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1.
N Engl J Med ; 382(3): 256-265, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31940699

RESUMO

Deficiency of ubiquitin-specific peptidase 18 (USP18) is a severe type I interferonopathy. USP18 down-regulates type I interferon signaling by blocking the access of Janus-associated kinase 1 (JAK1) to the type I interferon receptor. The absence of USP18 results in unmitigated interferon-mediated inflammation and is lethal during the perinatal period. We describe a neonate who presented with hydrocephalus, necrotizing cellulitis, systemic inflammation, and respiratory failure. Exome sequencing identified a homozygous mutation at an essential splice site on USP18. The encoded protein was expressed but devoid of negative regulatory ability. Treatment with ruxolitinib was followed by a prompt and sustained recovery. (Funded by King Saud University and others.).


Assuntos
Doenças Hereditárias Autoinflamatórias/tratamento farmacológico , Interferons/metabolismo , Interleucinas/metabolismo , Janus Quinase 1/antagonistas & inibidores , Inibidores de Janus Quinases/uso terapêutico , Mutação com Perda de Função , Pirazóis/uso terapêutico , Ubiquitina Tiolesterase/deficiência , Homozigoto , Humanos , Hidrocefalia/genética , Recém-Nascido , Masculino , Receptores de Interferon/metabolismo , Indução de Remissão , Choque Séptico/genética , Transdução de Sinais/genética , Ubiquitina Tiolesterase/genética , Sequenciamento Completo do Exoma
2.
Nat Med ; 25(12): 1873-1884, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31806906

RESUMO

Herpes simplex virus-1 (HSV-1) encephalitis (HSE) is typically sporadic. Inborn errors of TLR3- and DBR1-mediated central nervous system cell-intrinsic immunity can account for forebrain and brainstem HSE, respectively. We report five unrelated patients with forebrain HSE, each heterozygous for one of four rare variants of SNORA31, encoding a small nucleolar RNA of the H/ACA class that are predicted to direct the isomerization of uridine residues to pseudouridine in small nuclear RNA and ribosomal RNA. We show that CRISPR/Cas9-introduced bi- and monoallelic SNORA31 deletions render human pluripotent stem cell (hPSC)-derived cortical neurons susceptible to HSV-1. Accordingly, SNORA31-mutated patient hPSC-derived cortical neurons are susceptible to HSV-1, like those from TLR3- or STAT1-deficient patients. Exogenous interferon (IFN)-ß renders SNORA31- and TLR3- but not STAT1-mutated neurons resistant to HSV-1. Finally, transcriptome analysis of SNORA31-mutated neurons revealed normal responses to TLR3 and IFN-α/ß stimulation but abnormal responses to HSV-1. Human SNORA31 thus controls central nervous system neuron-intrinsic immunity to HSV-1 by a distinctive mechanism.


Assuntos
Encefalite por Herpes Simples/genética , Herpesvirus Humano 1/genética , Neurônios/imunologia , RNA Nucleolar Pequeno/genética , Adulto , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/virologia , Pré-Escolar , Encefalite por Herpes Simples/imunologia , Encefalite por Herpes Simples/patologia , Encefalite por Herpes Simples/virologia , Feminino , Predisposição Genética para Doença , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 1/patogenicidade , Humanos , Imunidade/genética , Lactente , Masculino , Metagenoma/genética , Metagenoma/imunologia , Pessoa de Meia-Idade , Neurônios/virologia , RNA Nucleolar Pequeno/imunologia
3.
J Clin Immunol ; 39(4): 414-420, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31041574

RESUMO

BACKGROUND: One of the limiting factors for successful hematopoietic stem cell transplantation (HSCT) is graft versus host disease (GVHD). The EBMT/ESID guidelines for HSCT in severe combined immunodeficiency (SCID) recommend no GVHD prophylaxis for a matched sibling donor (MSD). OBJECTIVE: To determine the risk of GVHD in MSD HSCT for SCID patients compared to matched related donor (MRD). METHODS: This retrospective cohort study compares MSD with MRD and the outcome of GVHD in all SCID patients who underwent HSCT between 1993 and 2013. All statistical analyses were done using IBM SPSS statistics software. RESULTS: One hundred forty-five SCID patients underwent 152 HSCTs while 82 (54%) received GVHD prophylaxis. GVHD occurred in 48 patients (31.5%); 20/48 (42%) had GVHD prophylaxis compared to 28/48 (58%) that did not, P = 0.022. Acute GVHD occurred at a higher trend in MSD, 37/120 (30.8%), compared to MRD, 6/32 (18.8%), P = 0.17. We also analyzed the outcome according to the period of HSCT. The first period was 1993 to 2003, 48 HSCTs, 43 MSD, 5 MRD; all patients had GVHD prophylaxis, and there was no difference in GVHD. The second period was 2004 to 2013: of 104 HSCTs, 77 had MSD and 27 had MRD; GVHD prophylaxis was used in 22.1% of MSD and 63% of MRD, P = 0.000. GVHD was significantly higher in the MSD (40.2%) compared to MRD (18.5%) patients, P = 0.041. CONCLUSION: GVHD prophylaxis in MSD transplant should be considered in SCID patients.

