RESUMO
We study a patient with the human papilloma virus (HPV)-2-driven "tree-man" phenotype and two relatives with unusually severe HPV4-driven warts. The giant horns form an HPV-2-driven multifocal benign epithelial tumor overexpressing viral oncogenes in the epidermis basal layer. The patients are unexpectedly homozygous for a private CD28 variant. They have no detectable CD28 on their T cells, with the exception of a small contingent of revertant memory CD4+ T cells. T cell development is barely affected, and T cells respond to CD3 and CD2, but not CD28, costimulation. Although the patients do not display HPV-2- and HPV-4-reactive CD4+ T cells in vitro, they make antibodies specific for both viruses in vivo. CD28-deficient mice are susceptible to cutaneous infections with the mouse papillomavirus MmuPV1. The control of HPV-2 and HPV-4 in keratinocytes is dependent on the T cell CD28 co-activation pathway. Surprisingly, human CD28-dependent T cell responses are largely redundant for protective immunity.
Assuntos
Antígenos CD28/deficiência , Padrões de Herança/genética , Papillomaviridae/fisiologia , Pele/virologia , Linfócitos T/imunologia , Adulto , Sequência de Aminoácidos , Animais , Sequência de Bases , Antígenos CD28/genética , Antígenos CD28/metabolismo , Linfócitos T CD4-Positivos/imunologia , Criança , Endopeptidases/metabolismo , Feminino , Genes Recessivos , Células HEK293 , Homozigoto , Humanos , Imunidade Humoral , Memória Imunológica , Células Jurkat , Queratinócitos/patologia , Masculino , Camundongos Endogâmicos C57BL , Oncogenes , Papiloma/patologia , Papiloma/virologia , Linhagem , Sinais Direcionadores de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Most cases of herpes simplex virus 1 (HSV-1) encephalitis (HSE) remain unexplained1,2. Here, we report on two unrelated people who had HSE as children and are homozygous for rare deleterious variants of TMEFF1, which encodes a cell membrane protein that is preferentially expressed by brain cortical neurons. TMEFF1 interacts with the cell-surface HSV-1 receptor NECTIN-1, impairing HSV-1 glycoprotein D- and NECTIN-1-mediated fusion of the virus and the cell membrane, blocking viral entry. Genetic TMEFF1 deficiency allows HSV-1 to rapidly enter cortical neurons that are either patient specific or derived from CRISPR-Cas9-engineered human pluripotent stem cells, thereby enhancing HSV-1 translocation to the nucleus and subsequent replication. This cellular phenotype can be rescued by pretreatment with type I interferon (IFN) or the expression of exogenous wild-type TMEFF1. Moreover, ectopic expression of full-length TMEFF1 or its amino-terminal extracellular domain, but not its carboxy-terminal intracellular domain, impairs HSV-1 entry into NECTIN-1-expressing cells other than neurons, increasing their resistance to HSV-1 infection. Human TMEFF1 is therefore a host restriction factor for HSV-1 entry into cortical neurons. Its constitutively high abundance in cortical neurons protects these cells from HSV-1 infection, whereas inherited TMEFF1 deficiency renders them susceptible to this virus and can therefore underlie HSE.
Assuntos
Encéfalo , Encefalite por Herpes Simples , Herpesvirus Humano 1 , Proteínas de Membrana , Internalização do Vírus , Animais , Feminino , Humanos , Masculino , Encéfalo/citologia , Encéfalo/metabolismo , Encéfalo/virologia , Encefalite por Herpes Simples/virologia , Encefalite por Herpes Simples/metabolismo , Herpesvirus Humano 1/patogenicidade , Herpesvirus Humano 1/fisiologia , Homozigoto , Interferon Tipo I/metabolismo , Interferon Tipo I/imunologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Nectinas/genética , Nectinas/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Neurônios/virologia , Células-Tronco Pluripotentes/citologia , Replicação Viral , Pré-Escolar , Adulto Jovem , LinhagemRESUMO
Genome-wide approaches, such as whole-exome sequencing (WES), are widely used to decipher the genetic mechanisms underlying inter-individual variability in disease susceptibility. We aimed to dissect inborn monogenic determinants of idiopathic liver injury in otherwise healthy children. We thus performed WES for 20 patients presented with paediatric-onset recurrent elevated transaminases (rELT) or acute liver failure (ALF) of unknown aetiology. A stringent variant screening was undertaken on a manually-curated panel of 380 genes predisposing to inherited human diseases with hepatobiliary involvement in the OMIM database. We identified rare nonsynonymous variants in nine genes in six patients (five rELT and one ALF). We next performed a case-level evaluation to assess the causal concordance between the gene mutated and clinical symptoms of the affected patient. A genetic diagnosis was confirmed in four rELT patients (40%), among whom two carried novel mutations in ACOX2 or PYGL, and two had previously-reported morbid variants in ABCB4 or PHKA2. We also detected rare variants with uncertain clinical significance in CDAN1, JAG1, PCK2, SLC27A5 or VPS33B in rELT or ALF patients. In conclusion, implementation of WES improves diagnostic yield and enables precision management in paediatric cases of liver injury with unknown aetiology, in particular recurrent hypertransaminasemia.
