Search

Mycobacterium tuberculosis causes widespread, persistent infection, often residing in macrophages that neither sterilize the bacilli nor allow them to cause disease. How macrophages restrict growth of pathogens is one of many aspects of human phagocyte biology whose study relies largely on macrophages differentiated from monocytes in vitro. However, such cells fail to recapitulate the phenotype of tissue macrophages in key respects, including that they support early, extensive replication of M. tuberculosis and die in several days. Here we found that human macrophages could survive infection, kill Mycobacterium bovis BCG, and severely limit the replication of M. tuberculosis for several weeks if differentiated in 40% human plasma under 5%-10% (physiologic) oxygen in the presence of GM-CSF and/or TNF-αfollowed by IFN-γ.Control was lost with fetal bovine serum, 20% oxygen, M-CSF, higher concentrations of cytokines, or premature exposure to IFN-γ.We believe that the new culture method will enable inquiries into the antimicrobial mechanisms of human macrophages., Introdution Tuberculosis is a major global health problem. Mycobacterium tuberculosis has humans as its only known natural host and macro-phages as its predominant cellular niche. However, the study of M. [...]

Many anti-infectives inhibit the synthesis of bacterial proteins, but none selectively inhibits their degradation. Most anti-infectives kill replicating pathogens, but few preferentially kill pathogens that have been forced into a non-replicating state by conditions in the host. To explore these alternative approaches we sought selective inhibitors of the proteasome of Mycobacterium tuberculosis. Given that the proteasome structure is extensively conserved, it is not surprising that inhibitors of all chemical classes tested have blocked both eukaryotic and prokaryotic proteasomes, and no inhibitor has proved substantially more potent on proteasomes of pathogens than of their hosts. Here we show that certain oxathiazol-2-one compounds kill non-replicating M. tuberculosis and act as selective suicide-substrate inhibitors of the M. tuberculosis proteasome by cyclocarbonylating its active site threonine. Major conformational changes protect the inhibitor-enzyme intermediate from hydrolysis, allowing formation of an oxazolidin-2-one and preventing regeneration of active protease. Residues outside the active site whose hydrogen bonds stabilize the critical loop before and after it moves are extensively non-conserved. This may account for the ability of oxathiazol-2-one compounds to inhibit the mycobacterial proteasome potently and irreversibly while largely sparing the human homologue., Proteasomes are structurally conserved from archaea to eukaryotes and are essential in eukaryotes (1). The cytotoxicity of proteasome inhibitors has been exploited for cancer therapy (2) and suggested for the [...]

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-[gamma]1 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-[gamma]1, and IL-27). It should be considered in patients with unexplained, severe, but curable intracellular bacterial and viral infections., Introduction STAT1 is a key signaling component of IFN responses. There are 2 STAT1 mRNAs, STAT1[alpha] and STAT1B, which use distinct polyadenylation sites. STAT1[alpha] is transcribed and spliced from 25 [...]

Background Interferon-c receptor 1 (IFN-γR1) deficiency is a life-threatening inherited disorder, conferring predisposition to mycobacterial diseases. Haematopoietic stem cell transplantation (HSCT) is the only curative treatment available, but is hampered by a very high rate of graft rejection, even with intra-familial HLA-identical transplants. This high rejection rate is not seen in any other congenital disorders and remains unexplained. We studied the underlying mechanism in a mouse model of HSCT for IFN-γR1 deficiency. Methods and Findings We demonstrated that HSCT with cells from a syngenic C57BL/6 Ifngr[1.sup.+/+] donor engrafted well and restored anti-mycobacterial immunity in naive, non-infected C57BL/6 Ifngr[1.sup.-/-] recipients. However, Ifngr[1.sup.-/-] mice previously infected with Mycobacterium bovis bacillus Calmette-Guerin (BCG) rejected HSCT. Like infected IFN-γR1-deficient humans, infected Ifngr[1.sup.-/-] mice displayed very high serum IFN-γ levels before HSCT. The administration of a recombinant IFN-γ-expressing AAV vector to Ifngr[1.sup.-/-] naive recipients also resulted in HSCT graft rejection. Transplantation was successful in Ifngr[1.sup.-/-] 3 [Ifng.sup.-/-] double-mutant mice, even after BCG infection. Finally, efficient antibody-mediated IFN-γ depletion in infected Ifngr[1.sup.-/-] mice in vivo allowed subsequent engraftment. Conclusions High serum IFN-γ concentration is both necessary and sufficient for graft rejection in IFN-γR1-deficient mice, inhibiting the development of heterologous, IFN-γR1-expressing, haematopoietic cell lineages. These results confirm that IFN-γ is an anti-haematopoietic cytokine in vivo. They also pave the way for HSCT management in IFN-γR1-deficient patients through IFN-γ depletion from the blood. They further raise the possibility that depleting IFN-γ may improve engraftment in other settings, such as HSCT from a haplo-identical or unrelated donor. The Editors' Summary of this article follows the references., Introduction Complete deficiency of the ligand-binding chain of the interferon-c receptor (IFN-γR1) is an autosomal recessive disorder described in 1996 as the first genetic aetiology of the syndrome of Mendelian [...]

