Clinical, immunological and molecular findings of 8 patients with typical and atypical severe combined immunodeficiency: identification of 7 novel mutations by whole exome sequencing.

Severe combined immunodeficiency (SCID) is one of the severe inborn errors of the immune system associated with life-threatening infections. Variations in SCID phenotypes, especially atypical SCID, may cause a significant delay in diagnosis. Therefore, SCID patients need to receive an early diagnosis. Here, we describe the clinical manifestations and genetic results of four SCID and atypical SCID patients. All patients (4 males and 4 females) in early infancy presented with SCID phenotypes within 6 months of birth. The mutations include RAG2 (p.I273T,p.G44X), IL7R (p.F361WfsTer17), ADA (c.780+1G>A), JAK3 (p.Q228Ter), LIG4 (p.G428R), and LAT (p.Y207fsTer33), as well as a previously reported missense mutation in RAG1 (p.A444V). The second report of LAT deficiency in SCID patients is presented in this study. Moreover, all variants were confirmed in patients and their parents as a heterozygous state by Sanger sequencing. The results of our study expand the clinical and molecular spectrum associated with SCID and leaky SCID phenotypes and provide valuable information for the clinical management of the patients.

Asthma phenotypes and T-bet protein expression in cells treated with Fluticasone Furoate/Vilanterol.

Asthma is a complex disease with diverse clinical manifestations ranging from mild to severe. Despite existing guidelines for asthma recognition and treatment, still a proportion of patients stay uncontrolled. Combinational therapy which comprises inhaled corticosteroids (ICS) and a long acting B2 adrenreceptor agonist (LABA) has been suggested to control asthma. In this study T-bet expression was attested in CD4 T cells treated with Fluticasone Furoate (FF), Vilanterol (V) and FF/V combination in severe asthmatic patients compared to patients with moderate asthma and healthy controls using Immunocytochemistry (ICC). First, CD4 T cells were isolated from PBMCs of 12 patients and controls using CD4 T cell isolation kit. Subsequently, isolated CD4 T cells were cultured with FF, V and FF/V for 1 h. To accomplish ICC, cells were incubated with anti-T-bet antibody, and then stained with HRP-bound secondary antibody. T-bet expression was evaluated using light microscopy. Statistical analyses were performed using R 3.5.2 software and visualized by ggplot2 3.1.0 package. Significant increasing in T-bet expression was seen in CD4 T cells from patients with moderate asthma treated with FF and FF/V. Suggesting conclusion would be distinct mechanisms responsible for severe asthma and moderate asthma in the patients and the needs for novel therapies. Further molecular studies in different asthma phenotypes would be instructive for asthma treatment.

Genetic Analysis of Patients with Two Different Types of Hyper IgM Syndrome.

BACKGROUND: Hyper IgM Syndrome (HIGM) is a rare primary immunodeficiency in which impairment of class switching recombination (CSR) and somatic hyper-mutation (SHM) leads to recurrent infections. OBJECTIVES: The aim of this study is to report the clinical and genetic features of six Iranian HIGM patients. METHODS: Six patients, who suspected to have HIGM based on two clinical findings, including recurrent infections and low levels of IgG and IgA and normal or elevated levels of IgM, were entered this study to undergo genetic studies. Sanger sequencing was applied to detect pathogenic mutations in CD40L and AID genes causing two most common forms of HIGM, which known as HIGM type 1 and 2, respectively. RESULTS: All patients who entered the study were males from unrelated families with a median age of 3.8 years. The most frequent clinical manifestation was recurrent pneumonia. Genetic studies of the patients revealed six different mutations, including five mutations in CD40L besides one mutation in AID. Two mutations in CD40L (p.F31fsX5 and p.C84S) were novel and three mutations (p. G219R, p.D62fsX18, and p.Q186X) have been previously reported. The mutation found in AID (p.E122X) was also previously described. CONCLUSION: The study results may provide valuable information for prenatal diagnosis and also for genetic counseling especially for those who have a history of primary immunodeficiency in their family.

New cell sources for CAR-based immunotherapy.

Chimeric antigen receptor (CAR) T cell therapy, in which a patient's own T lymphocytes are engineered to recognize and kill cancer cells, has achieved striking success in some hematological malignancies in preclinical and clinical trials, resulting in six FDA-approved CAR-T products currently available in the market. Despite impressive clinical outcomes, concerns about treatment failure associated with low efficacy or high cytotoxicity of CAR-T cells remain. While the main focus has been on improving CAR-T cells, exploring alternative cellular sources for CAR generation has garnered growing interest. In the current review, we comprehensively evaluated other cell sources rather than conventional T cells for CAR generation.

CAR-T cell potency: from structural elements to vector backbone components.

Chimeric antigen receptor (CAR) T cell therapy, in which a patient's own T lymphocytes are engineered to recognize and kill cancer cells, has achieved remarkable success in some hematological malignancies in preclinical and clinical trials, resulting in six FDA-approved CAR-T products currently available in the market. Once equipped with a CAR construct, T cells act as living drugs and recognize and eliminate the target tumor cells in an MHC-independent manner. In this review, we first described all structural modular of CAR in detail, focusing on more recent findings. We then pointed out behind-the-scene elements contributing to CAR expression and reviewed how CAR expression can be drastically affected by the elements embedded in the viral vector backbone.

