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1.  IL-17 Enhances Chemotaxis of Primary Human B Cells during Asthma 
PLoS ONE  2014;9(12):e114604.
IL-17 is a pro-inflammatory mediator that is believed to play a critical role in regulating tissue inflammation during asthma, COPD, as well as other inflammatory disorders. The level of expression of IL-17 has been shown to be upregulated in lung bronchial tissue of asthmatic patients. Several reports have provided further evidence that this cytokine could play a key role in enhancing the migration of inflammatory as well as structural cells of the bronchial lung tissue during asthma and COPD. B cell infiltration to sites of inflammation during inflammatory disorders such as bowel disease, asthma and COPD has been reported. Accordingly, in this study we hypothesized that IL-17 may exert a chemotactic effect on primary B cells during asthma. We observed that B cells from asthmatic patients expressed significantly higher levels of IL-17RA and IL-17RC, compared to those of healthy subjects. Using an in-vitro migration assay, B cells were shown to migrate towards both IL-17A and IL-17F. Interestingly, blocking IL-17A and IL-17F signaling using either anti-IL-17R antibodies or MAP kinase inhibitors prevented in vitro migration of B cell towards IL-17. These observations indicate a direct chemotactic effect of IL-17 cytokines on primary peripheral blood B cells with higher effect being on asthmatic B cells. These findings revealed a key role for IL-17 in enhancing the migration of B cells to the lung tissue during asthma or COPD.
doi:10.1371/journal.pone.0114604
PMCID: PMC4262428  PMID: 25494178
2.  IL-4 receptor alpha single-nucleotide polymorphisms rs1805010 and rs1801275 are associated with increased risk of asthma in a Saudi Arabian population 
Annals of Thoracic Medicine  2014;9(2):81-86.
BACKGROUND:
The IL-4 receptor alpha subunit (IL-4Rα), when associated with the common gamma chain receptor, or the IL-13Rα1 subunit, transduces signals to STAT6 in response to IL-4 and IL-13 stimulations. This results in a number of cell-specific responses including Th2 differentiation, lymphocyte proliferation and IgE production. Given the prominent role of IL-4Rα in allergic disorders, several single-nucleotide polymorphisms (SNPs) have been found associated with asthma and other atopic disorders, including rs1805010 (I75V) and rs1801275 (Q576R) SNPs; however, lack of significant association have also been reported for some ethnic groups. The objective of this study was to determine whether IL-4Rα rs1805010 and rs1801275 polymorphisms are associated with asthma in patients from Saudi Arabia.
MATERIALS AND METHODS:
One hundred and ninety severe asthmatic patients (11-70 years old) and 194 healthy subjects of equivalent age range were recruited for blood donation. DNA was purified and genotyping for rs1801275 and rs1805010 polymorphisms in the IL-4Rα gene was performed by PCR amplification, followed by cycle sequencing of the purified PCR fragments using BigDye chain terminator and capillary electrophoresis.
RESULTS:
Pearson's Chi-square tests showed that the minor alleles, G, for both rs1805010 and rs1801275 SNPs, were significantly more frequent in asthmatics than in the healthy group (Yates’ P < 0.05); conversely, the major alleles, A, were significantly more frequent in healthy than in asthmatics (P < 0.05). Concerning association analysis, odds for A/G-G/G genotypes were significantly higher to be associated with asthma predisposition (rs1801275: OR = 2.12; 95% CI = 1.39-3.22; P < 0.001*; rs1805010: OR = 1.6; 95% CI = 1.01-2.53; P < 0.05*; dominant model). Analysis of gender-genotype interactions, with genders nested within A/G-G/G, indicated higher odds for females than males of significant association with asthma (rs1801275: OR = 5.19, 95% CI = 2.09-12.94*; rs1805010: OR = 3.73, 95% CI = 2.06-6.74*). Rs1805010 and rs1801275 were in linkage disequilibrium (D’ = 0.27; P < 0.0004*), with G-G haplotype being more frequent in asthmatics than in healthy subjects (OR = 2.43, 95% CI = 1.59-3.71*).
CONCLUSIONS:
The risk alleles, G, of IL-4Rα rs1805010 and rs1801275 SNPs and corresponding A/G-G/G genotypes were significantly associated with asthma predisposition in asthmatics from Saudi Arabia.
doi:10.4103/1817-1737.128849
PMCID: PMC4005166  PMID: 24791170
Asthma; allergy; association; genotype; IL-4 receptor alpha; susceptibility; STAT6
3.  lnherited IL-12p40 deficiency: genetic, immunological, and clinical features of 49 patients from 30 kindreds 
Medicine  2013;92(2):10.1097/MD.0b013e31828a01f9.
Autosomal recessive interleukin (IL)-12 p40 (IL-12p40) deficiency is thought to be a rare genetic etiology of Mendelian susceptibility to mycobacterial disease (MSMD). We report the genetic, immunological and clinical features of 49 patients from 30 kindreds originating from 5 countries (India, Iran, Pakistan, Saudi Arabia and Tunisia). There are only 9 different mutant alleles of the IL12B gene: including 2 small insertions, 3 small deletions, 2 splice site mutations and 1 large deletion, all frameshift and leading to a premature stop codon, and 1 nonsense mutation. Four of these 9 variants are recurrent, affecting 25 of the 30 reported kindreds, due to founder effects in specific countries. All patients are homozygous and display complete IL-12p40 deficiency. As a result, the patients lack detectable IL-12p70 and IL-12p40 and have low levels of interferon gamma (IFN-γ). The clinical features are characterized by childhood onset of bacillus Calmette-Guérin (attenuated Mycobacterium bovis strain) (BCG) and Salmonella infections, with recurrences of salmonellosis (36.4%) more common than recurrences of mycobacterial disease (25%). BCG vaccination led to BCG disease in 40 of the 41 patients vaccinated (97.5%). Multiple mycobacterial infections were rare, observed in only 3 patients, whereas the association of salmonellosis and mycobacteriosis was observed in 9 patients. A few other infections were diagnosed, including chronic mucocutaneous candidiasis (CMC) (n=3), nocardiosis (n=2), and klebsiellosis (n=1). IL-12p40 deficiency has a high but incomplete clinical penetrance, with 33.3% of genetically affected relatives of index cases showing no symptoms. However, the prognosis is poor, with mortality rates of up to 28.6%. Overall, the clinical phenotype of IL-12p40 deficiency closely resembles that of interleukin 12 receptor β1 (IL12R-β1) deficiency. In conclusion, IL-12p40 deficiency is more common than initially thought and should be considered worldwide in patients with MSMD and other intra-macrophagic infectious diseases, salmonellosis in particular.
doi:10.1097/MD.0b013e31828a01f9
PMCID: PMC3822760  PMID: 23429356
4.  Haploinsufficiency at the human IFNGR2 locus contributes to mycobacterial disease 
Human Molecular Genetics  2012;22(4):769-781.
