GIMAP5 deficiency reveals a mammalian ceramide-driven longevity assurance pathway.

Preserving cells in a functional, non-senescent state is a major goal for extending human healthspans. Model organisms reveal that longevity and senescence are genetically controlled, but how genes control longevity in different mammalian tissues is unknown. Here, we report a new human genetic disease that causes cell senescence, liver and immune dysfunction, and early mortality that results from deficiency of GIMAP5, an evolutionarily conserved GTPase selectively expressed in lymphocytes and endothelial cells. We show that GIMAP5 restricts the pathological accumulation of long-chain ceramides (CERs), thereby regulating longevity. GIMAP5 controls CER abundance by interacting with protein kinase CK2 (CK2), attenuating its ability to activate CER synthases. Inhibition of CK2 and CER synthase rescues GIMAP5-deficient T cells by preventing CER overaccumulation and cell deterioration. Thus, GIMAP5 controls longevity assurance pathways crucial for immune function and healthspan in mammals.

Latest Advances in Laboratory Detection of Mycoplasma genitalium.

Mycoplasma genitalium is an important sexually transmitted pathogen affecting both men and women. Its extremely slow growth in vitro and very demanding culture requirements necessitate the use of molecular-based diagnostic tests for its detection in clinical specimens. The recent availability of U.S. Food and Drug Administration (FDA)-cleared commercial molecular-based assays has enabled diagnostic testing to become more widely available in the United States and no longer limited to specialized reference laboratories. Advances in the knowledge of the epidemiology and clinical significance of M. genitalium as a human pathogen made possible by the availability of molecular-based testing have led to updated guidelines for diagnostic testing and treatment that have been published in various countries. This review summarizes the importance of M. genitalium as an agent of human disease, explains the necessity of obtaining a microbiological diagnosis, describes currently available diagnostic methods, and discusses how the emergence of antimicrobial resistance has complicated treatment alternatives and influenced the development of diagnostic tests for resistance detection, with an emphasis on developments over the past few years.

Long-term efficacy, safety, and tolerability of a subcutaneous immunoglobulin 16.5% (cutaquig®) in the treatment of patients with primary immunodeficiencies.

A prospective study and its long-term extension examined whether weekly treatment of patients with primary immunodeficiencies (PIDs) with a 16.5% subcutaneous immunoglobulin (SCIg; cutaquig®) confers acceptable efficacy, safety, and tolerability over a follow-up of up to 238 weeks (>4 years). Seventy-five patients received 4462 infusions during up to 70 weeks of follow-up in the main study and 27 patients received 2777 infusions during up to 168 weeks of follow-up in the extension. In the main study, there were no serious bacterial infections (SBIs), and the annual rate of other infections was 3.3 (95% CI 2.4, 4.5). One SBI was recorded in the extension, for an SBI rate of 0.02 (upper 99% CI 0.19). The annual rate of all infections over the duration of the extension study was 2.2 (95% CI 1.2, 3.9). Only 15.0% (1085) of 7239 infusions were associated with infusion site reactions (ISRs), leaving 85.0% (6153) of infusions without reactions. The majority of ISRs were mild and transient. ISR incidence decreased over time, from 36.9% to 16% during the main study and from 9% to 2.3% during the extension. The incidence of related systemic adverse events was 14.7% in the main study and 7.4% in the extension. In conclusion, this prospective, long-term study with cutaquig showed maintained efficacy and low rates of local and systemic adverse reactions in PID patients over up to 238 weeks of follow-up.

Omadacycline Is Highly Active In Vitro against Mycoplasma genitalium.

Here, we performed in vitro susceptibility testing on 10 Mycoplasma genitalium isolates against omadacycline, minocycline, tetracycline, doxycycline, moxifloxacin, levofloxacin, and azithromycin. Omadacycline was the most potent agent, with all MICs of ≤0.5 µg/mL. MICs were not affected by resistance to other agents, including resistance to other tetracycline class drugs. Omadacycline may be a potential treatment option for M. genitalium infection. IMPORTANCE There are very few clinical isolates of Mycoplasma genitalium available for in vitro susceptibility testing. We studied 10 isolates and determined that the new semisynthetic aminomethylcycline omadacycline is active against isolates that are resistant to tetracyclines, macrolides, and quinolones. These data suggest that clinical studies should be performed in order to see if omadacycline may be useful to treat urogenital infections caused by M. genitalium.

Sequential Stem Cell-Kidney Transplantation in Schimke Immuno-osseous Dysplasia.