Assuntos
Sequenciamento do Exoma , Predisposição Genética para Doença , Mutação , Humanos , Masculino , Criança , Feminino , Pré-Escolar , Lactente , Adolescente , Falência Hepática Aguda/genética , Falência Hepática Aguda/diagnóstico , Transaminases/genética , Hepatopatias/genética , Hepatopatias/diagnósticoRESUMO
Insufficient dietary folate intake, hereditary malabsorption, or defects in folate transport may lead to combined immunodeficiency (CID). Although loss of function mutations in the major intestinal folate transporter PCFT/SLC46A1 was shown to be associated with CID, the evidence for pathogenic variants of RFC/SLC19A1 resulting in immunodeficiency was lacking. We report two cousins carrying a homozygous pathogenic variant c.1042 G > A, resulting in p.G348R substitution who showed symptoms of immunodeficiency associated with defects of folate transport. SLC19A1 expression by peripheral blood mononuclear cells (PBMC) was quantified by real-time qPCR and immunostaining. T cell proliferation, methotrexate resistance, NK cell cytotoxicity, Treg cells and cytokine production by T cells were examined by flow cytometric assays. Patients were treated with and benefited from folinic acid. Studies revealed normal NK cell cytotoxicity, Treg cell counts, and naive-memory T cell percentages. Although SLC19A1 mRNA and protein expression were unaltered, remarkably, mitogen induced-T cell proliferation was significantly reduced at suboptimal folic acid and supraoptimal folinic acid concentrations. In addition, patients' PBMCs were resistant to methotrexate-induced apoptosis supporting a functionally defective SLC19A1. This study presents the second pathogenic SLC19A1 variant in the literature, providing the first experimental evidence that functionally defective variants of SLC19A1 may present with symptoms of immunodeficiency.
Assuntos
Síndromes de Imunodeficiência , Leucovorina , Proteína Carregadora de Folato Reduzido , Humanos , Ácido Fólico/genética , Ácido Fólico/metabolismo , Leucovorina/uso terapêutico , Leucovorina/metabolismo , Leucócitos Mononucleares/metabolismo , Metotrexato/farmacologia , Metotrexato/uso terapêutico , Transportador de Folato Acoplado a Próton/genética , Transportador de Folato Acoplado a Próton/metabolismo , Proteína Carregadora de Folato Reduzido/genética , Síndromes de Imunodeficiência/tratamento farmacológico , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/metabolismoRESUMO
Human herpesvirus-6 (HHV-6) infection can rarely cause life-threatening conditions, such as encephalitis, in otherwise healthy children, with unclear pathogenesis. We studied a child who presented with acute HHV-6 encephalitis at the age of 10 months and who was homozygous for a novel missense mutation in IRAK4, encoding interleukin-1 receptor-associated kinase 4, identified by whole-exome sequencing. We tested the damaging impact of this mutation in silico by molecular dynamics simulations and in vitro by biochemical and functional experiments utilizing cell lines and patient's cells. We found that the mutation is severely hypomorphic, impairing both the expression and function of IRAK-4. Patient's leukocytes had barely detectable levels of IRAK-4 and diminished anti-viral immune responses to various stimuli inducing different Toll-like receptors and cytosolic nucleic acid sensors. Overall, these findings suggest that acute HHV-6 encephalitis can result from inborn errors of immunity to virus. This study represents the first report of isolated acute HHV-6 infection causing encephalitis in an inherited primary immunodeficiency, notably autosomal recessive (AR) partial IRAK-4 deficiency, and the first report of AR IRAK-4 deficiency presenting with a severe viral disease, notably HHV-6 encephalitis upon an acute infection, thereby expanding the clinical spectrum of IRAK-4 deficiency.