4.
J Clin Immunol ; 38(3): 278-282, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29589181

RESUMO

PURPOSE: Mendelian susceptibility to mycobacterial disease (MSMD) is a rare primary immunodeficiency predisposing congenitally affected individuals to diseases caused by weakly virulent mycobacteria, such as Bacillus Calmette-Guérin (BCG) vaccine strains and environmental mycobacteria. IL-12p40 deficiency is a genetic etiology of MSMD resulting in impaired IL-12- and IL-23-dependent IFN-γ immunity. Most of the reported patients with IL-12p40 deficiency originate from Saudi Arabia (30 of 52) and carry the recurrent IL12B mutation c.315insA (27 of 30). METHODS: Whole-exome sequencing was performed on three patients from two unrelated kindreds from Saudi Arabia with disseminated disease caused by a BCG vaccine substrain. RESULTS: Genetic analysis revealed a homozygous mutation, p.W60X, in exon 3 of the IL12B gene, resulting in complete IL12p40 deficiency. This mutation is recurrent due to a new founder effect. CONCLUSIONS: This report provides evidence for a second founder effect for recurrent mutations of IL12B in Saudi Arabia.


Assuntos
Efeito Fundador , Estudos de Associação Genética , Predisposição Genética para Doença , Subunidade p40 da Interleucina-12/genética , Mutação , Infecções por Mycobacterium/etiologia , Pré-Escolar , Análise Mutacional de DNA , Exoma , Feminino , Humanos , Lactente , Masculino , Infecções por Mycobacterium/diagnóstico , Infecções por Mycobacterium/terapia , Linhagem , Arábia Saudita , Sequenciamento Completo do Exoma
5.
Cell ; 172(5): 952-965.e18, 2018 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-29474921

RESUMO

Viruses that are typically benign sometimes invade the brainstem in otherwise healthy children. We report bi-allelic DBR1 mutations in unrelated patients from different ethnicities, each of whom had brainstem infection due to herpes simplex virus 1 (HSV1), influenza virus, or norovirus. DBR1 encodes the only known RNA lariat debranching enzyme. We show that DBR1 expression is ubiquitous, but strongest in the spinal cord and brainstem. We also show that all DBR1 mutant alleles are severely hypomorphic, in terms of expression and function. The fibroblasts of DBR1-mutated patients contain higher RNA lariat levels than control cells, this difference becoming even more marked during HSV1 infection. Finally, we show that the patients' fibroblasts are highly susceptible to HSV1. RNA lariat accumulation and viral susceptibility are rescued by wild-type DBR1. Autosomal recessive, partial DBR1 deficiency underlies viral infection of the brainstem in humans through the disruption of tissue-specific and cell-intrinsic immunity to viruses.


Assuntos
Encefalopatias Metabólicas Congênitas/genética , Tronco Encefálico/metabolismo , Tronco Encefálico/virologia , RNA/química , RNA/metabolismo , Alelos , Sequência de Aminoácidos , Animais , Encefalopatias Metabólicas Congênitas/patologia , Tronco Encefálico/patologia , Encefalite Viral/genética , Escherichia coli/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/virologia , Herpesvirus Humano 1 , Humanos , Interferons/metabolismo , Íntrons/genética , Masculino , Camundongos , Proteínas Mutantes/metabolismo , Mutação/genética , Fases de Leitura Aberta/genética , Linhagem , RNA Nucleotidiltransferases/química , RNA Nucleotidiltransferases/deficiência , RNA Nucleotidiltransferases/genética , Receptor 3 Toll-Like/metabolismo , Replicação Viral
6.
J Asthma ; 55(11): 1157-1165, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29211635

