Search

HIRA is a histone chaperone known to modulate gene expression through the deposition of H3.3. Conditional knockout of Hira in embryonic mouse hearts leads to cardiac septal defects. Loss of function mutation in HIRA, together with other chromatin modifiers, was found in patients with congenital heart diseases. However, the effects of HIRA on gene expression at earlier stages of cardiogenic mesoderm differentiation have not yet been studied. Differentiation of mouse embryonic stem cells (mESCs) towards cardiomyocytes mimics some of these early events and is an accepted model of these early stages. We performed RNA-Seq and H3.3-HA ChIP-seq on both WT and Hira-null mESCs and early cardiomyocyte progenitors of both genotypes. Analysis of RNA-seq data showed differential down regulation of cardiovascular development-related genes in Hira-null cardiomyocytes compared to WT cardiomyocytes. We found HIRA-dependent H3.3 deposition at these genes. In particular, we observed that HIRA influenced directly the expression of the transcription factors Gata6, Meis1 and Tbx2, essential for cardiac septation, through H3.3 deposition. We therefore identified new direct targets of HIRA during cardiac differentiation.

Chromatin remodelling is essential for cardiac development. Interestingly, the role of histone chaperones has not been investigated in this regard. HIRA is a member of the HUCA (HIRA/UBN1/CABIN1/ASF1a) complex that deposits the variant histone H3.3 on chromatin independently of replication. Lack of HIRA has general effects on chromatin and gene expression dynamics in embryonic stem cells and mouse oocytes. Here we describe the conditional ablation of Hira in the cardiogenic mesoderm of mice. We observed surface oedema, ventricular and atrial septal defects and embryonic lethality. We identified dysregulation of a subset of cardiac genes, notably upregulation of troponins Tnni2 and Tnnt3, involved in cardiac contractility and decreased expression of Epha3, a gene necessary for the fusion of the muscular ventricular septum and the atrioventricular cushions. We found that HIRA binds GAGA rich DNA loci in the embryonic heart, and in particular a previously described enhancer of Tnni2/Tnnt3 (TTe) bound by the transcription factor NKX2.5. HIRA-dependent H3.3 enrichment was observed at the TTe in embryonic stem cells (ESC) differentiated toward cardiomyocytes in vitro. Thus, we show here that HIRA has locus-specific effects on gene expression and that histone chaperone activity is vital for normal heart development, impinging on pathways regulated by an established cardiac transcription factor.

We report a series of 14 patients from 11 kindreds with recessive partial (RP)-interferon (IFN)-γR1 deficiency. The I87T mutation was found in nine homozygous patients from Chile, Portugal and Poland, and the V63G mutation was found in five homozygous patients from the Canary Islands. Founder effects accounted for the recurrence of both mutations. The most recent common ancestors of the patients with the I87T and V63G mutations probably lived 1600 (875–2950) and 500 (200–1275) years ago, respectively. The two alleles confer phenotypes that are similar but differ in terms of IFN-γR1 levels and residual response to IFN-γ. The patients suffered from bacillus Calmette-Guérin-osis (n= 6), environmental mycobacteriosis (n= 6) or tuberculosis (n= 1). One patient did not suffer from mycobacterial infections but had disseminated salmonellosis, which was also present in two other patients. Age at onset of the first environmental mycobacterial disease differed widely between patients, with a mean value of 11.25 ± 9.13 years. Thirteen patients survived until the age of 14.82 ± 11.2 years, and one patient died at the age of 7 years, 9 days after the diagnosis of long-term Mycobacterium avium infection and the initiation of antimycobacterial treatment. Up to 10 patients are currently free of infection with no prophylaxis. The clinical heterogeneity of the 14 patients was not clearly related to either IFNGR1 genotype or the resulting cellular phenotype. RP-IFN-γR1 deficiency is, thus, more common than initially thought and should be considered in both children and adults with mild or severe mycobacterial diseases.