Assuntos
Encefalite Viral , Herpesvirus Humano 6 , Doenças da Imunodeficiência Primária , Infecções por Roseolovirus , Criança , Humanos , Lactente , Quinases Associadas a Receptores de Interleucina-1/genética , Receptores Toll-Like/metabolismo , Doenças da Imunodeficiência Primária/genética , Encefalite Viral/diagnóstico , Encefalite Viral/genética , Herpesvirus Humano 6/genética , Infecções por Roseolovirus/diagnóstico , Infecções por Roseolovirus/genéticaRESUMO
Fulminant viral hepatitis (FVH) caused by hepatitis A virus (HAV) is a life-threatening disease that typically strikes otherwise healthy individuals. The only known genetic etiology of FVH is inherited IL-18BP deficiency, which unleashes IL-18-dependent lymphocyte cytotoxicity and IFN-γ production. We studied two siblings who died from a combination of early-onset inflammatory bowel disease (EOIBD) and FVH due to HAV. The sibling tested was homozygous for the W100G variant of IL10RB previously described in an unrelated patient with EOIBD. We show here that the out-of-frame IL10RB variants seen in other EOIBD patients disrupt cellular responses to IL-10, IL-22, IL-26, and IFN-λs in overexpression conditions and in homozygous cells. By contrast, the impact of in-frame disease-causing variants varies between cases. When overexpressed, the W100G variant impairs cellular responses to IL-10, but not to IL-22, IL-26, or IFN-λ1, whereas cells homozygous for W100G do not respond to IL-10, IL-22, IL-26, or IFN-λ1. As IL-10 is a potent antagonist of IFN-γ in phagocytes, these findings suggest that the molecular basis of FVH in patients with IL-18BP or IL-10RB deficiency may involve excessive IFN-γ activity during HAV infections of the liver. Inherited IL-10RB deficiency, and possibly inherited IL-10 and IL-10RA deficiencies, confer a predisposition to FVH, and patients with these deficiencies should be vaccinated against HAV and other liver-tropic viruses.
Assuntos
Hepatite Viral Humana , Interleucina-10 , Humanos , Interleucina-10/genética , Irmãos , Interferon gama/genéticaRESUMO
BACKGROUND: Cytomegalovirus (CMV) can cause severe disease in children and adults with a variety of inherited or acquired T-cell immunodeficiencies, who are prone to multiple infections. It can also rarely cause disease in otherwise healthy persons. The pathogenesis of idiopathic CMV disease is unknown. Inbred mice that lack the gene encoding nitric oxide synthase 2 (Nos2) are susceptible to the related murine CMV infection. METHODS: We studied a previously healthy 51-year-old man from Iran who after acute CMV infection had an onset of progressive CMV disease that led to his death 29 months later. We hypothesized that the patient may have had a novel type of inborn error of immunity. Thus, we performed whole-exome sequencing and tested candidate mutant alleles experimentally. RESULTS: We found a homozygous frameshift mutation in NOS2 encoding a truncated NOS2 protein that did not produce nitric oxide, which determined that the patient had autosomal recessive NOS2 deficiency. Moreover, all NOS2 variants that we found in homozygosity in public databases encoded functional proteins, as did all other variants with an allele frequency greater than 0.001. CONCLUSIONS: These findings suggest that inherited NOS2 deficiency was clinically silent in this patient until lethal infection with CMV. Moreover, NOS2 appeared to be redundant for control of other pathogens in this patient. (Funded by the National Center for Advancing Translational Sciences and others.).