Exosomal MicroRNAs as Biomarkers in Allergic Asthma.

Exosomes are extracellular vesicles that are involved in intracellular communication and different biological processes. Recently, the importance of microRNAs (miRNAs) in exosomes has been considered as biomarkers in asthma diagnosis. This study aimed to determine the expression of selective miRNAs from plasma-derived exosomes in moderate and severe asthmatic patients compared with healthy controls. Forty-six subjects including 22 patients with severe and mild to moderate allergic asthma and 24 healthy controls have entered this study. MiRNAs were extracted from the plasma exosomes and selective miRNAs (miR-21, miR-16, miR-Let7, miR-148a, miR-155, miR-125, miR-150, miR-146a, miR-223, miR-126) expressions levels were determined; using quantitative polymerase chain reaction (qPCR). In this study, we found a significant up-regulation of miR-223 and miR-21 in moderate asthmatic patients compared to the healthy controls (p=0.002, p=0.006). MiR-223 and miR-21 had the probability of 83% and 76% diagnosis estimation in moderate asthmatic patients respectively. Therefore, they could be used as biomarkers in these patients.  No expression of miR-125, miR-126, and miR-155 was found in plasma exosomes by qPCR in this study. The other miRNAs had no significant expression between different groups. Based on our findings,miR-223 and miR-21 may be considered biomarkers or used for targeted immunotherapies in asthma.

Clinical and Genetic Study of X-linked Agammaglobulinemia Patients (The Benefit of Early Diagnosis).

X-linked agammaglobulinemia (XLA) is a primary immunodeficiency caused by genetic defects in the Bruton tyrosine kinase (Btk) gene. XLA is characterized as an antibody deficiency by recurrent bacterial infections, the absence of peripheral B cells, and profound reductions in all immunoglobulin isotypes. This study aims to report the clinical and genetic features of five Iranian patients with XLA. Five male cases with recurrent bacterial infection entered this study based on clinical evaluation and Immunological screening tests. The levels of T-cell receptor excision circle (TREC) and kappa-deleting recombination excision circle (KREC) were also measured in dried blood spot (DBS) samples. Sanger sequencing was applied to PCR products of DNA samples of the patients for genetic studies. All patients were from unrelated families with a mean age of 6.7 years (2.5-11) at the time of diagnosis with 4.8 mean years of delay in diagnosis. The most frequent clinical manifestations were recurrent respiratory infections and arthritis. In these patients, five previously reported mutations were found including four mutations (p.Q496X, p.Q497X, p.R520X, and p.R641H) in the Kinase domain besides one mutation (p.L37P) in the pleckstrin homology (PH) domain. Evaluations of KREC and TREC level in patients' DBS showed low-to-undetectable copies of KREC (0-2 copies/3.2mm DBS) with normal copies of TREC. As patients with XLA have complete immunoglobulin defects and develop severe and recurrent infections, early diagnosis would be beneficial for the improvement of their quality of life. The study results may provide valuable information for the diagnosis, genetic counseling and prenatal diagnosis for the patients and their family members and emphasize performing KREC as an early diagnostic test in patients with XLA.

In Vitro Analysis of Nine MicroRNAs in CD8+ T Cells of Asthmatic Patients and the Effects of Two FDA-approved Drugs.

In this study, we first tried to determine whether the expression level of 9 miRNAs in the peripheral blood CD8+ T cells of asthmatic patients varies from that of controls, and secondly, we investigated the effects of fluticasone furoate and vilanterol on the expression level of these miRNAs. Fifteen subjects including 8 healthy individuals and 7 asthmatic patients were included in this study. CD8+T cells were isolated from participants' peripheral blood by a negative selection method using magnetic-activated cell sorting (MACS). The expression of 9 miRNAs was examined between the healthy individuals and asthmatic patients. Then the expression level of 9 miRNAs before and after treatment with the drugs was examined by quantitative real-time PCR. No significant changes in the expression level of 9 miRNAs were observed in asthmatic patients compared to the healthy controls. Fluticasone and vilanterol, in combination, had the greatest effect on miRNA expression. MiR-150 and miR-106a were the most and the least miRNAs, respectively, present in CD8+ T cells of patients and controls. MiR-106a and miR-126 had a positive correlation in CD8+ cells of asthmatic patients. Although no significant difference in the expression level of studies miRNAs was observed, the correlations among miRNAs were significant. Therefore, we suggest that the correlation between miRNAs would be a very important factor in physiological and pathological conditions in healthy individuals and asthmatic patients. Such a miRNA-miRNA correlation network can be even more critical than any changes in the variation of their expression in the CD8+ T cells.

Clinical and Genetic Analysis of Nine Suspected Familial Haemophagocytic Lymphohistiocytosis Patients for MUNC13-4 Deficiency and Introducing Four Novel Mutations in UNC13D.