RESUMO

OBJECTIVE: Interleukin 13 (IL-13) plays a critical pro-inflammatory role in asthma. Several single nucleotide polymorphisms (SNPs) are associated with asthma susceptibility in specific populations; however, further replicative studies in other ethnic groups are mandatory. METHODS: The association between IL-13 SNPs rs762534, rs20541, rs1295686, and rs1800925 (risk alleles A, A, T, and A, respectively) and asthma predisposition in a Saudi Arabian cohort was examined via a case-control cross-sectional study. RESULTS: The frequencies of alleles between asthmatics and control populations were significantly different for rs20541 and rs1295686 SNPs (p < 0.001), whereas the frequencies of genotypes between asthmatics and controls were significantly different only for rs20541. The association of the risk (minor) alleles with asthma was examined using the dominant genetic model. Individuals with at least one copy of the risk alleles A (for rs20541) and T (for rs1295686) had significantly greater odds of being asthmatic (OR = 2.13, 95% CI = 1.39-3.26, p < 0.0001; OR = 1.69, 95% CI = 1.12-2.54, p = 0.008) relative to their most common homozygous genotypes. On the other hand, the minor A alleles for rs762534 and rs1800925 were not significantly associated with asthma risk. Regarding haplotype association analysis, individuals with at least one copy of the minor "risk" allele for both rs20541 and rs1295686 (CATG and CATA, respectively) had greater odds of being asthmatic relative to CGCG haplotype; however, this trend was not statistically significant (p > 0.3). CONCLUSIONS: IL-13 minor T and A alleles for rs1295686 and rs20541, respectively, were associated with significantly higher risk of asthma in the Saudi Arabian population.


Assuntos
Asma/genética , Interleucina-13/genética , Adolescente , Adulto , Fatores Etários , Idoso , Alelos , Asma/epidemiologia , Criança , Pré-Escolar , Estudos Transversais , Feminino , Genótipo , Humanos , Hipersensibilidade Imediata/epidemiologia , Imunoglobulina E/sangue , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Arábia Saudita/epidemiologia , Fatores Sexuais , Fatores Socioeconômicos , Adulto Jovem
7.
J Asthma ; 54(9): 893-904, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28635548

RESUMO

BACKGROUND: In a subset of severe asthma patients, chronic airway inflammation is associated with infiltration of neutrophils, Th-17 cells and elevated expression of Th-17-derived cytokines (e.g., interleukin [IL]-17, IL-21, IL-22). Peripheral neutrophils from allergic asthmatics are known to express higher IL-17 cytokine levels than those from healthy subjects, but the regulatory mechanisms involved are not well understood. We hypothesize that Th-17 regulatory cytokines could modulate IL-17 expression in neutrophils. METHODS: Peripheral blood neutrophils isolated from asthmatics were stimulated with IL-21, IL-23, and IL-6 cytokines and their ability to produce IL-17A and IL-17F was determined relative to healthy controls. Signal transducer and activator of transcription 3 (STAT3) phosphorylation levels were measured in stimulated neutrophil using flow cytometry. The requirement for STAT3 phosphorylation was determined by blocking its activation using a specific chemical inhibitor. RESULTS: Stimulating asthmatic neutrophils with IL-21, 23, and 6 enhanced the production of IL-17A and IL-17F at significantly higher levels comparatively to healthy controls. Stimulating neutrophils with IL-21, IL-23, and IL-6 cytokines enhanced STAT3 phosphorylation, in all cases. Interestingly, inhibiting STAT3 phosphorylation using a specific chemical inhibitor dramatically blocked the ability of neutrophils to produce IL-17, demonstrating that STAT3 activation is the major factor mediating IL-17 gene expression. CONCLUSIONS: These findings suggest that neutrophil infiltration in lungs of severe asthmatics may represent an important source of pro-inflammatory IL-17A and -F cytokines, a production enhanced by Th-17 regulatory cytokines, and thus providing a feedback mechanism that sustains inflammation. Our results suggest that STAT3 pathway could be a potential target for regulating neutrophilic inflammation during severe asthma.


Assuntos
Asma/imunologia , Interleucina-16/imunologia , Interleucina-17/imunologia , Interleucina-23/imunologia , Interleucinas/imunologia , Neutrófilos/imunologia , Células Th17/imunologia , Adulto , Feminino , Humanos , Interleucina-16/biossíntese , Interleucina-17/biossíntese , Interleucina-23/biossíntese , Interleucinas/biossíntese , Masculino , Neutrófilos/metabolismo
8.
Saudi Pharm J ; 25(2): 224-230, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28344472