Assuntos
Infecções por Citomegalovirus , Mutação da Fase de Leitura , Óxido Nítrico Sintase Tipo II/deficiência , Evolução Fatal , Feminino , Genótipo , Homozigoto , Humanos , Mutação com Perda de Função , Masculino , Pessoa de Meia-Idade , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Linhagem , Sequenciamento do ExomaRESUMO
INTRODUCTION: Pentraxin-3 (PTX3) is a soluble long pentraxin molecule that regulates inflammatory responses. This study aimed to determine the plasma levels of plasma PTX-3 as an inflammation marker in chronic spontaneous urticaria (CSU) and whether the PTX3 levels correlate with disease activity and other clinical parameters, including acute phase reactants and biomarkers. METHODS: The study included 70 CSU patients and 30 healthy controls. Plasma PTX3 levels were measured by ELISA. CSU disease activity was evaluated with the urticaria activity score summed over 7 days. Complete blood count, C-reactive protein (CRP), transaminases, total IgE, antinuclear antibody, anti-thyroid peroxidase, anti-thyroglobulin, and D-dimer levels were recorded. RESULTS: Of the 70 patients, 52 (74.3%) were female, with a mean age of 37.51 ± 11.80 years. Disease activity was severe in 43, moderate in 15, and mild in 12 patients. Mean PTX3 levels were elevated in CSU patients compared to healthy controls (0.81 vs. 0.55 ng/mL, p = 0.031). The mean CRP levels were higher in patients than in the controls (4.26 vs. 1.57 mg/L, p = 0.023). Patients also had higher D-dimer levels than the controls (5.96 vs. 0.59 mg/L, p < 0.001). A significant positive correlation was found between PTX3 and CRP levels (r = 0.508, p < 0.001) and between D-dimer levels and UAS7 (r = 0.338, p = 0.004) and CRP (r = 0.213, p = 0.034) levels. A multivariable stepwise regression analysis showed that the one-unit increase in the CRP level increased to 38.19 units in the PTX3 level (95% confidence interval [17.40-58.98], p < 0.001). CONCLUSION: Circulating levels of CRP and PTX3, two members of the pentraxin family, are significantly correlated and elevated in CSU patients with increasing disease activity, indicating their utility as inflammatory markers in CSU.
Assuntos
Urticária Crônica , Urticária , Humanos , Feminino , Adulto , Pessoa de Meia-Idade , Masculino , Proteína C-Reativa/análise , Doença Crônica , BiomarcadoresRESUMO
Bovine tuberculosis might be seen in low-income countries, especially in children fed with raw milk. The most common transmission route is fecal-oral way, and it is most likely through unpasteurized dairy products. Although clinical and radiological findings are like non-zoonotic tuberculosis, treatment approaches may differ in individuals with zoonotic tuberculosis. Prevention of zoonotic diseases requires multidisciplinary approaches. These approaches include the development of veterinary and surveillance studies for the detection of communicable diseases in farm animals, as well as informing the public about raw milk consumption. In this case report, a patient with zoonotic pulmonary tuberculosis related to Mycobacterium bovis because of consumption of raw milk was presented. A five-month-old male was admitted to the hospital due to a persistent, feverless, non-productive cough since birth. Empirical antibiotic treatment was started with a preliminary diagnosis of pneumonia because of left upper lobe and right pericardial infiltration on chest X-ray. However, after two weeks of antimicrobial therapy, the patient's clinical and laboratory findings did not improve. This led to the referral for a computed tomography imaging, which revealed tracheomalacia, consolidation on the right upper lobe, an indistinguishable mass or consolidation on the left middle lobe of the lung, peribronchial thickening on the basal segment of the lower lobe, and mediastinal lymphadenopathy. Three consecutive days of fasting gastric lavage fluid was sent to the reference laboratory for acid-resistant bacillus examination, polymerase chain reaction (PCR) and culture studies. As the clinical findings were compatible and PCR was positive, the patient was started on quadruple antituberculous therapy. After initiation of anti-tuberculosis drugs, the patient's findings radiologically and clinically were improved. Mycobacterium bovis was grown in the culture. In the meantime, it was discovered that the patient was fed with raw milk. Due to the patient's clinical symptoms and the growth of Mycobacterium bovis in the gastric lavage fluid culture, the diagnosis of bovine tuberculosis was made. The culprit was that the milk of the cow belonging to the patient's family, which was later found to be infected with M.bovis, was milked and given to the patient without boiling. Today, unpasteurized dairy products continue to be consumed, especially in rural areas. One of the most important steps to prevent zoonotic diseases is to raise awareness about not consuming raw milk and undercooked meat. To elucidate the epidemiological link in childhood, taking a good anamnesis, including questioning raw milk consumption, is essential in the diagnosis of tuberculosis.