We present here a new method for bioethics: systemic modelling. In this method, the complex phenomenon being studied (e.g. personalized medicine, genetic testing, gene therapy, genetically modified organisms) is modelled as a whole, to shed light on its organization and functioning, and major (bio)ethical issues and solutions for their resolution are then identified. This systemic modelling method is ideal for use in the identification of solutions, rather than their validation, with other methods then used to test the solutions found. We provide a description and reproducible instructions for the application of systemic modelling in bioethics, together with a brief example of the application of this method to the study of the impact of personalized medicine on French society. [ABSTRACT FROM AUTHOR]

Networks for the exchange and/or sharing of genetic data are developing in many countries. We focus here on the situations in the US and France. We highlight some recent and remarkable differences between these two countries concerning the mode of access to, and the storage and use of genetic data, particularly as concerns two-sided markets and dynamic consent or dynamic electronic informed consent (e-IC). This brief overview suggests that, even though the organization and function of these two-sided markets remain open to criticism, dynamic e-IC should be more widely used, especially in France, if only to determine its real effectiveness. [ABSTRACT FROM AUTHOR]

In the digital age, a genetics cohort has become much more than a simple means of determining the cause of a disease. Two‐sided markets, of which 23andMe, Ancestry DNA and MyHeritage are the best known, have showed this perfectly over the last few years: a cohort has become a means of producing massive amounts of data for medical, scientific and commercial exploitation, and for genetic use in particular. French law does not currently allow these foreign private companies to develop on French national territory and also forbids the creation of similar entities in France. However, at least in theory, this same law does not preclude the creation of new types of cohorts in France inspired by the success of two‐sided markets but retaining features specific to the French healthcare management system. We propose an optimal solution for France, for genomic studies associated with multi‐subject questionnaires, still purely theoretical for the moment: the development, with no need for any change in the law, of France's own version of "Genetics v.2.0": "e‐CohortE." [ABSTRACT FROM AUTHOR]

The practice and development of modern medicine requires large amounts of data, particularly in the domain of cancer. The future of personalized medicine lies neither with “genomic medicine” nor with “precision medicine”, but with “data medicine” (DM) (big data, data mining). The establishment of this DM has required far-reaching changes, to establish four essential elements connecting patients and doctors: biobanks, databases, bioinformatic platforms and genomic platforms. The “transformation” of scientific research areas, such as genetics, bioinformatics and biostatistics, into clinical specialties has generated a new vision of care. Molecular tumor boards (MTB) are one response to these changes and are now providing better access to next-generation sequencing (NGS) and new cancer treatments to patients with inoperable or metastatic cancers, and those for whom the usual treatment has failed. However, MTB face a crucial ethical challenge: maintaining and improving the trust of patients, clinicians, researchers and industry in academic medical centers supported by private or public funding rather than providing genetic data directly to private companies. We believe that, in this era of DM, appropriate modern digital communication networks will be required to maintain this trust and to improve the organization and effectiveness of the system. There is, therefore, a need to reconsider the form and content of informed consent (IC) documents at all academic medical centers and to introduce dynamic and electronic informed consent (e-IC). [ABSTRACT FROM AUTHOR]

Background: Since 2006, the genetic testing company 23andMe has collected biological samples, self-reported information, and consent documents for biobanking and research from more than 1,000,000 individuals (90% participating in research), through a direct-to-consumer (DTC) online genetic-testing service providing a genetic ancestry report and a genetic health report. However, on November 22, 2013, the Food and Drug Administration (FDA) halted the sale of genetic health testing, on the grounds that 23andMe was not acting in accordance with federal law, by selling tests of undemonstrated reliability as predictive tests for medical risk factors. Consumers could still obtain the genetic ancestry report, but they no longer had access to the genetic health report in the United States (US). However, this did not prevent the company from continuing its health research, with previously obtained and future samples, provided that consent had been obtained from the consumers concerned, or with health reports for individuals from other countries. Furthermore, 23andMe was granted FDA authorization on February 19, 2015, first to provide reports about Bloom syndrome carrier status, and, more recently, to provide consumers with "carrier status" information for 35 genes known (with high levels of confidence) to cause disease.Discussion: In this Debate, we highlight the likelihood that the primary objective of the company was probably two-fold: promoting itself within the market for predictive testing for human genetic diseases and ancestry at a low cost to consumers, and establishing a high-value database/biobank for research (one of the largest biobanks of human deoxyribonucleic acid (DNA) and personal information). By dint of this marketing approach, a two-sided market has been established between the consumer and the research laboratories, involving the establishment of a database/DNA biobank for scientific and financial gain. We describe here the profound ethical issues raised by this setup. [ABSTRACT FROM AUTHOR]