The incorporation of histone H3 variants has been implicated in the epigenetic memory of cellular state. Using genome editing with zinc finger nucleases to tag endogenous H3.3, we report genome-wide profiles of H3 variants in mammalian embryonic stem (ES) cells and neuronal precursor cells. Genome-wide patterns of H3.3 are dependent on amino acid sequence, and change with cellular differentiation at developmentally regulated loci. The H3.3 chaperone Hira is required for H3.3 enrichment at active and repressed genes. Strikingly, Hira is not essential for localization of H3.3 at telomeres and many transcription factor binding sites. Immunoaffinity purification and mass spectrometry reveal that the proteins Atrx and Daxx associate with H3.3 in a Hira-independent manner. Atrx is required for Hira-independent localization of H3.3 at telomeres, and for the repression of telomeric RNA. Our data demonstrate that multiple and distinct factors are responsible for H3.3 localization at specific genomic locations in mammalian cells.

Disseminated disease caused by non-tuberculous, environmental mycobacteria (EM) reflects impaired host immunity. Disseminated disease caused by Mycobacterium scrofulaceum has primarily been reported in patients with AIDS. Moreover, observing M. scrofulaceum as the agent of localized disease in childhood has become increasingly rare. We report the first case of disseminated disease caused by M. scrofulaceum in a child with inherited interferon-gamma receptor 1 (IFN-gammaR1) complete deficiency. As in this case, mycobacterial bone infections in IFN-gammaR1 deficiency can sometimes mimic the clinical picture of chronic recurrent multifocal osteomyelitis.

IFN-gammaR1 deficiency is a genetic etiology of Mendelian susceptibility to mycobacterial diseases, and includes two forms of complete recessive deficiency, with or without cell surface expression, and two forms of partial deficiency, dominant or recessive. We report here a novel form of partial and recessive Interferon gamma receptor 1 (IFN-gammaR1) deficiency, which is almost as severe as complete deficiency. The patient is homozygous for a mutation of the initiation codon (M1K). No detectable expression and function of IFN-gammaR1 were found in the patient's fibroblasts. However, IFN-gammaR1 expression was found to be impaired, but not abolished, on the EBV-transformed B cells, which could respond weakly to IFN-gamma. The mechanism underlying this weak expression involves leaky translation initiation at both non-AUG codons and the third AUG codon at position 19. It results in the residual expression of IFN-gammaR1 protein of normal molecular weight and function. The residual IFN-gamma signaling documented in this novel form of partial IFN-gammaR1 deficiency was not ubiquitous and was milder than that seen in other forms of partial IFN-gammaR1 deficiency, accounting for the more severe clinical phenotype of the patient, which was almost as severe as that of patients with complete deficiency.

Complete STAT1 deficiency is an autosomal recessive primary immunodeficiency caused by null mutations that abolish STAT1-dependent cellular responses to both IFN-alpha/beta and IFN-gamma. Affected children suffer from lethal intracellular bacterial and viral diseases. Here we report a recessive form of partial STAT1 deficiency, characterized by impaired but not abolished IFN-alpha/beta and IFN-gamma signaling. Two affected siblings suffered from severe but curable intracellular bacterial and viral diseases. Both were homozygous for a missense STAT1 mutation: g.C2086T (P696S). This STAT1 allele impaired the splicing of STAT1 mRNA, probably by disrupting an exonic splice enhancer. The misspliced forms were not translated into a mature protein. The allele was hypofunctional, because residual full-length mRNA production resulted in low but detectable levels of normally functional STAT1 proteins. The P696S amino acid substitution was not detrimental. The patients' cells, therefore, displayed impaired but not abolished responses to both IFN-alpha and IFN-gamma. We also show that recessive STAT1 deficiencies impaired the IL-27 and IFN-lambda1 signaling pathways, possibly contributing to the predisposition to bacterial and viral infections, respectively. Partial recessive STAT1 deficiency is what we believe to be a novel primary immunodeficiency, resulting in impairment of the response to at least 4 cytokines (IFN-alpha/beta, IFN-gamma, IFN-lambda1, and IL-27). It should be considered in patients with unexplained, severe, but curable intracellular bacterial and viral infections.