Lifelong immunosuppression is required for allograft survival after kidney transplantation but may not ultimately prevent allograft loss resulting from chronic rejection. We developed an approach that attempts to abrogate immune rejection and the need for post-transplantation immunosuppression in three patients with Schimke immuno-osseous dysplasia who had both T-cell immunodeficiency and renal failure. Each patient received sequential transplants of αß T-cell-depleted and CD19 B-cell-depleted haploidentical hematopoietic stem cells and a kidney from the same donor. Full donor hematopoietic chimerism and functional ex vivo T-cell tolerance was achieved, and the patients continued to have normal renal function without immunosuppression at 22 to 34 months after kidney transplantation. (Funded by the Kruzn for a Kure Foundation.).

B-assembler: a circular bacterial genome assembler.

BACKGROUND: Accurate bacteria genome de novo assembly is fundamental to understand the evolution and pathogenesis of new bacteria species. The advent and popularity of Third-Generation Sequencing (TGS) enables assembly of bacteria genomes at an unprecedented speed. However, most current TGS assemblers were specifically designed for human or other species that do not have a circular genome. Besides, the repetitive DNA fragments in many bacterial genomes plus the high error rate of long sequencing data make it still very challenging to accurately assemble their genomes even with a relatively small genome size. Therefore, there is an urgent need for the development of an optimized method to address these issues. RESULTS: We developed B-assembler, which is capable of assembling bacterial genomes when there are only long reads or a combination of short and long reads. B-assembler takes advantage of the structural resolving power of long reads and the accuracy of short reads if applicable. It first selects and corrects the ultra-long reads to get an initial contig. Then, it collects the reads overlapping with the ends of the initial contig. This two-round assembling procedure along with optimized error correction enables a high-confidence and circularized genome assembly. Benchmarked on both synthetic and real sequencing data of several species of bacterium, the results show that both long-read-only and hybrid-read modes can accurately assemble circular bacterial genomes free of structural errors and have fewer small errors compared to other assemblers. CONCLUSIONS: B-assembler provides a better solution to bacterial genome assembly, which will facilitate downstream bacterial genome analysis.

Fusobacterium necrophorum oral infections - A need for guidance.

F. necrophorum, a gram-negative obligate anaerobe, causes pharyngotonsillitis, peritonsillar abscess and the Lemierre Syndrome as well as other significant infections. Clinical information on this bacterium has increased dramatically over the past 20 years, yet no standard guidance exists for treating these infections. While data support F. necrophorum as a cause of pharyngotonsillitis, no consensus exists on the clinical importance of these findings especially in the 15-30 age group. Similarly, recent data find this bacterium the most frequent and most likely to recur in peritonsillar abscess for that age group. Should this impact how we treat these patients? Finally, we have no studies of either antibiotics or anticoagulation for the Lemierre Syndrome. Thus, each physician making the diagnosis of the Lemierre Syndrome chooses antibiotics (and their duration) and whether or not to anticoagulate without guidance. Infectious disease specialists and hospitalists would benefit from consensus expert opinions based on reviewing data on these infections.

Genetic Analysis of a Cohort of 275 Patients with Hyper-IgE Syndromes and/or Chronic Mucocutaneous Candidiasis.

Hyper-IgE syndromes and chronic mucocutaneous candidiasis constitute rare primary immunodeficiency syndromes with an overlapping clinical phenotype. In recent years, a growing number of underlying genetic defects have been identified. To characterize the underlying genetic defects in a large international cohort of 275 patients, of whom 211 had been clinically diagnosed with hyper-IgE syndrome and 64 with chronic mucocutaneous candidiasis, targeted panel sequencing was performed, relying on Agilent HaloPlex and Illumina MiSeq technologies. The targeted panel sequencing approach allowed us to identify 87 (32 novel and 55 previously described) mutations in 78 patients, which generated a diagnostic success rate of 28.4%. Specifically, mutations in DOCK8 (26 patients), STAT3 (21), STAT1 (15), CARD9 (6), AIRE (3), IL17RA (2), SPINK5 (3), ZNF341 (2), CARMIL2/RLTPR (1), IL12RB1 (1), and WAS (1) have been detected. The most common clinical findings in this cohort were elevated IgE (81.5%), eczema (71.7%), and eosinophilia (62.9%). Regarding infections, 54.7% of patients had a history of radiologically proven pneumonia, and 28.3% have had other serious infections. History of fungal infection was noted in 53% of cases and skin abscesses in 52.9%. Skeletal or dental abnormalities were observed in 46.2% of patients with a characteristic face being the most commonly reported feature (23.1%), followed by retained primary teeth in 18.9% of patients. Targeted panel sequencing provides a cost-effective first-line genetic screening method which allows for the identification of mutations also in patients with atypical clinical presentations and should be routinely implemented in referral centers.