Assuntos
Mycobacterium bovis , Tuberculose Bovina , Tuberculose Pulmonar , Tuberculose , Animais , Feminino , Bovinos , Masculino , Tuberculose Bovina/diagnóstico , Tuberculose Bovina/tratamento farmacológico , Tuberculose Bovina/epidemiologia , Tuberculose/microbiologia , Tuberculose Pulmonar/diagnóstico por imagem , Tuberculose Pulmonar/tratamento farmacológico , Zoonoses , AntituberculososRESUMO
Juvenile-onset recurrent respiratory papillomatosis (JRRP) is a rare and debilitating childhood disease that presents with recurrent growth of papillomas in the upper airway. Two common human papillomaviruses (HPVs), HPV-6 and -11, are implicated in most cases, but it is still not understood why only a small proportion of children develop JRRP following exposure to these common viruses. We report 2 siblings with a syndromic form of JRRP associated with mild dermatologic abnormalities. Whole-exome sequencing of the patients revealed a private homozygous mutation in NLRP1, encoding Nucleotide-Binding Domain Leucine-Rich Repeat Family Pyrin Domain-Containing 1. We find the NLRP1 mutant allele to be gain of function (GOF) for inflammasome activation, as demonstrated by the induction of inflammasome complex oligomerization and IL-1ß secretion in an overexpression system. Moreover, patient-derived keratinocytes secrete elevated levels of IL-1ß at baseline. Finally, both patients displayed elevated levels of inflammasome-induced cytokines in the serum. Six NLRP1 GOF mutations have previously been described to underlie 3 allelic Mendelian diseases with differing phenotypes and modes of inheritance. Our results demonstrate that an autosomal recessive, syndromic form of JRRP can be associated with an NLRP1 GOF mutation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/genética , Mutação com Ganho de Função , Homozigoto , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/patologia , Infecções Respiratórias/genética , Infecções Respiratórias/patologia , Pré-Escolar , Citocinas/metabolismo , Feminino , Humanos , Lactente , Inflamassomos , Queratinócitos/citologia , Queratinócitos/imunologia , Queratinócitos/metabolismo , Masculino , Proteínas NLR , Linhagem , Irmãos , SíndromeRESUMO
miR-185 is a microRNA (miR) that targets Bruton's tyrosine kinase in B cells, with reductions in miR-185 linked to B cell autoantibody production. In hippocampal neurons, miR-185 targets both sarcoplasmic/endoplasmic reticulum calcium ATPase 2 and a novel Golgi inhibitor. This miR is haploinsufficient in 90-95% of individuals with chromosome 22q11.2 deletion syndrome, patients who can present with immune, cardiac, and parathyroid problems, learning disorders, and a high incidence of schizophrenia in adults. The reduced levels of miR-185 in neurons cause presynaptic neurotransmitter release. Many of the 22q11.2 deletion syndrome patients have a thymic hypoplasia, which results in a peripheral T cell lymphopenia and unusual T helper cell skewing. The molecular targets of miR-185 in thymocytes are unknown. Using an miR-185 T cell transgenic approach, increasing levels of miR-185 attenuated T cell development at the T cell receptor ß (TCRß) selection checkpoint and during positive selection. This caused a peripheral T cell lymphopenia. Mzb1, Nfatc3, and Camk4 were identified as novel miR-185 targets. Elevations in miR-185 enhanced TCR-dependent intracellular calcium levels, whereas a knockdown of miR-185 diminished these calcium responses. These effects concur with reductions in Mzb1, an endoplasmic reticulum calcium regulator. Consistent with their haploinsufficiency of miR-185, Mzb1 levels were elevated in thymocyte extracts from several 22q11.2 deletion syndrome patients. Our findings indicate that miR-185 regulates T cell development through its targeting of several mRNAs including Mzb1.