Mendelian susceptibility to mycobacterial diseases (MSMD) is a rare syndrome, the known genetic etiologies of which impair the production of, or the response to interferon-gamma (IFN-γ). We report here a patient (P1) with MSMD whose cells display mildly impaired responses to IFN-γ, at levels, however, similar to those from MSMD patients with autosomal recessive (AR) partial IFN-γR2 or STAT1 deficiency. Whole-exome sequencing (WES) and Sanger sequencing revealed only one candidate variation for both MSMD-causing and IFN-γ-related genes. P1 carried a heterozygous frame-shift IFNGR2 mutation inherited from her father. We show that the mutant allele is intrinsically loss-of-function and not dominant-negative, suggesting haploinsufficiency at the IFNGR2 locus. We also show that Epstein-Barr virus transformed B lymphocyte cells from 10 heterozygous relatives of patients with AR complete IFN-γR2 deficiency respond poorly to IFN-γ, in some cases as poorly as the cells of P1. Naive CD4+ T cells and memory IL-4-producing T cells from these individuals also responded poorly to IFN-γ, whereas monocytes and monocyte-derived macrophages (MDMs) did not. This is consistent with the lower levels of expression of IFN-γR2 in lymphoid than in myeloid cells. Overall, MSMD in this patient is probably due to autosomal dominant (AD) IFN-γR2 deficiency, resulting from haploinsufficiency, at least in lymphoid cells. The clinical penetrance of AD IFN-γR2 deficiency is incomplete, possibly due, at least partly, to the variability of cellular responses to IFN-γ in these individuals.
doi:10.1093/hmg/dds484
PMCID: PMC3554203  PMID: 23161749
5.  Antibodies to gp120 and PD-1 Expression on Virus-Specific CD8+ T Cells in Protection from Simian AIDS 
Journal of Virology  2013;87(6):3526-3537.
We compared the relative efficacies against simian immunodeficiency virus (SIV) challenge of three vaccine regimens that elicited similar frequencies of SIV-specific CD4+ and CD8+ T-cell responses but differed in the level of antibody responses to the gp120 envelope protein. All macaques were primed with DNA plasmids expressing SIV gag, pol, env, and Retanef genes and were boosted with recombinant modified vaccinia Ankara virus (MVA) expressing the same genes, either once (1 × MVA) or twice (2 × MVA), or were boosted once with MVA followed by a single boost with replication-competent adenovirus (Ad) type 5 host range mutant (Ad5 h) expressing SIV gag and nef genes but not Retanef or env (1 × MVA/Ad5). While two of the vaccine regimens (1 × MVA and 1 × MVA/Ad5) protected from high levels of SIV replication only during the acute phase of infection, the 2 × MVA regimen, with the highest anti-SIV gp120 titers, protected during the acute phase and transiently during the chronic phase of infection. Mamu-A*01 macaques of this third group exhibited persistent Gag CD8+CM9+ effector memory T cells with low expression of surface Programmed death-1 (PD-1) receptor and high levels of expression of genes associated with major histocompatibility complex class I (MHC-I) and MHC-II antigen. The fact that control of SIV replication was associated with both high titers of antibodies to the SIV envelope protein and durable effector SIV-specific CD8+ T cells suggests the hypothesis that the presence of antibodies at the time of challenge may increase innate immune recruiting activity by enhancing antigen uptake and may result in improvement of the quality and potency of secondary SIV-specific CD8+ T-cell responses.
doi:10.1128/JVI.02686-12
PMCID: PMC3592123  PMID: 23325679
6.  Dominant-negative STAT1 SH2 domain mutations in unrelated patients with Mendelian susceptibility to mycobacterial disease 
Human mutation  2012;33(9):1377-1387.
Patients carrying two loss-of-function (or hypomorphic) alleles of STAT1 are vulnerable to intracellular bacterial and viral diseases. Heterozygosity for loss-of-function dominant-negative mutations in STAT1 is responsible for autosomal dominant (AD) Mendelian susceptibility to mycobacterial disease (MSMD), whereas heterozygosity for gain-of-function loss-of-dephosphorylation mutations causes AD chronic mucocutaneous candidiasis (CMC). The two previously reported types of AD MSMD-causing STAT1 mutations are located in the tail domain (p.L706S) or in the DNA-binding domain (p.E320Q and p.Q463H), whereas the AD CMC-causing mutations are located in the coiled-coil domain. We identified two cases with AD-STAT1 deficiency in two unrelated patients from Japan and Saudi Arabia carrying heterozygous missense mutations affecting the SH2 domain (p.K637E and p.K673R). p.K673R is a hypomorphic mutation that impairs STAT1 tyrosine phosphorylation, whereas the p.K637E mutation is null and affects both STAT1 phosphorylation and DNA-binding activity. Both alleles are dominant-negative and result in impaired STAT1-mediated cellular responses to IFN-γ and IL-27. By contrast, STAT1-mediated cellular responses against IFN-α and IFN-λ1 were preserved at normal levels in patients’ cells. We describe here the first dominant mutations in the SH2 domain of STAT1, revealing the importance of this domain for tyrosine phosphorylation and DNA-binding, as well as for anti-mycobacterial immunity.
doi:10.1002/humu.22113
PMCID: PMC3668973  PMID: 22573496
MSMD; STAT1; osteomyelitis; dominant-negative effect
7.  Distribution of selected gene polymorphisms of UGT1A1 in a Saudi population 
Introduction
Glucuronidation is an important phase II pathway responsible for the metabolism of many endogenous substances and drugs to less toxic metabolites, which undergo renal excretion. The aim of the current work was to evaluate genotype and allele frequencies of certain UDP-glucuronosyltransferase 1A1 (UGT1A1) variants in an Arab population.