Background Atypical mycobacteria can cause systemic infections in patients with certain types of immunodeficiency. Methods Clinical samples were decontaminated and cultured to assess the presence of mycobacterial species. Gene sequencing was performed to reveal interferon-gamma receptor 1 (IFN-gamma R1) deficiency. Results The index patient received a diagnosis of dominant IFN-gamma R1 deficiency during treatment for a serious infection due to atypical mycobacteria. She belongs to a Norwegian multiplex family comprising 3 generations and 5 patients with dominant IFN-gamma R1 deficiency. Four of these patients have been treated with tuberculostatics because of extensive infection due to atypical mycobacteria, such as Mycobacterium avium-intracellulare, Mycobacterium scrofulaceum, Mycobacterium bovis (bacille Calmette-Guerin), Mycobacterium bohemicum, and Mycobacterium gordonae. Two of the patients have also received subcutaneous injections of IFN-gamma. One family member with the deficiency has not received treatment and is still healthy at 13 years of age. Conclusions Serious infection due to atypical mycobacteria should initiate a search for primary immunodeficiencies, particularly IFN-gamma R1 deficiency. Treatment with IFN-gamma should be started when serious infection due to atypical mycobacteria is verified and dominant partial IFN-gamma R1 deficiency is suspected.

480 l’homme, et les nombreuses manipulations requises par l’extinction de cdx2 et sa reexpression posent en elles-memes des problemes ethiques difficiles. Quant a la technique proposee par Y. Chung, bien que l’isolement de blastomeres soit pratique pour le diagnostic genetique preimplantatoire (DPI) dans les familles ou il existe un risque de maladie genetique grave, leur capacite a etablir des CSE n’est pas connue, et une telle demarche souleverait aussi de tres nombreuses questions [7-9]. ◊ Derivation of embryonic stem cells REMERCIEMENTS Je remercie D.J. Taupin pour la relecture de ce manuscrit. P.T. est supporte par des subventions du NMRC, BMRC, et de la Juvenile Diabetes Research Foundation.

Summary Polycomb repressive complex 2 (PRC2) regulates gene expression during lineage specification through trimethylation of lysine 27 on histone H3 (H3K27me3). In Drosophila , polycomb binding sites are dynamic chromatin regions enriched with the histone variant H3.3. Here, we show that, in mouse embryonic stem cells (ESCs), H3.3 is required for proper establishment of H3K27me3 at the promoters of developmentally regulated genes. Upon H3.3 depletion, these promoters show reduced nucleosome turnover measured by deposition of de novo synthesized histones and reduced PRC2 occupancy. Further, we show H3.3-dependent interaction of PRC2 with the histone chaperone, Hira, and that Hira localization to chromatin requires H3.3. Our data demonstrate the importance of H3.3 in maintaining a chromatin landscape in ESCs that is important for proper gene regulation during differentiation. Moreover, our findings support the emerging notion that H3.3 has multiple functions in distinct genomic locations that are not always correlated with an "active" chromatin state.

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.

Sustainable water management is a global challenge for the 21st century. One key aspect remains protection against urban flooding. The main objective is to ensure or maintain an adequate level of service for all inhabitants. However, level of service is still difficult to assess and the high-risk locations difficult to identify. In this article, we propose a methodology, which (i) allows water managers to measure the service provided by the urban drainage system with regard to protection against urban flooding; and (ii) helps stakeholders to determine effective strategies for improving the service provided. One key aspect of this work is to use a database of sewer flood event records to assess flood risk. Our methodology helps urban water managers to assess the risk of sewer flooding; this approach does not seek to predict flooding but rather to inform decision makers on the current level of risk and on actions which need to be taken to reduce the risk. This work is based on a comprehensive definition of risk, including territorial vulnerability and perceptions of urban water stakeholders. This paper presents the results and the methodological contributions from implementing the methodology on two case studies: the cities of Lyon and Mulhouse.