Assuntos
Sinalização do Cálcio , Citocinas/biossíntese , MicroRNAs/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Timócitos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Cálcio/imunologia , Cálcio/metabolismo , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/imunologia , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/metabolismo , Citocinas/genética , Citocinas/imunologia , Humanos , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , MicroRNAs/imunologia , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/imunologia , Fatores de Transcrição NFATC/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/imunologia , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Timócitos/citologia , Timócitos/imunologia , Transgenes/genética , Transgenes/imunologiaRESUMO
IL37 plays important roles in the regulation of innate immunity and its oligomeric status is critical to these roles. In its monomeric state, IL37 can effectively inhibit the inflammatory response of IL18 by binding to IL18Rα, a capacity lost in its dimeric form, underlining the pivotal role of the oligomeric status of IL37 in its anti-inflammatory action. Until now, two IL37 dimer structures have been deposited in PDB, reflecting a substantial difference in their dimer interfaces. Given this discrepancy, we analyzed the PDB structures of the IL37 dimer (PDB IDs: 6ncu, 5hn1) along with a AF2-multimer prediction by molecular dynamics (MD) simulations. Results showed that the 5hn1 and AF2-predicted dimers have the same interface and stably maintained their conformations throughout simulations, while the recent IL37 dimer (PDB ID: 6ncu) with a different interface did not, proposing a possible issue with the recent IL37 dimer structure (6ncu). Next, focusing on the stable dimer structures, we have identified five critical positions of V71/Y85/I86/E89/S114, three new positions compared to the literature, that would reduce dimer stability without affecting the monomer structure. Two quintuple mutants were tested by MD simulations and showed partial or complete dissociation of the dimer. Overall, the insights gained from this study reinforce the validity of the 5hn1 and AF2 multimer structures, while also advancing our understanding of the IL37 dimer interface through the generation of monomer-locked IL37 variants.
RESUMO
Patients with 22q11.2 deletion syndrome have heterogeneous clinical presentations including immunodeficiency, cardiac anomalies, and hypocalcemia. The syndrome arises from hemizygous deletions of up to 3Mb on chromosome 22q11.2, a region that contains 60 genes and 4 microRNAs. MicroRNAs are important post-transcriptional regulators of gene expression, with mutations in several microRNAs causal to specific human diseases. We characterized the microRNA expression patterns in the peripheral blood of patients with 22q11.2 deletion syndrome (n=31) compared to normal controls (n=22). Eighteen microRNAs had a statistically significant differential expression (p<0.05), with miR-185 expressed at 0.4× normal levels. The 22q11.2 deletion syndrome cohort exhibited microRNA expression hyper-variability and group dysregulation. Selected microRNAs distinguished patients with cardiac anomalies, hypocalcemia, and/or low circulating T cell counts. In summary, microRNA profiling of chromosome 22q11.2 deletion syndrome/DiGeorge patients revealed a signature microRNA expression pattern distinct from normal controls with clinical relevance.
Assuntos
Síndrome de DiGeorge/genética , Perfilação da Expressão Gênica , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Adolescente , Criança , Pré-Escolar , Deleção Cromossômica , Cromossomos Humanos Par 22/genética , Estudos de Coortes , Feminino , Cardiopatias Congênitas/genética , Humanos , Hipocalcemia/genética , Lactente , Contagem de Linfócitos , Masculino , Linfócitos T/metabolismoRESUMO
T cell activation involves a cascade of TCR-mediated signals that are regulated by three distinct intracellular signaling motifs located within the cytoplasmic tails of the CD3 chains. Whereas all the CD3 subunits possess at least one ITAM, the CD3 ε subunit also contains a proline-rich sequence and a basic-rich stretch (BRS). The CD3 ε BRS complexes selected phosphoinositides, interactions that are required for normal cell surface expression of the TCR. The cytoplasmic domain of CD3 ζ also contains several clusters of arginine and lysine residues. In this study, we report that these basic amino acids enable CD3 ζ to complex the phosphoinositides PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,5)P(2), and PtdIns(3,4,5)P(3) with high affinity. Early TCR signaling pathways were unaffected by the targeted loss of the phosphoinositide-binding functions of CD3 ζ. Instead, the elimination of the phosphoinositide-binding function of CD3 ζ significantly impaired the ability of this invariant chain to accumulate stably at the immunological synapse during T cell-APC interactions. Without its phosphoinositide-binding functions, CD3 ζ was concentrated in intracellular structures after T cell activation. Such findings demonstrate a novel functional role for CD3 ζ BRS-phosphoinositide interactions in supporting T cell activation.