Material and methods
Genomic DNA was isolated from 192 healthy unrelated Saudi males of various geographic regions and genotyping of UGT1A1*6, *27, *36, *28, *37, and *60 was carried out using polymerase chain reaction (PCR) amplification followed by direct sequencing.
Results
The most common allele for (TA) repeats was the wild type (TA)6 with a frequency of 74.3% followed by the mutant (TA)7 (i.e., UGT1A1*28) with a frequency of 25.7%. The distribution of UGT1A1*60 allele was 62.4% among subjects with the homozygous mutant genotype of 35.4%, while the wild type variant represents 10.6% only. Both UGT1A1*6 and *27 were not detected as all screened subjects showed a homozygous wild type pattern. Similarly, UGT1A1*36* and *37 were either not present or rarely found, respectively. In comparison to other populations, the frequency of UGT1A1*60 and *28 in the studied population was less than that of African Americans but higher than Asians. The geographical origin of the study subjects also implied some differences in genotype distribution of (TA) repeats and UGT1A1*60.
Conclusions
Our data indicate that Saudis harbor some important UGT1A1 mutations known to affect enzyme activity. Additional studies are warranted to assess the clinical implications of these gene polymorphisms in this ethnic group.
doi:10.5114/aoms.2013.37012
PMCID: PMC3776187  PMID: 24049537
glucuronidation; UDP-glucuronosyltransferase 1A1; gene polymorphism; Saudi Arabians
8.  LPS-responsive beige-like anchor (LRBA) gene mutation in a family with inflammatory bowel disease and combined immunodeficiency 
Background
Clinical immunology has traditionally relied on accurate phenotyping of the patient’s immune dysfunction for the identification of a candidate gene or genes for sequencing and molecular confirmation. Although this is also true for other branches of medicine, the marked variability in immune-related phenotypes and the highly complex network of molecules that confer normal host immunity are challenges that clinical immunologists often face in their quest to establish a specific genetic diagnosis.
Objective
We sought to identify the underlying genetic cause in a consanguineous family with chronic inflammatory bowel disease–like disorder and combined immunodeficiency.
Methods
We performed exome sequencing followed by autozygome filtration.
Results
A truncating mutation in LPS-responsive beige-like anchor (LRBA), which abolished protein expression, was identified as the most likely candidate variant in this family.
Conclusion
The combined exome sequencing and autozygosity mapping approach is a powerful tool in the study of atypical immune dysfunctions. We identify LRBA as a novel immunodeficiency candidate gene the precise role of which in the immune system requires future studies.
doi:10.1016/j.jaci.2012.05.043
PMCID: PMC3582381  PMID: 22721650
LPS-responsive beige-like anchor (LRBA); chronic diarrhea; common variable immunodeficiency; autoimmunity
9.  Impaired intrinsic immunity to HSV-1 in human iPSC-derived TLR3-deficient CNS cells 
Nature  2012;491(7426):769-773.
In the course of primary infection with herpes simplex virus 1 (HSV-1), children with inborn errors of TLR3 immunity are prone to HSV-1 encephalitis (HSE) 1–3. We tested the hypothesis that the pathogenesis of HSE involves non hematopoietic central nervous system (CNS)-resident cells. We derived induced pluripotent stem cells (iPSCs) from the dermal fibroblasts of TLR3- and UNC-93B-deficient patients and from controls. These iPSCs were differentiated into highly purified populations of neural stem cells (NSCs), neurons, astrocytes and oligodendrocytes. The induction of IFN-β and/or IFN-γ1 in response to poly(I:C) stimulation was dependent on TLR3 and UNC-93B in all cells tested. However, the induction of IFN-β and IFN-γ1 in response to HSV-1 infection was impaired selectively in UNC-93B-deficient neurons and oligodendrocytes. These cells were also much more susceptible to HSV-1 infection than control cells, whereas UNC-93B-deficient NSCs and astrocytes were not. TLR3-deficient neurons were also found to be susceptible to HSV-1 infection. The rescue of UNC-93B- and TLR3-deficient cells with the corresponding wild-type allele demonstrated that the genetic defect was the cause of the poly(I:C) and HSV-1 phenotypes. The viral infection phenotype was further rescued by treatment with exogenous IFN-α/β, but not IFN-γ1.Thus, impaired TLR3- and UNC-93B-dependent IFN-α/β intrinsic immunity to HSV-1 in the CNS, in neurons and oligodendrocytes in particular, may underlie the pathogenesis of HSE in children with TLR3 pathway deficiencies.
doi:10.1038/nature11583
PMCID: PMC3527075  PMID: 23103873
10.  Th17 cytokines induce pro-fibrotic cytokines release from human eosinophils 
Respiratory Research  2013;14(1):34.
Background
Subepithelial fibrosis is one of the most critical structural changes affecting bronchial airway function during asthma. Eosinophils have been shown to contribute to the production of pro-fibrotic cytokines, TGF-β and IL-11, however, the mechanism regulating this process is not fully understood.
Objective
In this report, we investigated whether cytokines associated with inflammation during asthma may induce eosinophils to produce pro-fibrotic cytokines.
Methods
Eosinophils were isolated from peripheral blood of 10 asthmatics and 10 normal control subjects. Eosinophils were stimulated with Th1, Th2 and Th17 cytokines and the production of TGF-β and IL-11 was determined using real time PCR and ELISA assays.