Las mutaciones de la linea germinal en CYBB, el gen humano que codifica la subunidad gp91phox del fagocito NADPH oxidasa, perjudican el estallido respiratorio de todos los tipos de fagocitos y dan como resultado la enfermedad granulomatosa cronica ligada al cromosoma X (CGD). Presentamos aqui dos familias en las que, por lo demas, hombres adultos sanos desarrollaron sindromes recesivos mendelianos ligados a X a sindromes de micobacterias (MSMD). Estos pacientes tenian anteriormente mutaciones desconocidas en CYBB que dio lugar a una explosion respiratoria deteriorada en macrofagos derivados de monocitos, pero no en monocitos o granulocitos. Las consecuencias funcionales especificas de los macrofagos de la mutacion de la linea germinal resultaron de un deterioro especifico de la celula en el montaje de NADPH oxidasa. Este experimento de la naturaleza indica que el CYBB se asocia con MSMD y demuestra que la explosion respiratoria en los macrofagos humanos es un mecanismo crucial para la inmunidad protectora a las micobacterias tuberculosas.

WOS: 000283840300002, PubMed ID: 21057261, Interleukin-12 receptor beta 1 (IL-12R beta 1) 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-Guerin (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-12R beta 1 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., Rockefeller University Center for Clinical and Translational Science [5UL1RR024143-03]; Rockefeller University; ANRFrench National Research Agency (ANR); PHRC; EUEuropean Union (EU) [LHSP-CT-2005-018736]; BNP Paribas Foundation; Dana Foundation; March of DimesMarch of Dimes; Fondation pour la Recherche MedicaleFondation pour la Recherche Medicale; Howard Hughes Medical InstituteHoward Hughes Medical Institute, The St. Giles Laboratory of Human Genetics of Infectious Diseases is supported by grants from The Rockefeller University Center for Clinical and Translational Science grant number 5UL1RR024143-03, and The Rockefeller University, ANR, PHRC, EU (LHSP-CT-2005-018736), BNP Paribas Foundation, the Dana Foundation, and the March of Dimes. LDB is supported by the Fondation pour la Recherche Medicale as part of the PhD program of Pierre et Marie Curie University (Paris, France). JLC was an International Scholar of the Howard Hughes Medical Institute.

Mendelian susceptibility to mycobacterial disease (MSMD) is a rare primary immunodeficiency associated with clinical disease caused by weakly virulent mycobacterial species. Interferon gamma receptor 1 (IFN-gammaR1) deficiency is a genetic etiology of MSMD. We describe the clinical and genetic features of a 7-year-old Italian boy suffering from MSMD associated with a complex phenotype, including neonatal hyperglycemia, neuromuscular disease, and dysmorphic features. The child also developed necrotizing pneumonia caused by Rhodococcus equi. The child is homozygous for a nonsense mutation in exon 3 of IFNGR1 as a result of paternal uniparental disomy (UPD) of the entire chromosome 6. This is the first reported case of uniparental disomy resulting in a complex phenotype including MSMD.

International audience; Some Toll and Toll-like receptors (TLRs) provide immunity to experimental infections in animal models, but their contribution to host defense in natural ecosystems is unknown. We report a dominant-negative TLR3 allele in otherwise healthy children with herpes simplex virus 1 (HSV-1) encephalitis. TLR3 is expressed in the central nervous system (CNS), where it is required to control HSV-1, which spreads from the epithelium to the CNS via cranial nerves. TLR3 is also expressed in epithelial and dendritic cells, which apparently use TLR3-independent pathways to prevent further dissemination of HSV-1 and to provide resistance to other pathogens in TLR3-deficient patients. Human TLR3 appears to be redundant in host defense to most microbes but is vital for natural immunity to HSV-1 in the CNS, which suggests that neurotropic viruses have contributed to the evolutionary maintenance of TLR3.

Patients presenting with genetic deficiencies in IFNGR1, IFNGR2, IL-12B, and IL-12RB1 display increased susceptibility to mycobacterial infections. We analyzed in this group of patients the cross-talk between human CD4+ T lymphocytes and dendritic cells (DCs) that leads to maturation of DC into producers of bioactive IL-12 and to activation of T cells into IFN-γ producers. We found that this cross-talk is defective in all patients from this group. Unraveling the mechanisms underlying this deficiency, we showed that IL-12 signaling in T cells is required to induce expression of costimulatory molecules and secretion of IL-12 by DCs and that IFNGR expression is required on both DCs and CD4+ T cells to induce IL-12 secretion by DCs. These data suggest that CD4+ T cell-mediated activation of DCs plays a critical role in the defense against mycobacterial infections in humans.