Assuntos
Complexo CD3/metabolismo , Sinapses Imunológicas , Fosfatidilinositóis/metabolismo , Complexo Receptor-CD3 de Antígeno de Linfócitos T/metabolismo , Aminoácidos Básicos , Animais , Sítios de Ligação/imunologia , Complexo CD3/química , Complexo CD3/imunologia , Linhagem Celular , Humanos , Ativação Linfocitária/imunologia , Camundongos , Fosfatidilinositóis/imunologia , Ligação Proteica/imunologia , Transdução de Sinais/imunologia , TransfecçãoRESUMO
Interleukin 18 (IL18) is a pro-inflammatory cytokine that modulates innate and adaptive immune responses. IL18 activity is tightly controlled by the constitutively secreted IL18 binding protein (IL18BP). PDB structures of human IL18 showed that a short stretch of amino acids between 68 and 81 adopted a disordered conformation in all IL18-IL18BP complexes while adopting a 310 helical structure in other IL18 structures, including the receptor complexes. The C74 of human IL18, which was reported to form a novel intermolecular disulfide bond in the human tetrameric assembly, is also located in this short epitope. These observations reflected the importance of this short surface epitope for the structure and dynamics of the IL18-IL18BP heterodimers. We have analyzed all known IL18-IL18BP complexes in the PDB by all-atom MD simulations. The analysis also included two computed complex models adopting a helical structure for the surface epitope. Heterodimer simulations showed a stabilizing impact of the small surface region at the helical form by reducing flexibility of the complex backbone. Analysis of the symmetry-related human IL18-IL18BP tetramer showed that the unfolding of this small surface region also contributed to the IL18-IL18BP stability through a completely exposed C74 sidechain to form an intermolecular disulfide bond in the self-assembled human IL18-IL18BP dimer. Our findings showed how the conformation of the short IL18 epitope between amino acids 68 and 81 would affect IL18 activity by mediating the intermolecular interactions of IL18.
RESUMO
We hypothesize that low-level efflux pump expression is the first step in the development of high-level drug resistance in mycobacteria. We performed 28-day azithromycin dose-effect and dose-scheduling studies in our hollow-fiber model of disseminated Mycobacterium avium-M. intracellulare complex. Both microbial kill and resistance emergence were most closely linked to the within-macrophage area under the concentration-time curve (AUC)/MIC ratio. Quantitative PCR revealed that subtherapeutic azithromycin exposures over 3 days led to a 56-fold increase in expression of MAV_3306, which encodes a putative ABC transporter, and MAV_1406, which encodes a putative major facilitator superfamily pump, in M. avium. By day 7, a subpopulation of M. avium with low-level resistance was encountered and exhibited the classic inverted U curve versus AUC/MIC ratios. The resistance was abolished by an efflux pump inhibitor. While the maximal microbial kill started to decrease after day 7, a population with high-level azithromycin resistance appeared at day 28. This resistance could not be reversed by efflux pump inhibitors. Orthologs of pumps encoded by MAV_3306 and MAV_1406 were identified in Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium abscessus, and Mycobacterium ulcerans. All had highly conserved protein secondary structures. We propose that induction of several efflux pumps is the first step in a general pathway to drug resistance that eventually leads to high-level chromosomal-mutation-related resistance in mycobacteria as ordered events in an "antibiotic resistance arrow of time."
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Antituberculosos/farmacologia , Azitromicina/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Proteínas Fúngicas/genética , Mycobacterium avium/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Área Sob a Curva , Sequência Conservada , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mycobacterium avium/efeitos dos fármacos , Mycobacterium avium/metabolismo , Reação em Cadeia da Polimerase , Estrutura Secundária de Proteína , Alinhamento de Sequência , Fatores de TempoRESUMO
Advanced non-alcoholic fatty liver disease (NAFLD) is a rapidly emerging global health problem associated with pre-disposing genetic polymorphisms, most strikingly an isoleucine to methionine substitution in patatin-like phospholipase domain-containing protein 3 (PNPLA3-I148M). Here, we study how human hepatocytes with PNPLA3 148I and 148M variants engrafted in the livers of broadly immunodeficient chimeric mice respond to hypercaloric diets. As early as four weeks, mice developed dyslipidemia, impaired glucose tolerance, and steatosis with ballooning degeneration selectively in the human graft, followed by pericellular fibrosis after eight weeks of hypercaloric feeding. Hepatocytes with the PNPLA3-148M variant, either from a homozygous 148M donor or overexpressed in a 148I donor background, developed microvesicular and severe steatosis with frequent ballooning degeneration, resulting in more active steatohepatitis than 148I hepatocytes. We conclude that PNPLA3-148M in human hepatocytes exacerbates NAFLD. These models will facilitate mechanistic studies into human genetic variant contributions to advanced fatty liver diseases.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Aciltransferases , Animais , Hepatócitos/metabolismo , Humanos , Lipase/genética , Lipase/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Hepatopatia Gordurosa não Alcoólica/genética , Fosfolipases A2 Independentes de CálcioRESUMO