RESUMO

Glucuronidation is an important phase II pathway responsible for many endogenous substances and drug metabolism. The present work evaluated allele frequencies of certain UDP-glucuronosyl-transferases (UGT 1A6∗2, A7∗12, A8∗3, A9∗3, 2B7∗2, and 2B15∗2) in Saudi Arabians that could provide essential ethnic information. Blood samples from 192 healthy unrelated Saudi males of various geographic regions were collected. Genomic DNA was isolated and genotyping of various UGTs was carried out using polymerase chain reaction (PCR) followed by direct sequencing. For UGT1A6∗2 A/G genotype, the most common variant was the homozygous repeat (AA) and the most common allele was (A) with a frequency of 46.5% and 67.3%, respectively. Similarly, the most common variant for UGT1A7∗12 T/C genotype was the heterozygous repeat (TC) with a frequency of 78.7% while the mutant allele (C) was present in 60.6% of the study population. Both UGT1A8∗3 (G/A) and UGT1A9∗3 (T/C) showed only a wild homozygous pattern in all screened subjects. For UGT2B7∗2, the heterozygous repeat (TC) was found with a frequency of 57.3% and the alleles (A) showed a frequency of 50.8%. In contrast, for UGT2B15∗2 (G253T), the heterozygous repeat (TG) presented 62.3% of the subjects where the most common allele (G) was with a frequency of 66.2%. In conclusion, our data indicate that Saudis harbor some important UGT mutations known to affect enzyme activity. Additional studies are therefore, warranted to assess the clinical implications of these gene polymorphisms in this ethnic group.

9.
Pediatr Int ; 59(6): 661-668, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28218986

RESUMO

BACKGROUND: Maintaining good control of asthma symptoms can help to prevent exacerbations and its associated complications. The Asthma Control Test (ACT) can rapidly assess the effectiveness of asthma management plan and therapy. The aim of this study was therefore to identify risk factors associated with uncontrolled asthma symptoms in young Saudi asthmatic children (3-17 years old). METHODS: In this cross-sectional hospital-based survey, the ACT was administered to 297 asthmatic children/adolescents, recruited at the emergency department (ED) of two major hospitals. RESULTS: Most recruited patients had intermittent (63.5%) and mild persistent (27.6%) asthma; few had moderate persistent (8.9%) and none had severe asthma. These patients visited the ED four times (3.9 ± 3.2), on average. Almost half of the patients stated that they had not received education about asthma (47%) or education about medication use (43%). Most patients (60.3%) had uncontrolled symptoms (ACT score ≤19), of whom the intermittent asthma patients had better scores than those with more severe symptoms. Children ≤6 years old, with symptoms diagnosed <5 years previously and who were not attending school, had significantly worse control than older patients. Poor medication compliance and inappropriate inhaler device use were ascribed to younger patients (<12 years old) and worse scores; particularly in relation to stopping inhaled corticosteroid therapy when their symptoms improve. Patients with poor control also stated that they had not received education about inhaler device use. CONCLUSIONS: Most Saudi asthmatic children/adolescents visiting the ED had poor control of symptoms; indeed, none achieved complete control, which is related to deficient medication compliance and improper medication inhaler device use; deficient knowledge about asthma was also another factor hindering control.


Assuntos
Antiasmáticos/uso terapêutico , Asma/tratamento farmacológico , Conhecimentos, Atitudes e Prática em Saúde , Adesão à Medicação , Administração por Inalação , Adolescente , Asma/psicologia , Criança , Pré-Escolar , Estudos Transversais , Progressão da Doença , Serviço Hospitalar de Emergência , Feminino , Humanos , Masculino , Nebulizadores e Vaporizadores , Fatores de Risco , Arábia Saudita , Índice de Gravidade de Doença , Resultado do Tratamento
12.
Proc Natl Acad Sci U S A ; 113(51): E8277-E8285, 2016 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-27930337

RESUMO

Chronic mucocutaneous candidiasis (CMC) is defined as recurrent or persistent infection of the skin, nails, and/or mucosae with commensal Candida species. The first genetic etiology of isolated CMC-autosomal recessive (AR) IL-17 receptor A (IL-17RA) deficiency-was reported in 2011, in a single patient. We report here 21 patients with complete AR IL-17RA deficiency, including this first patient. Each patient is homozygous for 1 of 12 different IL-17RA alleles, 8 of which create a premature stop codon upstream from the transmembrane domain and have been predicted and/or shown to prevent expression of the receptor on the surface of circulating leukocytes and dermal fibroblasts. Three other mutant alleles create a premature stop codon downstream from the transmembrane domain, one of which encodes a surface-expressed receptor. Finally, the only known missense allele (p.D387N) also encodes a surface-expressed receptor. All of the alleles tested abolish cellular responses to IL-17A and -17F homodimers and heterodimers in fibroblasts and to IL-17E/IL-25 in leukocytes. The patients are currently aged from 2 to 35 y and originate from 12 unrelated kindreds. All had their first CMC episode by 6 mo of age. Fourteen patients presented various forms of staphylococcal skin disease. Eight were also prone to various bacterial infections of the respiratory tract. Human IL-17RA is, thus, essential for mucocutaneous immunity to Candida and Staphylococcus, but otherwise largely redundant. A diagnosis of AR IL-17RA deficiency should be considered in children or adults with CMC, cutaneous staphylococcal disease, or both, even if IL-17RA is detected on the cell surface.