The transcription factor signal transducer and activator of transcription-1 (STAT1) plays a key role in immunity against mycobacterial and viral infections. Here, we characterize three human STAT1 germline alleles from otherwise healthy patients with mycobacterial disease. The previously reported L706S, like the novel Q463H and E320Q alleles, are intrinsically deleterious for both interferon gamma (IFNG)–induced gamma-activating factor–mediated immunity and interferon alpha (IFNA)–induced interferon-stimulated genes factor 3–mediated immunity, as shown in STAT1-deficient cells transfected with the corresponding alleles. Their phenotypic effects are however mediated by different molecular mechanisms, L706S affecting STAT1 phosphorylation and Q463H and E320Q affecting STAT1 DNA-binding activity. Heterozygous patients display specifically impaired IFNG-induced gamma-activating factor–mediated immunity, resulting in susceptibility to mycobacteria. Indeed, IFNA-induced interferon-stimulated genes factor 3–mediated immunity is not affected, and these patients are not particularly susceptible to viral disease, unlike patients homozygous for other, equally deleterious STAT1 mutations recessive for both phenotypes. The three STAT1 alleles are therefore dominant for IFNG-mediated antimycobacterial immunity but recessive for IFNA-mediated antiviral immunity at the cellular and clinical levels. These STAT1 alleles define two forms of dominant STAT1 deficiency, depending on whether the mutations impair STAT1 phosphorylation or DNA binding., Synopsis Mendelian susceptibility to mycobacterial disease is a rare syndrome. It is defined by the occurrence of severe disease caused by low virulence mycobacteria in otherwise healthy individuals, in whom antiviral immune response is not affected. Eleven known genetic defects, affecting five genes, have been involved in this type of deficient response to infection, involving immune-mediator molecules IL12 and interferon gamma: IL12B, IL12RB1, IFNGR1, IFNGR2, and STAT1. The signal transducer and activator of transcription-1 (STAT1) amino acid change L706S was previously shown to cause disease by impairing STAT1 phosphorylation. Here, we report two new STAT1 mutations that impair STAT1 DNA-binding activity. We show, by functional analysis of the three STAT1 mutant alleles, that they are intrinsically deleterious for both interferon gamma–induced antimycobacterial immunity, which is mediated through gamma-activated factor and for interferon alpha–induced antiviral immunity, which is mediated through interferon-stimulated genes factor 3. Interestingly, the three alleles are dominant for interferon gamma–induced gamma-activated factor–mediated antimycobacterial immunity, but recessive for interferon alpha–induced interferon-stimulated genes factor 3–mediated antiviral immunity at the cellular and clinical levels. These two new STAT1 alleles, which affect the binding of STAT1 to DNA, define distinct novel genetic causes of Mendelian susceptibility to mycobacterial disease and provide further insight into the molecular mechanism of disease.

We describe the case of a 2-year-old boy with disseminated infection by a rapidly growing, poorly pathogenic mycobacterial species that belonged to the Mycobacterium fortuitum-Mycobacterium peregrinum complex. He had a severe course characterized by a poor response to treatment and recurrent lymph node abscess formation. Sequencing of the interferon-gamma receptor 1 gene (IFN gamma R1) revealed that he was homozygous for a novel null mutation, 453delT. Patients presenting with disseminated infections by rapidly growing environmental mycobacteria must be investigated for complete IFN gamma R1 deficiency. The spectrum of IFN gamma R1 genotypes associated with this immunological disorder is expanding.