Familial haemophagocytic lymphohistiocytosis (FHL) is a rare disorder of immune dysregulation. FHL inherited in an autosomal recessive pattern is classified into five subtypes based on underlying genetic defects. Mutations in four genes including PRF1, UNC13D, STX11 and STXBP2 are responsible for FHL2 to FHL5 respectively. The cause of FHL1 is associated with mutations in an unknown gene located at 9q21.3-22. This study aims to report the clinical features and genetic results of nine Iranian patients suffering from -haemophagocytic lymphohistiocytosis. Nine patients (five males and four females) suspected to FHL whose genetic evaluation of PRF1 and STX11 revealed no mutations, were entered the study to investigate UNC13D mutations. Primers were designed to amplify all coding regions and exon-intron boundaries of the gene. PCR products were then sequenced and analyzed by sequence analysis tools including BLAST. The most frequent clinical manifestations observed in the patients were fever and hepatosplenomegaly. In this study, five mutations were detected in UNC13D including four novel mutations (c.1434_1446delACCCATGGTGCAGinsTGGTGCT, c.1933C>T, c.1389+1G>C and c.2091+1G>A) besides to a previously reported deletion (c.627delT). The pathogenicity of the missense mutation was assessed using online prediction tools including SIFT and PolyPhen2. The study results may provide valuable information for genetic counseling especially for those who have a history of immunodeficiency diseases in their family and can be used for prenatal diagnosis.

Chemical composition of PM10 and its effect on in vitro hemolysis of human red blood cells (RBCs): a comparison study during dust storm and inversion.

PURPOSE: This study aimed to investigate chemical composition of PM10 (particulate matter with aerodynamic diameter ≤ 10 µm) during dust storm and inversion in Tehran and hemolysis effects. METHODS: PM10 was sampled in Tehran, Iran, during dust storm and inversion conditions. Water soluble ions (F¯, Cl¯, NO2¯, NO3¯, SO4¯2, Na+, K+, NH4 +, Ca+2, Mg+2) and elements (Al, Ba, Cd, Co, Cr, Cu, Fe, Li, Mn, Mo, Ni, Pb, Se, Sn, Sr, V, Zn, Pt, Rh, Pd, As and Si) were analyzed by ion chromatograph (IC) and inductively coupled plasma optical emission spectrometer (ICP-OES), respectively. Hemolysis was examined as in vitro at PM10 concentrations of 50-300 µg/ml. RESULTS: Daily average of PM10 concentrations in dusty and inversion days were 348.40 and 220.54 µg/m3, respectively. Most prevalence ionic components were NO3¯, Cl¯, SO4¯2 and Ca+2 during dust storm and SO4¯2, NO3¯, Cl¯ and NH4 + during inversion. Si, Fe and Al had the maximum values in both conditions. Particles associated with both conditions induced hemolytic responses. PM10 from dusty day showed a higher hemolysis percent (10.24 ± 4.67%) than inversion (9.08 ± 5.47%), but this difference was not significant (p = 0.32). Hemolytic effects were significantly intensified by increased PM concentrations (p < 0.001) in a dose-response manner. CONCLUSIONS: As the results, chemical composition of sampled particles from inversion days and dust storm was different from each other. Hemolytic effects of particles during dust storm were more than inversion days. However, this difference was not statistically significant.

Correction to: Chemical composition of PM10 and its effect on in vitro hemolysis.

[This corrects the article DOI: 10.1007/s40201-018-00327-w.].

Glucocorticoid Receptor Nuclear Translocation in CD4 T Cells from Severe and Moderate Asthmatic Patients Treated with Fluticasone/Vilanterol.

Different phenotypes of asthma from mild to severe are categorized based on diverse clinical features. A guideline for the recognition and treatment of asthma has been provided by Global Initiative for Asthma (GINA). To control symptoms and prevent asthma exacerbation in most patients combinational therapy with inhaled corticosteroids (ICS) and a long acting B2-adrenreceptor agonist (LABA) are recommended. Understanding asthma phenotypes would be helpful to improve asthma diagnosis and treatment. The aim of this study was to verify glucocorticoid receptor glcococorticoid receptor (GR) nuclear translocation in CD4 T cells treated with fluticasone furoate (FF), vilanterol (V) and FF/V combination in severe asthmatic patients compare to patients with moderate asthma and healthy controls using Immunocytochemistry (ICC). After taking blood and separating PBMCs from each subject, CD4 T cells were isolated from PBMCs using CD4+ T cell isolation kit. Isolated CD4 T cells were cultured in presence of FF, V and FF/V combination for 1 hour and after cytocentrifugation, cells were incubated with anti GR-antibody and subsequently stained with FITC bound secondary antibody and GR nuclear translocation was observed under microscope. The results showed significant increasing in GR nuclear translocation in treated CD4 T cells from patients with moderate asthma and controls compare to those severe asthmatic patients, along with treating cells with FF/V combination no significant GR nuclear translocation was observed compare to that of using mono treatment of cells with FF and V. Based on our findings, it can be concluded different mechanisms are responsible for severe asthma and moderate asthma.