Results
The basal expression levels of eosinophil derived TGF-β and IL-11 cytokines were comparable between asthmatic and healthy individuals. Stimulating eosinophils with Th1 and Th2 cytokines did not induce expression of pro-fibrotic cytokines. However, stimulating eosinophils with Th17 cytokines resulted in the enhancement of TGF-β and IL-11 expression in asthmatic but not healthy individuals. This effect of IL-17 on eosinophils was dependent on p38 MAPK activation as inhibiting the phosphorylation of p38 MAPK, but not other kinases, inhibited IL-17 induced pro-fibrotic cytokine release.
Conclusions
Th17 cytokines might contribute to airway fibrosis during asthma by enhancing production of eosinophil derived pro-fibrotic cytokines. Preventing the release of pro-fibrotic cytokines by blocking the effect of Th17 cytokines on eosinophils may prove to be beneficial in controlling fibrosis for disorders with IL-17 driven inflammation such as allergic and autoimmune diseases.
doi:10.1186/1465-9921-14-34
PMCID: PMC3602055  PMID: 23496774
Asthma; Eosinophils; Th17 cytokines; Pro-fibrotic cytokines; TGF-β; IL-11
11.  Improper inhaler technique is associated with poor asthma control and frequent emergency department visits 
Background
Uncontrolled asthma remains a frequent cause of emergency department (ED) visits and hospital admissions. Improper asthma inhaler device use is most likely one of the major causes associated with uncontrolled asthma and frequent ED visits.
Objectives
To evaluate the inhaler technique among asthmatic patients seen in ED, and to investigate the characteristics of these patients and factors associated with improper use of inhaler devices and its relationship with asthma control and ED visits.
Methods
A cross-sectional study of all the patients who visited the ED with bronchial asthma attacks over a 9-month period was undertaken at two major academic hospitals in Saudi Arabia. Information was collected about demographic data and asthma management and we assessed the inhaler techniques for each patient using an inhaler technique checklist.
Results
A total of 450 asthma patients were included in the study. Of these, 176(39.1%) were males with a mean age of 42.3 ±16.7 years and the mean duration of asthma was 155.9 ± 127.1 weeks. The improper use of asthma inhaler devices was observed in 203(45%) of the patients and was associated with irregular clinic follow-ups (p = 0.0001), lack of asthma education (p = 0.0009), uncontrolled asthma ACT (score ≤ 15) (p = 0.001), three or more ED visits (p = 0.0497), and duration of asthma of less than 52 weeks (p = 0.005). Multiple logistic regression analysis revealed that a lack of education about asthma disease (OR =1.65; 95% CI: 1.07, 2.54) or a lack of regular follow-up (OR =1.73; 95% CI: 1.08, 2.76) was more likely to lead to the improper use of an asthma inhaler device.
Conclusion
Improper asthma inhaler device use is associated with poor asthma control and more frequent ED visits. We also identified many avoidable risk factors leading to the improper use of inhaler devices among asthma patients visiting the ED.
doi:10.1186/1710-1492-9-8
PMCID: PMC3605255  PMID: 23510684
Asthma control; Inhaled corticosteroid; Emergency department; Inhaler devices; Asthma education
12.  Factors associated with patient visits to the emergency department for asthma therapy 
Background
Acute asthma attacks remain a frequent cause of emergency department (ED) visits and hospital admission. Many factors encourage patients to seek asthma treatment at the emergency department. These factors may be related to the patient himself or to a health system that hinders asthma control. The aim of this study was to identify the main factors that lead to the frequent admission of asthmatic patients to the ED.
Methods
A cross-sectional survey of all the patients who visited the emergency room with bronchial asthma attacks over a 9-month period was undertaken at two major academic hospitals. The following data were collected: demographic data, asthma control in the preceding month, where and by whom the patients were treated, whether the patient received education about asthma or its medication and the patients’ reasons for visiting the ED.
Result
Four hundred fifty (N = 450) patients were recruited, 39.1% of whom were males with a mean age of 42.3 ± 16.7. The mean duration of asthma was 155.90 ± 127.13 weeks. Approximately half of the patients did not receive any information about bronchial asthma as a disease, and 40.7% did not receive any education regarding how to use asthma medication. Asthma was not controlled or partially controlled in the majority (97.7%) of the patients preceding the admission to ED. The majority of the patients visited the ED to receive a bronchodilator by nebuliser (86.7%) and to obtain oxygen (75.1%). Moreover, 20.9% of the patients believed that the ED managed them faster than the clinic, and 21.1% claimed that their symptoms were severe enough that they could not wait for a clinic visit. No education about asthma and uncontrolled asthma are the major factors leading to frequent ED visits (three or more visits/year), p-value = 0.0145 and p-value = 0.0003, respectively. Asthma control also exhibited a significant relationship with inhaled corticosteroid ICS use (p-value =0.0401) and education about asthma (p-value =0.0117).
Conclusion
This study demonstrates that many avoidable risk factors lead to uncontrolled asthma and frequent ED visits.
doi:10.1186/1471-2466-12-80
PMCID: PMC3534524  PMID: 23244616
Asthma; Control; Inhaled cortisone; Emergency department
13.  Mycobacterial disease and impaired IFN-γ immunity in humans with inherited ISG15 deficiency 
Science (New York, N.Y.)  2012;337(6102):1684-1688.
ISG15 is an interferon (IFN)-α/β-inducible, ubiquitin-like intracellular protein. Its conjugation to various proteins (ISGylation) contributes to antiviral immunity in mice. We describe human patients with inherited ISG15 deficiency and mycobacterial, but not viral diseases. The lack of intracellular ISG15 production and protein ISGylation was not associated with cellular susceptibility to any viruses tested, consistent with the lack of viral diseases in these patients. By contrast, the lack of mycobacterium-induced ISG15 secretion by leukocytes — granulocytes in particular — reduced the production of IFN-γ by lymphocytes, including natural killer cells, probably accounting for the enhanced susceptibility to mycobacterial disease. This experiment of Nature shows that human ISGylation is largely redundant for antiviral immunity, but that ISG15 plays an essential role as an IFN-γ-inducing secreted molecule for optimal antimycobacterial immunity.
doi:10.1126/science.1224026
PMCID: PMC3507439  PMID: 22859821
15.  Germline CYBB mutations that selectively affect macrophages in kindreds with X-linked predisposition to tuberculous mycobacterial disease 
Nature immunology  2011;12(3):213-221.
