD25+FOXP3+ LPLs were significantly increased in patients with active CD, proliferation and IFN-γ production of intestinal T lymphocytes were significantly less inhibited by autologous or heterologous Tregs in CD patients than in controls (P < 0.01). In all tested CD patients, IEL were unable to respond to Tregs. Resistance of LPLs and PBLs to Treg suppression was observed in patients with villous atrophy who had significantly enhanced serum levels of IL-15 compared with patients without villous atrophy and controls. CONCLUSIONS: Our results indicate that effector T lymphocytes from active CD become resistant to suppression by Tregs. This resistance might cause loss of tolerance to gluten, but also to self-antigens.

Multi-Criteria Decision Analysis to Prioritize People for COVID-19 Vaccination When Vaccines Are in Short Supply.

COVID-19 pandemic underscored the need for a rapid tool supporting decision-makers in prioritizing patients in the immediate and overwhelming context of pandemics, where shortages in different healthcare resources are faced. We have proposed Multi-Criteria Decision Analysis (MCDA) to create a system of criteria and weights to prioritize uses of COVID-19 vaccines in groups of people at significantly higher risk of severe COVID-19 disease or death, when vaccines are in short supply, for use in Tunisia. The prioritization criteria and the levels within each criterion were identified based on available COVID-19 evidence with a focus on the criteria selected by Tunisian scientific committees. To determine the weights for the criteria and levels, reflecting their relative importance, a panel of frontline physicians treating COVID-19 were invited to participate in an online survey using 1,000 minds MCDA software (www.1000minds.com) which implements the PAPRIKA (Potentially All Pairwise RanKings of all possible Alternatives) method. Ten criteria and twenty-three levels have been selected for prioritizing the uses of COVID-19 vaccines in groups of people at significantly higher risk of severe disease or death. Among the invited physicians, sixty have completed the survey. The obtained scores were, in decreasing order of importance (mean weights in parentheses, summing to 100%). Obesity (16.2%), Age (12.7%), Chronic pulmonary diseases (10.8%), Chronic cardiovascular conditions (10.3%), Bone marrow or organ transplantation (10.1%), Immunodeficiency or Immunosuppression (9.6%), Diabetes (9%), Renal failure (8.4%), evolutive cancer (6.9%), and high blood pressure (6%). MCDA-based prioritization scoring system comprising explicit criteria and weights provides an adaptable and multicriteria approach that can assist policy-makers to prioritize uses of COVID-19 vaccines.