Germline mutations in CYBB, the human gene encoding the gp91phox subunit of the phagocyte NADPH oxidase, impair the respiratory burst of all types of phagocytes and result in X-linked chronic granulomatous disease (CGD). We report here two kindreds in which otherwise healthy male adults developed X-linked recessive Mendelian susceptibility to mycobacterial disease (MSMD) syndromes. These patients had previously unknown mutations in CYBB that resulted in an impaired respiratory burst in monocyte-derived macrophages but not in monocytes or granulocytes. The macrophage-specific functional consequences of the germline mutation resulted from cell-specific impairment in the assembly of the NADPH oxidase. This 'experiment of nature' indicates that CYBB is associated with MSMD and demonstrates that the respiratory burst in human macrophages is a crucial mechanism for protective immunity to tuberculous mycobacteria. [ABSTRACT FROM AUTHOR]

Many anti-infectives inhibit the synthesis of bacterial proteins, but none selectively inhibits their degradation. Most anti-infectives kill replicating pathogens, but few preferentially kill pathogens that have been forced into a non-replicating state by conditions in the host. To explore these alternative approaches we sought selective inhibitors of the proteasome of Mycobacterium tuberculosis. Given that the proteasome structure is extensively conserved, it is not surprising that inhibitors of all chemical classes tested have blocked both eukaryotic and prokaryotic proteasomes, and no inhibitor has proved substantially more potent on proteasomes of pathogens than of their hosts. Here we show that certain oxathiazol-2-one compounds kill non-replicating M. tuberculosis and act as selective suicide-substrate inhibitors of the M. tuberculosis proteasome by cyclocarbonylating its active site threonine. Major conformational changes protect the inhibitor-enzyme intermediate from hydrolysis, allowing formation of an oxazolidin-2-one and preventing regeneration of active protease. Residues outside the active site whose hydrogen bonds stabilize the critical loop before and after it moves are extensively non-conserved. This may account for the ability of oxathiazol-2-one compounds to inhibit the mycobacterial proteasome potently and irreversibly while largely sparing the human homologue. [ABSTRACT FROM AUTHOR]

Claire Soudais and colleagues investigated the mechanism of rejection of hematopoietic stem cell transplants in patients with interferon-gamma receptor 1 (IFN-R1) deficiency and show that IFN- is an anti-hematopoietic cytokine in vivo. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Germline mutations in five autosomal genes involved in interleukin (IL)-12-dependent, interferon (IFN)-γ-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 leu cine zipper (LZ) domain of the NF-κB 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-γ, secretion by T cells. IL-12 production was also impaired as the result of a specific defect in NEMO- and NF-κB/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-KB activators, such as tumor necrosis factor-α, IL-1β, and lipopolysaccharide. These two mutations in the NEMO 17 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-κB/c-Rel-mediated induction of IL-12 by monocyte-derived cells for protective immunity to mycobacteria in humans. [ABSTRACT FROM AUTHOR]

Disseminated BCG infection is a rare complication of vaccination that occurs in patients with impaired immunity. In recent years, a series of inherited disorders of the IL-12-IFN-gamma axis have been described that predispose affected individuals to disseminated disease caused by BCG, environmental Mycobacteria, and non-typhoidal Salmonella. The routine immunological work-up of these patients is normal and the diagnosis requires specific investigation of the IL-12-IFN-gamma circuit. We report here the first two such patients originating from and living in Iran. The first child is two years old and suffers from complete IFN-gamma receptor 2 deficiency and disseminated BCG infection. He is currently in clinical remission thanks to prolonged multiple antibiotic therapy. The other, a 28-year-old adult, suffers from IL-12p40 deficiency and presented with disseminated BCG infection followed by recurrent episodes of systemic salmonellosis. He is now doing well. A third patient of Iranian descent, living in North America, was reported elsewhere to suffer from IL-12Rbeta1 deficiency. These three patients thus indicate that various inherited defects of the IL-12-IFN-gamma circuit can be found in Iranian people. In conclusion we recommend to consider the disorders of the IL-12-IFN-gamma circuit in all patients with severe BCG infection, disseminated environmental mycobacterial disease, or systemic non-typhoidal salmonellosis, regardless of their ethnic origin and country of residence. [ABSTRACT FROM AUTHOR]