WOS: 000238940500017, PubMed ID: 16818673, Germline mutations in five autosomal genes involved in interleukin (IL)-12-dependent, interferon (IFN)-gamma-mediated immunity cause Mendelian susceptibility to mycobacterial diseases (MSMD). The molecular basis of X-linked recessive (XR)-MSMD remains unknown. We report here mutations in the leucine zipper (LZ) domain of the NF-kappa B essential modulator (NEMO) gene in three unrelated kindreds with XR-MSMD. The mutant proteins were produced in normal amounts in blood and fibroblastic cells. However, the patients' monocytes presented an intrinsic defect in T cell-dependent IL-12 production, resulting in defective IFN-gamma secretion by T cells. IL-12 production was also impaired as the result of a specific defect in NEMO- and NF-kappa B/c-Rel-mediated CD40 signaling after the stimulation of monocytes and dendritic cells by CD40L-expressing T cells and fibroblasts, respectively. However, the CD40-dependent up-regulation of costimulatory molecules of dendritic cells and the proliferation and immunoglobulin class switch of B cells were normal. Moreover, the patients' blood and fibroblastic cells responded to other NF-kappa B activators, such as tumor necrosis factor-alpha, IL-beta, and lipopolysaccharide. These two mutations in the NEMO LZ domain provide the first genetic etiology of XR-MSMD. They also demonstrate the importance of the T cell- and CD40L-triggered, CD40-, and NEMO/NF-kappa B/c-Rel-mediated induction of IL-12 by monocyte-derived cells for protective immunity to mycobacteria in humans., Intramural NIH HHSUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA; Medical Research CouncilMedical Research Council UK (MRC) [G0900867]

Germline mutations in five autosomal genes involved in interleukin (IL)-12-dependent, interferon (IFN)-gamma-mediated immunity cause Mendelian susceptibility to mycobacterial diseases (MSMD). The molecular basis of X-linked recessive (XR)-MSMD remains unknown. We report here mutations in the leucine zipper (LZ) domain of the NF-kappa B essential modulator (NEMO) gene in three unrelated kindreds with XR-MSMD. The mutant proteins were produced in normal amounts in blood and fibroblastic cells. However, the patients’ monocytes presented an intrinsic defect in T cell-dependent IL-12 production, resulting in defective IFN-gamma secretion by T cells. IL-12 production was also impaired as the result of a specific defect in NEMO- and NF-kappa B/c-Rel-mediated CD40 signaling after the stimulation of monocytes and dendritic cells by CD40L-expressing T cells and fibroblasts, respectively. However, the CD40-dependent up-regulation of costimulatory molecules of dendritic cells and the proliferation and immunoglobulin class switch of B cells were normal. Moreover, the patients’ blood and fibroblastic cells responded to other NF-kappa B activators, such as tumor necrosis factor-alpha, IL-beta, and lipopolysaccharide. These two mutations in the NEMO LZ domain provide the first genetic etiology of XR-MSMD. They also demonstrate the importance of the T cell- and CD40L-triggered, CD40-, and NEMO/NF-kappa B/c-Rel-mediated induction of IL-12 by monocyte-derived cells for protective immunity to mycobacteria in humans.

The IL-12/IFN-gamma axis is crucial for protective immunity to Mycobacterium in humans and mice. Our goal was to analyze the relative contribution of various human blood cell subsets and molecules to the production of, or response to IL-12 and IFN-gamma. We designed an assay for the stimulation of whole blood by live M. bovis Bacillus Calmette-Guerin (BCG) alone, or BCG plus IL-12 or IFN-gamma, measuring IFN-gamma and IL-12 levels. We studied patients with a variety of specific inherited immunodeficiencies resulting in a lack of leukocytes, or T, B, and/or NK lymphocytes, or polymorphonuclear cells, or a lack of expression of key molecules such as HLA class II, CD40L, NF-kappaB essential modulator (NEMO), and IL-1 receptor-associated kinase-4 (IRAK-4). Patients with deficiencies in IL-12p40, IL-12 receptor beta1 chain (IL-12Rbeta1), IFN-gammaR1, IFN-gammaR2, and STAT-1 were used as internal controls. We showed that monocytes were probably the main producers of IL-12, and that NK and T cells produced similar amounts of IFN-gamma. NEMO and IRAK-4 were found to be important for IL-12 production and subsequent IFN-gamma production, while a lack of CD40L or HLA class II had no major impact on the IL-12/IFN-gamma axis. The stimulation of whole blood by live BCG thus triggers the IL-12/IFN-gamma axis by an IRAK-4- and NEMO-dependent, non-cognate interaction between monocytes, NK, and T lymphocytes.