Germline mutations in CYBB, the human gene encoding the gp91phox subunit of the phagocyte NADPH oxidase, impair the respiratory burst of all types of phagocytes and result in X-linked chronic granulomatous disease (CGD). We report here two kindreds in which otherwise healthy male adults developed X-linked recessive Mendelian susceptibility to mycobacterial disease (MSMD) syndromes. These patients had previously unknown mutations in CYBB that resulted in an impaired respiratory burst in monocyte-derived macrophages but not in monocytes or granulocytes. The macrophage-specific functional consequences of the germline mutation resulted from cell-specific impairment in the assembly of the NADPH oxidase. This ‘experiment of nature’ indicates that CYBB is associated with MSMD and demonstrates that the respiratory burst in human macrophages is a crucial mechanism for protective immunity to tuberculous mycobacteria.
doi:10.1038/ni.1992
PMCID: PMC3097900  PMID: 21278736
16.  Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis 
The Journal of Experimental Medicine  2011;208(8):1635-1648.
Whole-exome sequencing reveals activating STAT1 mutations in some patients with autosomal dominant chronic mucocutaneous candidiasis disease.
Chronic mucocutaneous candidiasis disease (CMCD) may be caused by autosomal dominant (AD) IL-17F deficiency or autosomal recessive (AR) IL-17RA deficiency. Here, using whole-exome sequencing, we identified heterozygous germline mutations in STAT1 in 47 patients from 20 kindreds with AD CMCD. Previously described heterozygous STAT1 mutant alleles are loss-of-function and cause AD predisposition to mycobacterial disease caused by impaired STAT1-dependent cellular responses to IFN-γ. Other loss-of-function STAT1 alleles cause AR predisposition to intracellular bacterial and viral diseases, caused by impaired STAT1-dependent responses to IFN-α/β, IFN-γ, IFN-λ, and IL-27. In contrast, the 12 AD CMCD-inducing STAT1 mutant alleles described here are gain-of-function and increase STAT1-dependent cellular responses to these cytokines, and to cytokines that predominantly activate STAT3, such as IL-6 and IL-21. All of these mutations affect the coiled-coil domain and impair the nuclear dephosphorylation of activated STAT1, accounting for their gain-of-function and dominance. Stronger cellular responses to the STAT1-dependent IL-17 inhibitors IFN-α/β, IFN-γ, and IL-27, and stronger STAT1 activation in response to the STAT3-dependent IL-17 inducers IL-6 and IL-21, hinder the development of T cells producing IL-17A, IL-17F, and IL-22. Gain-of-function STAT1 alleles therefore cause AD CMCD by impairing IL-17 immunity.
doi:10.1084/jem.20110958
PMCID: PMC3149226  PMID: 21727188
17.  514 Role of TH-17 Cytokines in Steroid Insensitivity in Peripheral Blood Mononuclear Cells. Relationship to GR-alpha and GR-beta Expression 
Background
Inhaled corticosteroids represent the most common treatment for asthma. Although most asthmatic patients respond well, a significant proportion of severe asthmatics require higher doses or even fail to respond to oral or inhaled corticosteroids. We previously reported that glucocorticoid receptor-beta is associated with corticosteroid resistance in airway epithelial cells from asthmatic patients and that Th-17 cytokines increase steroid insensitivity via a mechanism involving GR-beta upregulation. We aim to investigate whether IL-17A and F cytokines enhance steroid unresponsiveness in PBMCs from normal subjects and severe asthmatics via the upregulation of GR-beta isoform.
Methods
PBMCs were cultured for 48 hours in the presence or absence of IL-2, IL-4, IL-17A, IL-17F or IL-23 cytokines. Expression of GR-alpha, GR-beta, GILZ and IL-6 was determined using Q-RT-PCR and/or Western blotting. Response to Dexamethasone was determined on the inhibition of PHA-induced proliferation by Dexamethasone (IC50) using either 3H-thymidine or CFSE-labelled cells. Response of the cells to Dexamethasone-induced apoptosis was determined by Annexin-V staining.
Results
Treatment of PBMCs with IL-17A+IL-17F combined significantly decreased the mRNA expression of GR-alpha while that of GR-beta was significantly upregulated. IL-2+IL-4 in combination significantly decreased GR-alpha expression but had no effect on GR-beta receptor expression. IL17A+IL17F+IL23 combined induced the highest ratio of GR-beta/GR-alpha in PBMC from normal subjects. Either IL-17A+F or IL-2+IL-4 combinations significantly decreased the inhibitory effect of Dexamethasone on PBMC proliferation (IL-17A+F IC50 = 190 nM Dex; IL-2+4 IC50 = 1060 nM Dex), when compared to the control without cytokine stimulation. In the presence of Dexamethasone, IL-2+IL-4 but not IL-17A+IL-17F, inhibited the expression of the glucocorticoid-inducible leucine zipper gene (GILZ) in PBMCs from both normal (60%) and asthmatics (45–50%), which was correlated with significantly higher apoptosis in cells stimulated with IL-2+IL-4.
Conclusions
IL-17A, IL-17F, IL-2, and IL-4, which are known to be upregulated in the blood and lung tissue of asthmatics, contribute to steroid insensitivity of severe asthmatic patients by modulating the expression of GR-alpha and GR-beta receptors on peripheral blood PBMCs. GR-beta could protect PBMCs from Dex-induced apoptosis. Furthermore, the increased GR-beta/GR-alpha ratios by both IL-17A+F and IL-2+4 cytokines correlates with the decreased inhibitory effect of Dexamethasone on PHA-induced PBMC proliferation.
doi:10.1097/01.WOX.0000411629.24667.df
PMCID: PMC3512825
18.  513 IL-17 Role in the Regulatory Function of B Cells 
Background
B lymphocytes are known to be important cytokine sources in inflammation and play a pathogenic role by producing autoantibodies in a number of chronic immunological diseases. However, B cell depletion therapy induced an exacerbation of symptoms in some patients with autoimmune disorders, revealing that B cells play a critical anti-inflammatory role mediated by IL-10 release. We therefore investigated the human B cell regulatory subset producing IL-10 in response to stimulation.
