BACKGROUND & AIMS Chronic hepatitis C virus infection activates an intrahepatic immune response, leading to increased expression of interferon (IFN)-stimulated genes and activation of natural killer (NK) cells—the most prevalent innate immune cell in the liver. We investigated whether the elimination of HCV with direct-acting antiviral agents normalizes expression of IFN-stimulated genes and NK cell function. METHODS We used multicolor flow cytometry to analyze NK cells from liver and blood of 13 HCV-infected patients who did not respond to treatment with pegylated interferon and ribavirin. Samples were collected before and during IFN-free treatment with daclatasvir and asunaprevir therapy and compared with those from blood of 13 healthy individuals (controls). Serum levels of CXCL10 and CXCL11 were measured by ELISA. RESULTS Before treatment, all patients had increased levels of CXCL10 or CXCL11 and a different NK cell phenotype from controls, characterized by increased expression of HLA-DR, NKp46, NKG2A, CD85j, pSTAT1, STAT1, and TNF-related apoptosis-inducing ligand (TRAIL). NK cells from patients also had increased degranulation and decreased production of IFNγ and TNFα compared with NK cells from controls. Nine patients had an end-of-treatment response (undetectable virus) and 4 had virologic breakthrough between weeks 4 and 12 of therapy. A rapid decrease in viremia and level of inflammatory cytokines in all patients was associated with decreased activation of intrahepatic and blood NK cells; it was followed by restoration of a normal NK cell phenotype and function by week 8 in patients with undetectable viremia. This normalized NK cell phenotype was maintained until week 24 (EOT). CONCLUSIONS DAA-mediated clearance of HCV is associated with loss of intrahepatic immune activation by IFNα, indicated by decreased levels of CXCL10 and CXCL11 and normalization of NK cell phenotype and function.

Wide-spread protozoan parasites carry endosymbiotic dsRNA viruses with uncharted implications to the human host. Among them, Trichomonas vaginalis, a parasite adapted to the human genitourinary tract, infects globally ∼250 million each year rendering them more susceptible to devastating pregnancy complications (especially preterm birth), HIV infection and HPV-related cancer. While first-line antibiotic treatment (metronidazole) commonly kills the protozoan pathogen, it fails to improve reproductive outcome. We show that endosymbiotic Trichomonasvirus, highly prevalent in T. vaginalis clinical isolates, is sensed by the human epithelial cells via Toll-like receptor 3, triggering Interferon Regulating Factor -3, interferon type I and proinflammatory cascades previously implicated in preterm birth and HIV-1 susceptibility. Metronidazole treatment amplified these proinflammatory responses. Thus, a new paradigm targeting the protozoan viruses along with the protozoan host may prevent trichomoniasis-attributable inflammatory sequelae.

Ribavirin is an important component of interferon-based and direct antiviral treatment regimens for hepatitis C virus (HCV) infection. Immunomodulation, in particular improvement of the host interferon (IFN) response, has been proposed as ribavirin’s mechanism of action. Natural killer (NK) cells are sensitive biomarkers for IFN-α/β receptor signaling, as NK cell cytotoxicity and IFN-γ production are regulated by STAT1- and STAT4-phosphorylation, respectively. Specifically, pSTAT1-dependent NK cell cytotoxicity increases and pSTAT4-dependent IFN-γ production decreases in response to endogenous, virus-induced IFN-α and during IFN-α-based therapy. To assess whether ribavirin has a direct effect on NK cells and/or improves the IFN-γ response of NK cells in the presence of IFN-α, we prospectively studied 22 HCV patients with and 32 patients without 4 weeks of ribavirin pretreatment, who all received subsequent PegIFN/ribavirin combination therapy. During ribavirin pretreatment, the frequency of CD56dim NK cells with cytotoxic effector functions decreased (p=0.049) as did the frequency of CD56bright NK cells with the capacity to produce IFN-γ (p=0.001). In vitro or in vivo exposure of NK cells to ribavirin improved the pSTAT4 (p<0.01) but not pSTAT1 response of NK cells to subsequent stimulation with IFN-α. This was associated with an increase in IFN-γ production but not cytotoxicity of NK cells during subsequent IFN-α-based therapy. The frequency of IFN-γ-producing NK cells was greater in fast second-phase virological responders than in slow responders. Conclusion Ribavirin enhances the pSTAT4 and IFN-γ response of NK cells to IFN-α–stimulation.

Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed enzymes that ligate amino acids onto tRNA molecules. Genes encoding ARSs have been implicated in myriad dominant and recessive disease phenotypes. Glycyl-tRNA synthetase (GARS) is a bi-functional ARS that charges tRNAGly in the cytoplasm and mitochondria. GARS variants have been associated with dominant Charcot-Marie-Tooth disease but have not been convincingly implicated in recessive phenotypes. Here we describe a patient from the NIH Undiagnosed Diseases Program with a multi-system, developmental phenotype. Whole-exome sequence analysis revealed that the patient is compound heterozygous for one frameshift (p.Glu83Ilefs*6) and one missense (p.Arg310Gln) GARS variant. Using in vitro and in vivo functional studies, we show that both GARS variants cause a loss-of-function effect: the frameshift variant results in depleted protein levels and the missense variant reduces GARS tRNA charging activity. In support of GARS variant pathogenicity, our patient shows striking phenotypic overlap with other patients having ARS-related recessive diseases, including features associated with variants in both cytoplasmic and mitochondrial ARSs; this observation is consistent with the essential function of GARS in both cellular locations. In summary, our clinical, genetic, and functional analyses expand the phenotypic spectrum associated with GARS variants.

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Ca 2þ signaling is vital for various cellular processes including synaptic vesicle exocytosis, muscle contraction, regulation of secretion, gene transcription, and cellular proliferation. The endoplasmic reticulum (ER) is the largest intracellular Ca 2þ store, and dysregulation of ER Ca 2þ signaling and homeostasis contributes to the pathogenesis of various complex disorders and Mendelian disease traits. We describe four unrelated individuals with a complex multisystem disorder characterized by woolly hair, liver dysfunction, pruritus, dysmorphic features, hypotonia, and global developmental delay. Through whole-exome sequencing and family-based genomics, we identified bi-allelic variants in CCDC47 that encodes the Ca 2þ-binding ER transmembrane protein CCDC47. CCDC47, also known as calumin, has been shown to bind Ca 2þ with low affinity and high capacity. In mice, loss of Ccdc47 leads to embryonic lethality, suggesting that Ccdc47 is essential for early development. Characterization of cells from individuals with predicted likely damaging alleles showed decreased CCDC47 mRNA expression and protein levels. In vitro cellular experiments showed decreased total ER Ca 2þ storage, impaired Ca 2þ signaling mediated by the IP 3 R Ca 2þ release channel, and reduced ER Ca 2þ refilling via store-operated Ca 2þ entry. These results, together with the previously described role of CCDC47 in Ca 2þ signaling and development, suggest that bi-allelic loss-offunction variants in CCDC47 underlie the pathogenesis of this multisystem disorder. Ca 2þ signaling is a multipurpose intracellular signaling system that regulates a number of cellular processes including synaptic vesicle exocytosis, muscle contraction, regulation of secretion, transcription, and cellular proliferation. 1 The endoplasmic reticulum (ER), or the sarcoplasmic reticulum (SR) in muscle cells, is the largest store of intracellular Ca 2þ. 2 ER Ca 2þ depletion is also observed in a number of genetic disorders due to variants in Ca 2þ channels and sensors. For example, Brody myopathy (MIM: 601003) is caused by recessive variants in ATP2A1 (MIM: 611974), which encodes the fast-twitch skeletal muscle sarcoplasmic reticulum Ca 2þ ATPase (SERCA1), 3 while Darier disease (MIM: 124200) occurs due to variants in ATP2A2 (MIM: 108740), which encodes another sarcoplasmic reticulum Ca 2þ ATPase, SERCA2. 4 Minicore myopathy (MIM: 255320) and central core disease (MIM: 117000) result from variants in RYR1 (MIM: 180901), which encodes a major Ca 2þ release channel, 5 and autosomal centronuclear myopathy (MIM: 160150) is associated with variants in MTMR14 (MIM: 611089), which encodes a muscle-specific inositol phosphatase. 6 Stormorken syndrome (MIM: 185070), tubular aggregate myopathy 1 (MIM: 160565), and immunodeficiency 10 (MIM: 612783) are caused by variants in STIM1 (MIM: 605921), 7-9 which encodes a Ca 2þ sensor. Tubular aggregate myopathy 2 (MIM: 615883) and immunodeficiency 9 (MIM: 612782) are caused by variants in ORAI1 (MIM: 610277), 10,11 which encodes a Ca 2þ channel that...

The genetic etiologies of many rare disorders, including early infantile epileptic encephalopathies, are largely undiagnosed. A 6-year old girl was admitted to the National Institutes of Health Undiagnosed Diseases Program with profound intellectual disability, infantile onset seizures, chronic respiratory failure, facial dysmorphisms, skeletal abnormalities and atrial septum defect. A large region of homozygosity was discovered on chromosome 16, spanning 16q22.1–16q24.3 caused by uniparental disomy (UPD) that included a maternally inherited homozygous microdeletion covering exon 6 of WWOX (NM_016373.3). mRNA expression analysis revealed that the deletion led to nonsense-mediated decay of the NM_016373.3 transcript; the exon 6 of an alternative transcript (NM_130791.3), lacking the short-chain dehydrogenase, was utilized. The microdeletion in WWOX explains the seizures and intellectual disability, while pathogenic variants in another gene, HSPG2, are likely responsible for the patient’s skeletal abnormalities. This report describes a rare autosomal recessive disorder with multiple genetic etiologies, one of which involves UPD.

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