Assuntos
Infecções Bacterianas/imunologia , Candidíase/imunologia , Micoses/imunologia , Receptores de Interleucina-17/deficiência , Receptores de Interleucina-17/genética , Alelos , Candida , Membrana Celular , Criança , Pré-Escolar , Saúde da Família , Feminino , Fibroblastos/metabolismo , Genes Recessivos , Estudo de Associação Genômica Ampla , Células HEK293 , Homozigoto , Humanos , Imunofenotipagem , Lactente , Recém-Nascido , Interleucina-17/metabolismo , Masculino , Mutação , Fases de Leitura Aberta , Linhagem , Receptores de Interleucina-17/metabolismo , Pele/microbiologia , Linfócitos T/citologia
13.
Exp Mol Med ; 48(10): e262, 2016 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-27713399

RESUMO

Drug resistance and the harmful side effects accompanying the prolonged corticosteroid treatment of chronic pulmonary diseases prompted the development of more specific anti-inflammatory approaches. Several strategies aiming to block IL4Rα, the receptor for a key pro-inflammatory pathway, were investigated. However, their efficiency was limited, mostly due to the systemic or subcutaneous route of administrations. In this paper, we examined the ability of an intranasal treatment with biocompatible nanoparticles targeting IL4Rα to control lung inflammation in ovalbumin (OVA)-sensitized mice. OVA-sensitized mice were treated with anti-IL4Rα-conjugated nanoparticles. The levels of pro-inflammatory cytokines in the lungs and broncho-alveolar lavage fluid (BALF) were determined using a cytokine array assay. The effects of nanoparticle treatment on the activation of lung inflammatory cells and their ability to proliferate and produce cytokines were determined using fluorescence-activated cell sorting (FACS) analysis. Lung inflammation was also monitored using immunohistochemical staining. Treatment with the anti-IL4Rα nanoparticles significantly decreased pro-inflammatory cytokine expression and release in BALF and airway lung tissue in mice. The numbers of lung tissue lymphocytes, neutrophils and eosinophils were also decreased. Interestingly, anti-IL4Rα nanoparticles deactivated CD4 and CD8 T cells in lung tissue and inhibited their ability to produce pro-inflammatory cytokines to a significantly lower level than the treatment with free anti-IL4Rα. Moreover, they induced a sustained low level of lung inflammation for 1 week following the last instillation compared with the treatment with free anti-IL4Rα antibodies. Together, this data suggested that the enhanced tissue penetrability and sustainability of these nanoparticles improved the strength and durability of the immunosuppressive effects of anti-IL4Rα.


Assuntos
Asma/terapia , Imunoconjugados/uso terapêutico , Nanoconjugados/uso terapêutico , Pneumonia/terapia , Animais , Asma/complicações , Asma/imunologia , Asma/patologia , Citocinas/imunologia , Feminino , Imunoglobulina E/imunologia , Imunoterapia , Pulmão/imunologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Pneumonia/complicações , Pneumonia/imunologia , Pneumonia/patologia , Receptores de Superfície Celular/imunologia
14.
J Allergy Clin Immunol ; 137(6): 1780-1787, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26915675

RESUMO

BACKGROUND: Molecular genetics techniques are an essential diagnostic tool for primary immunodeficiency diseases (PIDs). The use of next-generation sequencing (NGS) provides a comprehensive way of concurrently screening a large number of PID genes. However, its validity and cost-effectiveness require verification. OBJECTIVES: We sought to identify and overcome complications associated with the use of NGS in a comprehensive gene panel incorporating 162 PID genes. We aimed to ascertain the specificity, sensitivity, and clinical sensitivity of the gene panel and its utility as a diagnostic tool for PIDs. METHODS: A total of 162 PID genes were screened in 261 patients by using the Ion Torrent Proton NGS sequencing platform. Of the 261 patients, 122 had at least 1 known causal mutation at the onset of the study and were used to assess the specificity and sensitivity of the assay. The remaining samples were from unsolved cases that were biased toward more phenotypically and genotypically complicated cases. RESULTS: The assay was able to detect the mutation in 117 (96%) of 122 positive control subjects with known causal mutations. For the unsolved cases, our assay resulted in a molecular genetic diagnosis for 35 of 139 patients. Interestingly, most of these cases represented atypical clinical presentations of known PIDs. CONCLUSIONS: The targeted NGS PID gene panel is a sensitive and cost-effective diagnostic tool that can be used as a first-line molecular assay in patients with PIDs. The assay is an alternative choice to the complex and costly candidate gene approach, particularly for patients with atypical presentation of known PID genes.