Methods
Highly purified B cells were obtained from tonsils by using a multiple-step separation procedure which included rosette depletion, adherence depletion, CD3+ cell magnetic-activated depletion and CD19+ magnetic-activated positive cell selection. CD20+ purity was verified by flow cytometry. The CD19+CD20+ B cells were stimulated with CpG oligonucleotide, IL-4, IFN-gamma, anti-CD40, IL-17A and IL-17F, either alone or in combination. The expression of both IL-6 and IL-10 mRNA was analyzed by quantitative RT-PCR and by ELISA. B regulatory cell subsets expressing IL-10 and the markers CD5 and CD1d were quantified by FACS analysis. B cell proliferation was determined by 3H thymidine incorporation or CFSE labeling.
Results
Expression of IL-10 mRNA and protein in purified B cells from tonsils was weakly stimulated by anti-CD40 antibody, CpG oligonucleotide or with IL-17. When B cells were simultaneously stimulated with IL-17, anti-CD40 antibody and CpG oligonucleotide, the mRNA and protein expression of IL-10 was strongly increased (n = 3; P ≤ 0.001). B cells proliferation was also significantly increased. In contrast, stimulation with IL-4 alone or in combination with anti-CD40 antibody, decreased the expression of IL-10 (n = 3; P ≤ 0.001).
Conclusions
TLR9 receptor stimulation synergizes with CD40 and IL-17 receptors stimulation in the induced proliferation and potent release of IL-10 cytokine while decreasing IL-6 production in B cells. These novel findings provide evidence that B lymphocytes might be an important source of the anti-inflammatory IL-10 cytokine, and provide novel evidence that stimulation of B lymphocytes with IL-17 cytokine could be an important regulatory mechanism in immune responses.
doi:10.1097/01.WOX.0000411628.86548.fc
PMCID: PMC3512855
19.  127 Eosinophils Enhance Airway Smooth Muscle Cell Proliferation Via the Release of Cysteinyl Leukotrines 
The World Allergy Organization Journal  2012;5(Suppl 2):S59-S60.
Background
Asthma is a chronic inflammatory disorder of the lung airways that is associated with airway remodeling and hyperresponsiveness. Its is well documented that the smooth muscle mass in asthmatic airways is increased due to hypertrophy and hyperplasia of the ASM cells. Moreover, eosinophils have been proposed in different studies to play a major role in airway remodeling. Here, we hypothesized that eosinophils modulate the airways through enhancing ASM cell proliferation. The aim of this study is to examine the effect of eosinophils on ASM cell proliferation using eosinophils isolated from asthmatic and normal control subjects.
Methods
Eosinophils were isolated from peripheral blood of 6 mild asthmatics and 6 normal control subjects. ASM cells were incubated with eosinophils or eosinophil membranes and ASM proliferation was estimated using thymidine incorporation. The mRNA expression of extracellular matrix (ECM) in ASM cells was measured using quantitative real-time PCR. The effect of eosinophil-derived proliferative cytokines on ASM cells was determined using neutralizing antibodies. The role of eosinophil derived Cysteinyl Leukotrienes in enhancing ASM was also investigated.
Results
Co-culture with eosinophils significantly increased ASM cell proliferation. However, there was no significant difference in ASM proliferation following incubation with eosinophils from asthmatic versus normal control subjects. Co-culture with eosinophil membranes had no effect on ASM proliferation. Moreover, there was no significant change in the mRNA expression of ECM proteins in ASM cells following co-culture with eosinophils when compared with medium alone. Interestingly, blocking the activity of cysteinyl Leukotries using antagonists inhibited eosinophil-derived ASM proliferation.
Conclusions
Eosinophils enhances the proliferation of ASM cells. This role of eosinophil does not seem to depend on ASM derived ECM proteins nor on Eosinophil derived TGF-β or TNF-α. Eosinophil seems to induce ASM proliferation via the secretion of Cysteinyl Leukotrienes.
doi:10.1097/01.WOX.0000411872.81749.50
PMCID: PMC3513122
20.  Herpes simplex encephalitis in children with autosomal recessive and dominant TRIF deficiency 
The Journal of Clinical Investigation  2011;121(12):4889-4902.
Herpes simplex encephalitis (HSE) is the most common sporadic viral encephalitis of childhood. Autosomal recessive (AR) UNC-93B and TLR3 deficiencies and autosomal dominant (AD) TLR3 and TRAF3 deficiencies underlie HSE in some children. We report here unrelated HSE children with AR or AD TRIF deficiency. The AR form of the disease was found to be due to a homozygous nonsense mutation that resulted in a complete absence of the TRIF protein. Both the TLR3- and the TRIF-dependent TLR4 signaling pathways were abolished. The AD form of disease was found to be due to a heterozygous missense mutation, resulting in a dysfunctional protein. In this form of the disease, the TLR3 signaling pathway was impaired, whereas the TRIF-dependent TLR4 pathway was unaffected. Both patients, however, showed reduced capacity to respond to stimulation of the DExD/H-box helicases pathway. To date, the TRIF-deficient patients with HSE described herein have suffered from no other infections. Moreover, as observed in patients with other genetic etiologies of HSE, clinical penetrance was found to be incomplete, as some HSV-1–infected TRIF-deficient relatives have not developed HSE. Our results provide what we believe to be the first description of human TRIF deficiency and a new genetic etiology for HSE. They suggest that the TRIF-dependent TLR4 and DExD/H-box helicase pathways are largely redundant in host defense. They further demonstrate the importance of TRIF for the TLR3-dependent production of antiviral IFNs in the CNS during primary infection with HSV-1 in childhood.
doi:10.1172/JCI59259
PMCID: PMC3226004  PMID: 22105173
21.  Clinical Features and Outcome of Patients With IRAK-4 and MyD88 Deficiency 
Medicine  2010;89(6):403-425.