Mutations involving gains of glycosylation have been considered rare, and the pathogenic role of the new carbohydrate chains has never been formally established. We identified three children with mendelian susceptibility to mycobacterial disease who were homozygous with respect to a missense mutation in IFNGR2 creating a new N-glycosylation site in the IFNγR2 chain. The resulting additional carbohydrate moiety was both necessary and sufficient to abolish the cellular response to IFNγ. We then searched the Human Gene Mutation Database for potential gain-of-N-glycosylation missense mutations; of 10,047 mutations in 577 genes encoding proteins trafficked through the secretory pathway, we identified 142 candidate mutations (∼1.4%) in 77 genes (∼13.3%). Six mutant proteins bore new N-linked carbohydrate moieties. Thus, an unexpectedly high proportion of mutations that cause human genetic disease might lead to the creation of new N-glycosylation sites. Their pathogenic effects may be a direct consequence of the addition of N-linked carbohydrate. [ABSTRACT FROM AUTHOR]

Disseminated squamous cell carcinoma (SCC) of the skin is exceedingly rare in children. SCC occurs after immunodeficiency from immunosuppression in organ transplant recipients or patients with HIV infection or leukaemia, but has not been reported in primary immunodeficiencies other than epidermodysplasia verruciformis. Interferon γ receptor 2 (IFNγR2) deficiency is an exceedingly rare primary immunodeficiency, conferring almost selective predisposition to mycobacterial diseases. A disseminated, cutaneous SCC is described that occurred in a patient homozygous for a novel frameshift deletion at positions 949 and 950 (949delTG) in the IFNGR2 gene. The patient first presented at 1 year of age with disseminated Mycobacterium avium infection, with later infections of atypical mycobacteria (Mycobacterium fortuitum and Mycobacterium porcium). At 17 years of age, the patient developed multifocal SCC lesions on the face and both hands. Histopathological examination revealed well differentiated SCC. Despite local tumour excision, multiple lesions occurred and a large SCC on the right arm required amputation. The patient died at 20 years of age of disseminated SCC. Inherited disorders of IFNγ mediated immunity may predispose patients to SCC. [ABSTRACT FROM AUTHOR]

Background: Interferon-gamma receptor 1 (IFN-gammaR1) deficiency is a life-threatening inherited disorder, conferring predisposition to mycobacterial diseases. Haematopoietic stem cell transplantation (HSCT) is the only curative treatment available, but is hampered by a very high rate of graft rejection, even with intra-familial HLA-identical transplants. This high rejection rate is not seen in any other congenital disorders and remains unexplained. We studied the underlying mechanism in a mouse model of HSCT for IFN-gammaR1 deficiency.Methods and Findings: We demonstrated that HSCT with cells from a syngenic C57BL/6 Ifngr1+/+ donor engrafted well and restored anti-mycobacterial immunity in naive, non-infected C57BL/6 Ifngr1-/- recipients. However, Ifngr1-/- mice previously infected with Mycobacterium bovis bacillus Calmette-Guérin (BCG) rejected HSCT. Like infected IFN-gammaR1-deficient humans, infected Ifngr1-/- mice displayed very high serum IFN-gamma levels before HSCT. The administration of a recombinant IFN-gamma-expressing AAV vector to Ifngr1-/- naive recipients also resulted in HSCT graft rejection. Transplantation was successful in Ifngr1-/- x Ifng-/- double-mutant mice, even after BCG infection. Finally, efficient antibody-mediated IFN-gamma depletion in infected Ifngr1-/- mice in vivo allowed subsequent engraftment.Conclusions: High serum IFN-gamma concentration is both necessary and sufficient for graft rejection in IFN-gammaR1-deficient mice, inhibiting the development of heterologous, IFN-gammaR1-expressing, haematopoietic cell lineages. These results confirm that IFN-gamma is an anti-haematopoietic cytokine in vivo. They also pave the way for HSCT management in IFN-gammaR1-deficient patients through IFN-gamma depletion from the blood. They further raise the possibility that depleting IFN-gamma may improve engraftment in other settings, such as HSCT from a haplo-identical or unrelated donor. [ABSTRACT FROM AUTHOR]