Assuntos
Marcadores Genéticos , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Síndromes de Imunodeficiência/diagnóstico , Síndromes de Imunodeficiência/genética , Biologia Computacional , Variações do Número de Cópias de DNA , Análise Mutacional de DNA , Testes Genéticos , Estudo de Associação Genômica Ampla , Humanos , Síndromes de Imunodeficiência/imunologia , Mutação , Polimorfismo de Nucleotídeo Único , Fluxo de Trabalho
15.
AAPS PharmSciTech ; 17(4): 978-87, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26729529

RESUMO

In this study, we investigated whether tacrolimus extracted and purified from the commercial capsules (Prograf® 5 mg) have retained its original quality and activity beyond the capsules expiration date in order to be reused for research purposes after extraction. High-performance liquid chromatography (HPLC) assay method was developed and validated for the quantification of tacrolimus, using cyclosporine A as an internal standard (IS). Moreover, a combination of analytical methods, including nuclear magnetic resonance (NMR), gas chromatography-mass spectrometry (GC-MS), Fourier transform-infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), and differential scanning calorimetry (DSC) were used to assess the quality of extracted/purified tacrolimus. Suppression of murine peripheral-blood mononuclear cells (PBMC) proliferation and the levels of interleukin-2 (IL-2) and interferon gamma (IFN-γ) were also assessed. The data obtained showed no detectable differences in the quality profile between the authentic sample and extracted drug. Also, the results showed that the extracted/purified tacrolimus was able to suppress T cell proliferation, induced by concanavalin A, indicating the retained pharmacological activity. We proved that tacrolimus extracted/purified from expired Prograf® capsuled retains its purity and immunosuppressive activity and can be reused for research and possibly in pharmaceutical manufacturing.


Assuntos
Cápsulas/química , Cápsulas/farmacologia , Tacrolimo/química , Tacrolimo/farmacologia , Animais , Cromatografia Líquida de Alta Pressão/métodos , Ciclosporina/química , Cromatografia Gasosa-Espectrometria de Massas/métodos , Imunossupressores/química , Imunossupressores/farmacologia , Interferon gama/metabolismo , Interleucina-2/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Espectroscopia de Ressonância Magnética/métodos , Camundongos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Difração de Raios X/métodos
16.
Respir Res ; 17: 6, 2016 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-26772733

RESUMO

BACKGROUND: Although corticosteroid is a powerful anti-inflammatory drug that is used widely to control asthma, still severe asthmatics can develop steroid resistance. Airway fibroblasts are quite resistant to steroids during Idiopathic pulmonary fibrosis (IPF) and fibrosis in asthmatic lungs is not always controlled. Th-17 regulatory cytokine which are elevated in lung tissues of asthmatics were shown to enhance the survival of various types of cells. STAT factors are central to this anti-apoptotic function. However, it is not yet clear whether these cytokines contribute to steroid hypo-responsiveness in asthma. Therefore, in this study, we investigated the ability of Th-17 regulatory cytokines, specifically IL-21, IL22 and IL23, to protect structural airway cells against dexamethasone-induced apoptosis. METHODS: Primary human fibroblasts, ASM cells, and lung endothelial cells line were treated with IL-21, IL-22, and IL-23 cytokines before incubation with dexamethasone and the level of apoptosis was determined by measuring cellular Annexin-V using Flow cytometry. RESULTS: Our data indicated that treatment with Th-17 regulatory cytokines was effective in inhibiting induced apoptosis for both fibroblasts and endothelial cells but not ASM cells. STAT3 phosphorylation levels were also upregulated in fibroblasts and endothelial upon treatment with these cytokines. Interestingly, inhibiting STAT3 phosphorylation abrogated IL-21, IL-22, and IL-23 anti-apoptotic effect on fibroblasts and endothelial cells. CONCLUSIONS: This data suggest that Th-17 regulatory cytokines may play a critical role in regulating the survival of fibroblasts during asthma, IPF as well as other chronic lung inflammatory diseases leading to enhanced fibrosis. Accordingly, findings of this paper may pave the way for more extensive research on the role of these regulatory cytokines in fibrosis development in various chronic inflammatory diseases.