Autosomal recessive interleukin-1 receptor-associated kinase (IRAK)-4 and myeloid differentiation factor (MyD)88 deficiencies impair Toll-like receptor (TLR)- and interleukin-1 receptor-mediated immunity. We documented the clinical features and outcome of 48 patients with IRAK-4 deficiency and 12 patients with MyD88 deficiency, from 37 kindreds in 15 countries. The clinical features of IRAK-4 and MyD88 deficiency were indistinguishable. There were no severe viral, parasitic, and fungal diseases, and the range of bacterial infections was narrow. Noninvasive bacterial infections occurred in 52 patients, with a high incidence of infections of the upper respiratory tract and the skin, mostly caused by Pseudomonas aeruginosa and Staphylococcus aureus, respectively. The leading threat was invasive pneumococcal disease, documented in 41 patients (68%) and causing 72 documented invasive infections (52.2%). P. aeruginosa and Staph. aureus documented invasive infections also occurred (16.7% and 16%, respectively, in 25% and 25% of patients). Systemic signs of inflammation were usually weak or delayed. The first invasive infection occurred before the age of 2 years in 53 (88.3%) and in the neonatal period in 19 (32.7%) patients. Multiple or recurrent invasive infections were observed in most survivors (n = 36/50, 72%).
doi:10.1097/MD.0b013e3181fd8ec3
PMCID: PMC3103888  PMID: 21057262
22.  Revisiting Human IL-12Rβ1 Deficiency 
de Beaucoudrey, Ludovic | Samarina, Arina | Bustamante, Jacinta | Cobat, Aurélie | Boisson-Dupuis, Stéphanie | Feinberg, Jacqueline | Al-Muhsen, Saleh | Jannière, Lucile | Rose, Yoann | Desurenaim, Maylis | Kong, Xiao-Fei | Filipe-Santos, Orchidée | Chapgier, Ariane | Picard, Capucine | Fischer, Alain | Dogu, Figen | Ikinciogullari, Aydan | Tanir, Gonul | Hajjar, Sami Al | Jumaah, Suliman Al | Frayha, Husn H. | AlSum, Zobida | Ajaji, Sulaiman Al | Alangari, Abdullah | Al-Ghonaium, Abdulaziz | Adimi, Parisa | Mansouri, Davood | Mustapha, Imen Ben | Yancoski, Judith | Garty, Ben Zion | Rodriguez-Gallego, Carlos | Caragol, Isabel | Kutukculer, Necil | Kumararatne, Dinanthaka S. | Patel, Smita | Doffinger, Rainer | Exley, Andrew | Jeppsson, Olle | Reichenbach, Janine | Nadal, David | Boyko, Yaryna | Pietrucha, Barbara | Anderson, Suzanne | Levin, Michael | Schandené, Liliane | Schepers, Kinda | Efira, André | Mascart, Françoise | Matsuoka, Masao | Sakai, Tatsunori | Siegrist, Claire-Anne | Frecerova, Klara | Blüetters-Sawatzki, Renate | Bernhöft, Jutta | Freihorst, Joachim | Baumann, Ulrich | Richter, Darko | Haerynck, Filomeen | De Baets, Frans | Novelli, Vas | Lammas, David | Vermylen, Christiane | Tuerlinckx, David | Nieuwhof, Chris | Pac, Malgorzata | Haas, Walther H. | Müller-Fleckenstein, Ingrid | Fleckenstein, Bernhard | Levy, Jacob | Raj, Revathi | Cohen, Aileen Cleary | Lewis, David B. | Holland, Steven | Yang, Kuender D. | Wang, Xiaochuan | Jiang, Xiaohong Wang, Liping | Yang, Xiqiang | Zhu, Chaomin | Xie, Yuanyuan | Lee, Pamela Pui Wah | Chan, Koon Wing | Chen, Tong-Xin | Castro, Gabriela | Ivelisse, Natera | Codoceo, Ana | King, Alejandra | Bezrodnik, Liliana | Giovani, Daniela Di | Gaillard, Maria Isabel | de Moraes-Vasconcelos, Dewton | Grumach, Anete Sevciovic | Duarte, Alberto Jose da Silva | Aldana, Ruth | Espinosa-Rosales, Francisco Javier | Bejaoui, Mohammed | Bousfiha, Ahmed Aziz | El Baghdadi, Jamila | Özbek, Namik | Aksu, Guzide | Keser, Melike | Somer, Ayper | Hatipoglu, Nevin | Aydogmus, Çigdem | Asilsoy, Suna | Camcioglu, Yildiz | Gülle, Saniye | Ozgur, Tuba T. | Ozen, Meteran | Oleastro, Matias | Bernasconi, Andrea | Mamishi, Setareh | Parvaneh, Nima | Rosenzweig, Sergio | Barbouche, Ridha | Pedraza, Sigifredo | Lau, Yu Lung | Ehlayel, Mohammad S. | Fieschi, Claire | Abel, Laurent | Sanal, Ozden | Casanova, Jean-Laurent
Medicine  2010;89(6):381-402.
Interleukin-12 receptor β1 (IL-12Rβ1) deficiency is the most common form of Mendelian susceptibility to mycobacterial disease (MSMD). We undertook an international survey of 141 patients from 102 kindreds in 30 countries. Among 102 probands, the first infection occurred at a mean age of 2.4 years. In 78 patients, this infection was caused by Bacille Calmette-Guérin (BCG; n = 65), environmental mycobacteria (EM; also known as atypical or nontuberculous mycobacteria) (n = 9) or Mycobacterium tuberculosis (n = 4). Twenty-two of the remaining 24 probands initially presented with nontyphoidal, extraintestinal salmonellosis. Twenty of the 29 genetically affected sibs displayed clinical signs (69%); however 8 remained asymptomatic (27%). Nine nongenotyped sibs with symptoms died. Recurrent BCG infection was diagnosed in 15 cases, recurrent EM in 3 cases, recurrent salmonellosis in 22 patients. Ninety of the 132 symptomatic patients had infections with a single microorganism. Multiple infections were diagnosed in 40 cases, with combined mycobacteriosis and salmonellosis in 36 individuals. BCG disease strongly protected against subsequent EM disease (p = 0.00008). Various other infectious diseases occurred, albeit each rarely, yet candidiasis was reported in 33 of the patients (23%). Ninety-nine patients (70%) survived, with a mean age at last follow-up visit of 12.7 years ± 9.8 years (range, 0.5-46.4 yr). IL-12Rβ1 deficiency is characterized by childhood-onset mycobacteriosis and salmonellosis, rare recurrences of mycobacterial disease, and more frequent recurrence of salmonellosis. The condition has higher clinical penetrance, broader susceptibility to infections, and less favorable outcome than previously thought.
doi:10.1097/MD.0b013e3181fdd832
PMCID: PMC3129625  PMID: 21057261
23.  Human TRAF3 adaptor molecule deficiency leads to impaired Toll-like receptor 3 response and susceptibility to herpes simplex encephalitis 
Immunity  2010;33(3):400-411.
Tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3) functions downstream of multiple receptors that induce interferon-α (IFN-α), IFN–β and IFN-λ production, including Toll-like receptor 3 (TLR3), which is deficient in some patients with herpes simplex virus-1 encephalitis (HSE). Mice lacking TRAF3 die in the neonatal period, preventing direct investigation of the role of TRAF3 in immune responses and host defenses in vivo. Here we reported the autosomal dominant, human TRAF3 deficiency in a young adult with a history of HSE in childhood. The TRAF3 mutant allele was a loss-of-expression, loss-of-function, dominant-negative phenotype, and was associated with impaired, but not abolished TRAF3-dependent responses upon stimulation of both TNF receptors and receptors that induce IFN production. TRAF3 deficiency was associated with a clinical phenotype limited to HSE resulting from the impairment of TLR3-dependent induction of IFN. Thus, TLR3-mediated immunity against primary infection by HSV-1 in the central nervous system is critically dependent on TRAF3.
Highlight sentence
Autosomal dominant TRAF3 deficiency is a genetic etiology of herpes simplex encephalitis.
Highlight sentence
R118W TRAF3 allele is loss-of-function, loss-of-expression, and dominant-negative.
Highlight sentence
Human TRAF3 deficiency impairs the TLR3-dependent induction of anti-viral interferons.
doi:10.1016/j.immuni.2010.08.014
PMCID: PMC2946444  PMID: 20832341
Herpes simplex encephalitis (HSE); TRAF3; herpes simplex virus 1 (HSV-1); TLR3; interferon (IFN)
24.  Human metapneumovirus and human coronavirus infection and pathogenicity in Saudi children hospitalized with acute respiratory illness 
Annals of Saudi Medicine  2011;31(5):523-527.
BACKGROUND AND OBJECTIVES:
Human metapneumovirus (hMPV) and the Netherlands human coronavirus (HCoV-NL63) have been isolated from children with respiratory tract infection. The prevalence of these viruses has not been reported from Saudi Arabia. We sought to determine whether hMPV and HCoV-NL63 are responsible for acute respiratory illness and also to determine clinical features and severity of illness in the hospitalized pediatric patient population.
DESIGN AND SETTING:
Prospective hospital-based study from July 2007 to November 2008.
PATIENTS AND METHODS:
Nasopharyngeal specimens from children less than 16 years old who were suffering from acute respiratory diseases were tested for hMPV and HCoV-NL63 by reverse transcriptase–polymerase chain reaction. Samples were collected from July 2007 to November 2008.
RESULTS:
Both viruses were found among Saudi children with upper and lower respiratory tract diseases during the autumn and winter of 2007 and 2008, contributing to 11.1% of all viral diagnoses, with individual incidences of 8.3% (hMPV) and 2.8% (HCoV-NL63) among 489 specimens. Initial symptoms included fever, cough, and nasal congestion. Lower respiratory tract disease occurs in immunocompromised individuals and those with underlying conditions. Clinical findings of respiratory failure and culture-negative shock were established in 7 children infected with hMPV and having hematologic malignancies, myelofibrosis, Gaucher disease, and congenital immunodeficiency; 2 of the 7 patients died with acute respiratory failure. All children infected with HCoV-NL63 had underlying conditions; 1 of the 4 patients developed respiratory failure.
CONCLUSION:
hMPV and HCoV-NL63 are important causes of acute respiratory illness among hospitalized Saudi children. hMPV infection in the lower respiratory tract is associated with morbidity and mortality in immunocompromised children. HCoV-NL63 may cause severe lower respiratory disease with underlying conditions.
doi:10.4103/0256-4947.84633
PMCID: PMC3183689  PMID: 21911992
25.  Mutations in IRF8 and Human Dendritic Cell Immunodeficiency 
The New England journal of medicine  2011;365(2):127-138.
Background
The genetic analysis of human primary immunodeficiencies has defined the contribution of specific cell populations and molecular pathways in host defense against infections. Disseminated infection caused by BCG vaccines is an early manifestation of primary immunodeficiencies, such as severe combined immunodeficiency. In many affected individuals, the etiology of disseminated BCG disease is unexplained.
Methods
We investigated an infant presenting with features of severe immunodeficiency, including early-onset disseminated BCG disease, requiring hematopoietic stem cell transplantation. We also studied two otherwise healthy adults with a history of disseminated but curable BCG disease in childhood. We characterized the monocyte and dendritic cells compartments in these three persons and sequenced candidate genes, mutation of which could plausibly confer susceptibility to BCG disease.
Results
We detected two distinct disease-causing mutations affecting the transcriptional regulator IRF8. Both K108A and T80A mutations impair IRF8 transcriptional activity by disrupting IRF8 interaction with DNA. Mutation K108E was associated with an autosomal recessive severe immunodeficiency with a complete lack of circulating monocytes and dendritic cells. Mutation T80A was associated with an autosomal dominant milder immunodeficiency and a selective depletion of CD11c+ CD1c+ circulating dendritic cells.
Conclusions
These findings define a new class of human primary immunodeficiency, affecting the differentiation of mononuclear phagocytes. They also demonstrate that human IRF8 is critically required for the development of monocytes and dendritic cells and for anti-mycobacterial immunity.
doi:10.1056/NEJMoa1100066
PMCID: PMC3136554  PMID: 21524210

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