Successful hematopoietic stem cell transplantation in a child with active disseminated Mycobacterium fortuitum infection and interferon- γ receptor 1 deficiency

Germline mutations may cause human disease by various mechanisms. Missense and other in-frame mutations may be deleterious because the mutant proteins are not correctly targeted, do not function correctly, or both. We studied a child with mycobacterial disease caused by homozygosity for a novel in-frame microinsertion in IFNGR2. In cells transfected with the mutant allele, most of the interferon gamma receptor 2 (IFN-gamma R2) protein was retained within the cell, and that expressed on the cell surface had an abnormally high molecular weight (MW). The misfolding mutation was not gain-of-glycosylation, as it created no new N-glycosylation site. The mutant IFNGR2 allele was null, as the patient's cells did not respond to IFN-gamma. Based on the well-established relationship between protein N-glycosylation and protein quality control processes, we tested 29 compounds affecting maturation by N-glycosylation in the secretory pathway. Remarkably, up to 13 of these compounds reduced the MW of surface-expressed mutant IFN-gamma R2 molecules and restored cellular responsiveness to IFN-gamma. Modifiers of N-glycosylation may therefore complement human cells carrying in-frame and misfolding, but not necessarily gain-of-glycosylation, mutations in genes encoding proteins subject to trafficking via the secretory pathway. Some of these compounds are available for clinical use, paving the way for clinical trials of chemical complementation for various human genetic traits.

Background Mendelian susceptibility to mycobacterial disease (MSMD) is associated with infection caused by weakly virulent mycobacteria in otherwise healthy people. Causal germline mutations in five autosomal genes (IFNGR1, IFNGR2, STAT1, IL12RB1, IL12B) and one X-linked (NEMO) gene have been described. The gene products are physiologically related, as they are involved in interleukin 12/23-dependent, interferon gamma-mediated immunity. However, no genetic aetiology has yet been identified for about half the patients with MSMD. Methods A large kindred was studied, including four male maternal relatives with recurrent mycobacterial disease, suggesting X-linked recessive inheritance. Three patients had recurrent disease caused by the bacille Calmette-Guerin vaccine, and the fourth had recurrent tuberculosis. The infections showed tropism for the peripheral lymph nodes. Results Known autosomal and X-linked genetic aetiologies of MSMD were excluded through genetic and immunological investigations. Genetic linkage analysis of the X-chromosome identified two candidate regions, on Xp11.4-Xp21.2 and Xq25-Xq26.3, with a maximum LOD score of 2. Conclusion A new X-linked recessive form of MSMD is reported, paving the way for the identification of a new MSMD-causing gene.

The autosomal recessive form of human complete Stat-1 deficiency is a rare disorder, thus far reported in two unrelated patients, both of whom developed disseminated bacillus Calmette-Guérin (BCG) and subsequently died of viral illnesses before detailed studies of the condition could be performed. It is associated with impaired cellular responses to both IFN-γ and IFN-αβ via Stat-1-containing complexes. We describe a third patient with complete Stat-1 deficiency and disseminated BCG infection, who died 3 mo after bone marrow transplantation. The patient’s EBV-transformed B cells did not express Stat-1 protein and did not activate Stat-1-containing transcription factors. We also report the ex vivo responses of a Stat-1-deficient patient’s fresh blood cells to IFN-γ and the in vitro responses of a SV40-transformed fibroblastic cell line to IFN-γ and IFN-αβ. There was no response to IFN-γ in terms of IL-12 production and HLA class II induction, accounting for vulnerability to BCG. Moreover, IFN-αβ did not suppress HSV and vesicular stomatitis virus replication in fibroblasts, although in vivo the patient was able to successfully clear at least some viruses. This study broadens our understanding of complete Stat-1 deficiency, a severe form of innate immunodeficiency. Stat-1 deficiency should be suspected in children with severe infections, notably but not exclusively patients with mycobacterial or viral diseases.