Assuntos
Apoptose/imunologia , Citocinas/imunologia , Dexametasona/administração & dosagem , Pulmão/citologia , Pulmão/imunologia , Células Th17/imunologia , Corticosteroides/administração & dosagem , Anti-Inflamatórios/administração & dosagem , Apoptose/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Fatores Imunológicos/imunologia , Pulmão/efeitos dos fármacos , Células Th17/citologia , Células Th17/efeitos dos fármacos
17.
Contrast Media Mol Imaging ; 11(3): 172-83, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26708935

RESUMO

Simultaneous inhibition of IL4 and IL13 via the common receptor chain IL4Rα to block adequately their biologic effects presents a promising therapeutic approach to give the additional relief required for asthma patients. In this study, superparamagnetic iron oxide nanoparticles were conjugated with anti-IL4Rα blocking antibodies via polyethylene glycol (PEG) polymers. The delivery of these blocking antibodies to the inflammatory sites in the lung via the developed nanocarriers was assessed using noninvasive free-breathing pulmonary MRI. Biocompatibility assays confirmed the safety of the developed nanocarriers for pre-clinical investigations. For all the investigated formulations, nanocarriers were found to be very stable at neutral pH. However, the stability noticeably decreased with the PEG length in acidic environment and thus the loaded antibodies were preferentially released. Immunofluorescence and fluorimetry assays confirmed the binding of the nanocarriers to the IL4Rα asthma biomarker. Pulmonary MRI performed using an ultra-short echo time sequence allowed simultaneous noninvasive monitoring of inflammatory responses induced by ovalbumin challenge and tracking of the developed nanocarriers, which were found to colocalize with the inflammatory sites in the lung. Targeting of the developed nanocarriers to areas rich in IL4Rα positive inflammatory cells was confirmed using histological and flow cytometry analyses. The anti-IL4Rα-conjugated nanocarriers developed here have been confirmed to be efficient in targeting key inflammatory cells during chronic lung inflammation following intrapulmonary administration. Targeting efficiency was monitored using noninvasive MRI, allowing detection of the nanocarriers' colocalizations with the inflammatory sites in the lung of ovalbumin-challenged asthmatic mice. Copyright © 2015 John Wiley & Sons, Ltd.


Assuntos
Asma/diagnóstico por imagem , Portadores de Fármacos/química , Pulmão/diagnóstico por imagem , Imagem por Ressonância Magnética/métodos , Imagem Molecular/métodos , Animais , Asma/patologia , Biomarcadores/análise , Feminino , Imunoconjugados/química , Inflamação/diagnóstico por imagem , Isoanticorpos/administração & dosagem , Isoanticorpos/imunologia , Isoanticorpos/metabolismo , Pulmão/patologia , Nanopartículas de Magnetita , Camundongos , Camundongos Endogâmicos BALB C , Polietilenoglicóis , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo
18.
J Clin Immunol ; 35(7): 651-60, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26395454

RESUMO

PURPOSE: Primary immunodeficiencies (PID) are a group of heterogeneous diseases. Epidemiological studies from databases worldwide show geographical variation. In this study the objective is to determine the spectrum of PID in Saudi Arabia by analyzing the database in a referral tertiary hospital. METHODS: This is a prospective data collection by interviews and medical chart review for all PID patients followed at the King Faisal Specialist Hospital & Research Center (KFSH&RC) from May 2010 to April 2013. RESULTS: A total of 502 patients presented (53 % male and 47 % female). Combined immunodeficiencies were the most common (59.7 %), followed by predominantly antibody deficiencies (12.3 %), congenital defects of phagocyte (9.4 %), combined immunodeficiencies with associated or syndromic features (6.2 %), disease of immune dysregulation (6 %), complement deficiencies (5.8), and defects in innate immunity (0.6 %). The most common combined immunodeficiencies phenotype was T-B-SCID (17 %). The patients' ages ranged from less than 1 year old to 78 years, and 394 patients (78.2 %) are in the paediatrics age group (<14 years). The overall mean age of symptoms onset was 17 months and the overall mean delay in diagnosis was 21.6 months. Recurrent infections were the most common occurring clinical presentation (66 %), followed by family history (26 %). Consanguinity was found in 75 % of the patients. A total of 308 (61 %) patients had undergone stem cell transplantation (SCT). CONCLUSION: The study revealed that combined immunodeficiencies are not uncommon and are the most frequent occurring diagnosis in our patient population. This study is a prerequisite to establish a national registry of primary immunodeficiency in Saudi Arabia.