Septic polyarthritis with Mycoplasma salivarium in a patient with common variable immunodeficiency: case report and review of the literature.

Mycoplasma salivarium is a common mycoplasma usually isolated from human oropharynx, particularly from individuals with periodontal disease. It is also among the more common mycoplasmal contaminants of eukaryotic cell cultures. Although M. salivarium has been isolated occasionally from abscesses and other sterile sites, to our knowledge, only three cases of septic arthritis have been documented in the past due to this organism, all in patients with humoral immunodeficiency. We now report a fourth case of septic polyarthritis in a patient with profound hypoimmunoglobulinemia who had experienced dental abscesses within the preceding 2 years. Our case highlights the importance of considering invasive mycoplasmal infection in hypogammaglobulinemic patients. It is likely of significance that the patient had suffered recurrent dental abscesses as a source of infection with M. salivarium .

Constrained chromatin accessibility in PU.1-mutated agammaglobulinemia patients.

The pioneer transcription factor (TF) PU.1 controls hematopoietic cell fate by decompacting stem cell heterochromatin and allowing nonpioneer TFs to enter otherwise inaccessible genomic sites. PU.1 deficiency fatally arrests lymphopoiesis and myelopoiesis in mice, but human congenital PU.1 disorders have not previously been described. We studied six unrelated agammaglobulinemic patients, each harboring a heterozygous mutation (four de novo, two unphased) of SPI1, the gene encoding PU.1. Affected patients lacked circulating B cells and possessed few conventional dendritic cells. Introducing disease-similar SPI1 mutations into human hematopoietic stem and progenitor cells impaired early in vitro B cell and myeloid cell differentiation. Patient SPI1 mutations encoded destabilized PU.1 proteins unable to nuclear localize or bind target DNA. In PU.1-haploinsufficient pro-B cell lines, euchromatin was less accessible to nonpioneer TFs critical for B cell development, and gene expression patterns associated with the pro- to pre-B cell transition were undermined. Our findings molecularly describe a novel form of agammaglobulinemia and underscore PU.1's critical, dose-dependent role as a hematopoietic euchromatin gatekeeper.

Oncolytic HSV-1 G207 Immunovirotherapy for Pediatric High-Grade Gliomas.

BACKGROUND: Outcomes in children and adolescents with recurrent or progressive high-grade glioma are poor, with a historical median overall survival of 5.6 months. Pediatric high-grade gliomas are largely immunologically silent or "cold," with few tumor-infiltrating lymphocytes. Preclinically, pediatric brain tumors are highly sensitive to oncolytic virotherapy with genetically engineered herpes simplex virus type 1 (HSV-1) G207, which lacks genes essential for replication in normal brain tissue. METHODS: We conducted a phase 1 trial of G207, which used a 3+3 design with four dose cohorts of children and adolescents with biopsy-confirmed recurrent or progressive supratentorial brain tumors. Patients underwent stereotactic placement of up to four intratumoral catheters. The following day, they received G207 (107 or 108 plaque-forming units) by controlled-rate infusion over a period of 6 hours. Cohorts 3 and 4 received radiation (5 Gy) to the gross tumor volume within 24 hours after G207 administration. Viral shedding from saliva, conjunctiva, and blood was assessed by culture and polymerase-chain-reaction assay. Matched pre- and post-treatment tissue samples were examined for tumor-infiltrating lymphocytes by immunohistologic analysis. RESULTS: Twelve patients 7 to 18 years of age with high-grade glioma received G207. No dose-limiting toxic effects or serious adverse events were attributed to G207 by the investigators. Twenty grade 1 adverse events were possibly related to G207. No virus shedding was detected. Radiographic, neuropathological, or clinical responses were seen in 11 patients. The median overall survival was 12.2 months (95% confidence interval, 8.0 to 16.4); as of June 5, 2020, a total of 4 of 11 patients were still alive 18 months after G207 treatment. G207 markedly increased the number of tumor-infiltrating lymphocytes. CONCLUSIONS: Intratumoral G207 alone and with radiation had an acceptable adverse-event profile with evidence of responses in patients with recurrent or progressive pediatric high-grade glioma. G207 converted immunologically "cold" tumors to "hot." (Supported by the Food and Drug Administration and others; ClinicalTrials.gov number, NCT02457845.).

Rubella Virus Infected Macrophages and Neutrophils Define Patterns of Granulomatous Inflammation in Inborn and Acquired Errors of Immunity.