We studied a child with severe viral, bacterial, fungal, and parasitic diseases, who was homozygous for a loss-of-function mutation of REL, encoding c-Rel, which is selectively expressed in lymphoid and myeloid cells. The patient had low frequencies of NK, effector memory cells reexpressing CD45RA (Temra) CD8+ T cells, memory CD4+ T cells, including Th1 and Th1*, Tregs, and memory B cells, whereas the counts and proportions of other leukocyte subsets were normal. Functional deficits of myeloid cells included the abolition of IL-12 and IL-23 production by conventional DC1s (cDC1s) and monocytes, but not cDC2s. c-Rel was also required for induction of CD86 expression on, and thus antigen-presenting cell function of, cDCs. Functional deficits of lymphoid cells included reduced IL-2 production by naive T cells, correlating with low proliferation and survival rates and poor production of Th1, Th2, and Th17 cytokines by memory CD4+ T cells. In naive CD4+ T cells, c-Rel is dispensable for early IL2 induction but contributes to later phases of IL2 expression. The patient's naive B cells displayed impaired MYC and BCL2L1 induction, compromising B cell survival and proliferation and preventing their differentiation into Ig-secreting plasmablasts. Inherited c-Rel deficiency disrupts the development and function of multiple myeloid and lymphoid cells, compromising innate and adaptive immunity to multiple infectious agents.
Assuntos
Genes rel , Doenças da Imunodeficiência Primária/genética , Doenças da Imunodeficiência Primária/imunologia , Proteínas Proto-Oncogênicas c-rel/deficiência , Proteínas Proto-Oncogênicas c-rel/genética , Imunidade Adaptativa/genética , Imunidade Adaptativa/imunologia , Criança , Consanguinidade , Feminino , Transplante de Células-Tronco Hematopoéticas , Homozigoto , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Imunidade Inata/genética , Imunidade Inata/imunologia , Ativação Linfocitária , Linfócitos/classificação , Linfócitos/imunologia , Mutação , Células Mieloides/imunologia , Doenças da Imunodeficiência Primária/terapia , Isoformas de ProteínasRESUMO
Nearly one third of the world's population is infected with Mycobacterium tuberculosis (Mtb). While much work has focused on the role of different Mtb encoded proteins in pathogenesis, recent studies have revealed that Mtb also transcribes many noncoding RNAs whose functions remain poorly characterized. We performed RNA sequencing and identified a subset of Mtb H37Rv-encoded small RNAs (<30 nts in length) that were produced in infected macrophages. Designated as smaller noncoding RNAs (sncRNAs), three of these predominated the read counts. Each of the three, sncRNA-1, sncRNA-6, and sncRNA-8 had surrounding sequences with predicted stable secondary RNA stem loops. Site-directed mutagenesis of the precursor sequences suggest the existence of a hairpin loop dependent RNA processing mechanism. A functional assessment of sncRNA-1 suggested that it positively regulated two mycobacterial transcripts involved in oleic acid biosynthesis. Complementary loss- and gain- of-function approaches revealed that sncRNA-1 positively supports Mtb growth and survival in nutrient-depleted cultures as well as in infected macrophages. Overall, the findings reveal that Mtb produces sncRNAs in infected cells, with sncRNA-1 modulating mycobacterial gene expression including genes coupled to oleic acid biogenesis.
RESUMO
Fulminant viral hepatitis (FVH) is a devastating and unexplained condition that strikes otherwise healthy individuals during primary infection with common liver-tropic viruses. We report a child who died of FVH upon infection with hepatitis A virus (HAV) at age 11 yr and who was homozygous for a private 40-nucleotide deletion in IL18BP, which encodes the IL-18 binding protein (IL-18BP). This mutation is loss-of-function, unlike the variants found in a homozygous state in public databases. We show that human IL-18 and IL-18BP are both secreted mostly by hepatocytes and macrophages in the liver. Moreover, in the absence of IL-18BP, excessive NK cell activation by IL-18 results in uncontrolled killing of human hepatocytes in vitro. Inherited human IL-18BP deficiency thus underlies fulminant HAV hepatitis by unleashing IL-18. These findings provide proof-of-principle that FVH can be caused by single-gene inborn errors that selectively disrupt liver-specific immunity. They also show that human IL-18 is toxic to the liver and that IL-18BP is its antidote.