Assuntos
Linfócitos B/fisiologia , Síndromes de Imunodeficiência/epidemiologia , Linfócitos T/fisiologia , Centros de Atenção Terciária , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Humanos , Síndromes de Imunodeficiência/complicações , Síndromes de Imunodeficiência/diagnóstico , Lactente , /etiologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Recidiva , Arábia Saudita , Transplante de Células-Tronco , Adulto Jovem
19.
J Exp Med ; 212(10): 1641-62, 2015 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-26304966

RESUMO

Autosomal recessive, complete TYK2 deficiency was previously described in a patient (P1) with intracellular bacterial and viral infections and features of hyper-IgE syndrome (HIES), including atopic dermatitis, high serum IgE levels, and staphylococcal abscesses. We identified seven other TYK2-deficient patients from five families and four different ethnic groups. These patients were homozygous for one of five null mutations, different from that seen in P1. They displayed mycobacterial and/or viral infections, but no HIES. All eight TYK2-deficient patients displayed impaired but not abolished cellular responses to (a) IL-12 and IFN-α/ß, accounting for mycobacterial and viral infections, respectively; (b) IL-23, with normal proportions of circulating IL-17(+) T cells, accounting for their apparent lack of mucocutaneous candidiasis; and (c) IL-10, with no overt clinical consequences, including a lack of inflammatory bowel disease. Cellular responses to IL-21, IL-27, IFN-γ, IL-28/29 (IFN-λ), and leukemia inhibitory factor (LIF) were normal. The leukocytes and fibroblasts of all seven newly identified TYK2-deficient patients, unlike those of P1, responded normally to IL-6, possibly accounting for the lack of HIES in these patients. The expression of exogenous wild-type TYK2 or the silencing of endogenous TYK2 did not rescue IL-6 hyporesponsiveness, suggesting that this phenotype was not a consequence of the TYK2 genotype. The core clinical phenotype of TYK2 deficiency is mycobacterial and/or viral infections, caused by impaired responses to IL-12 and IFN-α/ß. Moreover, impaired IL-6 responses and HIES do not appear to be intrinsic features of TYK2 deficiency in humans.


Assuntos
Síndrome de Job/etiologia , TYK2 Quinase/deficiência , Adolescente , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Interferon gama/metabolismo , Interleucina-10/farmacologia , Interleucina-12/metabolismo , Interleucina-12/farmacologia , Interleucina-23/farmacologia , Interleucina-6/farmacologia , Síndrome de Job/complicações , Síndrome de Job/genética , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Masculino , Mutação , Infecções por Mycobacterium/etiologia , Linfócitos T/metabolismo , Linfócitos T/patologia , TYK2 Quinase/genética , TYK2 Quinase/metabolismo , Viroses/etiologia , Adulto Jovem
20.
Science ; 349(6248): 606-613, 2015 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-26160376

RESUMO

Human inborn errors of immunity mediated by the cytokines interleukin-17A and interleukin-17F (IL-17A/F) underlie mucocutaneous candidiasis, whereas inborn errors of interferon-γ (IFN-γ) immunity underlie mycobacterial disease. We report the discovery of bi-allelic RORC loss-of-function mutations in seven individuals from three kindreds of different ethnic origins with both candidiasis and mycobacteriosis. The lack of functional RORγ and RORγT isoforms resulted in the absence of IL-17A/F-producing T cells in these individuals, probably accounting for their chronic candidiasis. Unexpectedly, leukocytes from RORγ- and RORγT-deficient individuals also displayed an impaired IFN-γ response to Mycobacterium. This principally reflected profoundly defective IFN-γ production by circulating γδ T cells and CD4(+)CCR6(+)CXCR3(+) αß T cells. In humans, both mucocutaneous immunity to Candida and systemic immunity to Mycobacterium require RORγ, RORγT, or both.


Assuntos
Candida albicans/imunologia , Candidíase Mucocutânea Crônica/genética , Imunidade/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Imunodeficiência Combinada Severa/genética , Tuberculose Bovina/genética , Tuberculose Pulmonar/genética , Alelos , Animais , Candidíase Mucocutânea Crônica/complicações , Candidíase Mucocutânea Crônica/imunologia , Bovinos , Criança , Pré-Escolar , Análise Mutacional de DNA , Exoma/genética , Feminino , Rearranjo Gênico da Cadeia alfa dos Receptores de Antígenos dos Linfócitos T , Humanos , Interferon gama/imunologia , Interleucina-17/imunologia , Camundongos , Mutação , Mycobacterium bovis/imunologia , Mycobacterium bovis/isolamento & purificação , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/isolamento & purificação , Linhagem , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/imunologia , Timo/anormalidades , Timo/imunologia , Tuberculose Bovina/imunologia , Tuberculose Pulmonar/imunologia
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