Rubella virus (RuV) has recently been found in association with granulomatous inflammation of the skin and several internal organs in patients with inborn errors of immunity (IEI). The cellular tropism and molecular mechanisms of RuV persistence and pathogenesis in select immunocompromised hosts are not clear. We provide clinical, immunological, virological, and histological data on a cohort of 28 patients with a broad spectrum of IEI and RuV-associated granulomas in skin and nine extracutaneous tissues to further delineate this relationship. Combined immunodeficiency was the most frequent diagnosis (67.8%) among patients. Patients with previously undocumented conditions, i.e., humoral immunodeficiencies, a secondary immunodeficiency, and a defect of innate immunity were identified as being susceptible to RuV-associated granulomas. Hematopoietic cell transplantation was the most successful treatment in this case series resulting in granuloma resolution; steroids, and TNF-α and IL-1R inhibitors were moderately effective. In addition to M2 macrophages, neutrophils were identified by immunohistochemical analysis as a novel cell type infected with RuV. Four patterns of RuV-associated granulomatous inflammation were classified based on the structural organization of granulomas and identity and location of cell types harboring RuV antigen. Identification of conditions that increase susceptibility to RuV-associated granulomas combined with structural characterization of the granulomas may lead to a better understanding of the pathogenesis of RuV-associated granulomas and discover new targets for therapeutic interventions.

Mycoplasma genitalium Biofilms Contain Poly-GlcNAc and Contribute to Antibiotic Resistance.

Mycoplasma genitalium is an important etiologic agent of non-gonococcal urethritis (NGU), known for chronicity and multidrug resistance, in which biofilms may play an integral role. In some bacterial species capable of forming biofilms, extracellular polymeric substances (EPS) composed of poly-N-acetylglucosamine (PNAG) are a crucial component of the matrix. Monosaccharide analysis of M. genitalium strains revealed high abundance of GlcNAc, suggesting a biofilm-specific EPS. Chromatograms also showed high concentrations of galactose and glucose as observed in other mycoplasma species. Fluorescence microscopy of M. genitalium biofilms utilizing fluor-coupled lectins revealed differential staining of biofilm structures. Scanning electron microscopy (SEM) showed increasing maturation over time of bacterial "towers" seen in biofilm development. As seen with Mycoplasma pneumoniae, organisms within fully mature M. genitalium biofilms exhibited loss of cell polarization. Bacteria associated with disrupted biofilms exhibited decreased dose-dependent viability after treatment with antibiotics compared to bacteria with intact biofilms. In addition, growth index analysis demonstrated decreases in metabolism in cultures with disrupted biofilms with antibiotic treatment. Taken together, these data suggest that M. genitalium biofilms are a contributing factor in antibiotic resistance.

Evaluation of Commercial Molecular Diagnostic Methods for Detection and Determination of Macrolide Resistance in Mycoplasma pneumoniae.

We evaluated six commercial molecular tests targeting Mycoplasma pneumoniae, namely, the BioFire FilmArray respiratory panel (RP), the Meridian Alethia Mycoplasma Direct, the GenMark ePlex respiratory pathogen panel (RPP), the Luminex NxTAG RPP, the ELITech ELITe InGenius Mycoplasma MGB research use only (RUO) PCR, and the SpeeDx Resistance Plus MP assays. Laboratory-developed PCR assays at the University of Alabama at Birmingham and the Centers for Disease Control and Prevention were used as reference standards. Among 428 specimens, 212 were designated confirmed positives for M. pneumoniae The highest clinical sensitivities were found with the InGenius PCR (99.5%) and the FilmArray RP (98.1%). The Resistance Plus MP identified 93.3% of the confirmed-positive specimens, whereas 83.6, 64.6, and 55.7% were identified by the ePlex RPP, NxTAG RPP, and Mycoplasma Direct assays, respectively. There was no significant difference between the sensitivity of the reference methods and that of the FilmArray RP and InGenius assays, but the remaining four assays detected significantly fewer positive specimens (P < 0.05). Specificities of all assays were 99.5 to 100%. The Resistance Plus MP assay detected macrolide resistance in 27/33 specimens, resulting in a sensitivity of 81.8%. This study provides the first large-scale comparison of commercial molecular assays for detection of M. pneumoniae in the United States and identified clear differences among their performance. Additional studies are necessary to explore the impact of various test performances on patient outcome.

Benefit of Anakinra in Treating Pediatric Secondary Hemophagocytic Lymphohistiocytosis.

OBJECTIVE: To assess the benefit of the recombinant human interleukin-1 receptor antagonist anakinra in treating pediatric patients with secondary hemophagocytic lymphohistiocytosis (HLH)/macrophage activation syndrome (MAS) associated with rheumatic and nonrheumatic conditions. METHODS: A retrospective chart review of all anakinra-treated patients with secondary HLH/MAS was performed at Children's of Alabama from January 2008 through December 2016. Demographic, clinical, laboratory, and genetic characteristics, outcomes data, and information on concurrent treatments were collected from the records and analyzed using appropriate univariate statistical approaches to assess changes following treatment and associations between patient variables and outcomes. RESULTS: Forty-four patients with secondary HLH/MAS being treated with anakinra were identified in the electronic medical records. The median duration of hospitalization was 15 days. The mean pretreatment serum ferritin level was 33,316 ng/ml and dropped to 14,435 ng/ml (57% decrease) within 15 days of the start of anakinra treatment. The overall mortality rate in the cohort was 27%. Earlier initiation of anakinra (within 5 days of hospitalization) was associated with reduced mortality (P = 0.046), whereas thrombocytopenia (platelet count <100,000/µl) and STXBP2 mutations were both associated with increased mortality (P = 0.008 and P = 0.012, respectively). In considering patients according to their underlying diagnosis, those with systemic juvenile idiopathic arthritis (JIA) had the lowest mortality rate, with no deaths among the 13 systemic JIA patients included in the study (P = 0.006). In contrast, those with an underlying hematologic malignancy had the highest mortality rate, at 100% (n = 3). CONCLUSION: These findings suggest that anakinra appears to be effective in treating pediatric patients with non-malignancy-associated secondary HLH/MAS, especially when it is given early in the disease course and when administered to patients who have an underlying rheumatic disease.

Recurrent microdeletions at chromosome 2p11.2 are associated with thymic hypoplasia and features resembling DiGeorge syndrome.

BACKGROUND: Thymic hypoplasia/aplasia occurs as a part of DiGeorge syndrome, which has several known genetic causes, and with loss-of-function mutations in forkhead box N1 (FOXN1). OBJECTIVE: We sought to determine the cause of selective T-cell lymphopenia with inverted kappa/lambda ratio in several kindreds. METHODS: Patients were identified through newborn screening for severe combined immunodeficiency using the T-cell receptor excision circle assay. Those found to have selective T-cell lymphopenia underwent testing with chromosomal microarray analysis. Three-week-old mice heterozygous for a loss-of-function mutation in forkhead box I3 (FOXI3), a candidate gene within the common deleted region found in patients, were compared with wild-type littermates. Assessments included body and organ weights, flow cytometric analysis of thymocytes and splenocytes, and histologic/transcriptomic analyses of thymic tissue. RESULTS: Five kindreds with similar immunophenotypes that included selective T-cell lymphopenia had overlapping microdeletions at chromosome 2p11.2 that spanned FOXI3 and, in most cases, the immunoglobulin kappa light chain locus. Studies in a mouse knockout strain for FOXI3 revealed smaller body weights and relatively lower thymus weights in heterozygous compared with wild-type animals. Histology and flow cytometry on spleens and thymi from 3-week-old pups for T- and B-cell subsets and epithelial cells did not show any significant qualitative or quantitative differences. Transcriptomic analysis of thymic RNA revealed divergence in global transcriptomic signatures, and Ingenuity Pathway Analysis revealed predicted dysfunction in epithelial adherens junctions. CONCLUSIONS: Microdeletions at chromosome 2p11.2 are associated with T-cell lymphopenia and probable thymic hypoplasia in human subjects, and haploinsufficiency for FOXI3, a candidate gene within the deleted region, is the likely underlying cause.

A novel in situ multiplex immunofluorescence panel for the assessment of tumor immunopathology and response to virotherapy in pediatric glioblastoma reveals a role for checkpoint protein inhibition.

Immunotherapy with oncolytic herpes simplex virus-1 therapy offers an innovative, targeted, less-toxic approach for treating brain tumors. However, a major obstacle in maximizing oncolytic virotherapy is a lack of comprehensive understanding of the underlying mechanisms that unfold in CNS tumors/associated microenvironments after infusion of virus. We demonstrate that our multiplex biomarker screening platform comprehensively informs changes in both topographical location and functional states of resident/infiltrating immune cells that play a role in neuropathology after treatment with HSV G207 in a pediatric Phase 1 patient. Using this approach, we identified robust infiltration of CD8+ T cells suggesting activation of the immune response following virotherapy; however there was a corresponding upregulation of checkpoint proteins PD-1, PD-L1, CTLA-4, and IDO revealing a potential role for checkpoint inhibitors. Such work may ultimately lead to an understanding of the governing pathobiology of tumors, thereby fostering development of novel therapeutics tailored to produce optimal responses.