Impaired STING Activation Due to a Variant in the E3 Ubiquitin Ligase AMFR in a Patient with Severe VZV Infection and Hemophagocytic Lymphohistiocytosis.

Varicella zoster virus (VZV) is a neurotropic alphaherpesvirus exclusively infecting humans, causing two distinct pathologies: varicella (chickenpox) upon primary infection and herpes zoster (shingles) following reactivation. In susceptible individuals, VZV can give rise to more severe clinical manifestations, including disseminated infection, pneumonitis, encephalitis, and vasculopathy with stroke. Here, we describe a 3-year-old boy in whom varicella followed a complicated course with thrombocytopenia, hemorrhagic and necrotic lesions, pneumonitis, and intermittent encephalopathy. Hemophagocytic lymphohistiocytosis (HLH) was strongly suspected and as the condition deteriorated, HLH therapy was initiated. Although the clinical condition improved, longstanding hemophagocytosis followed despite therapy. We found that the patient carries a rare monoallelic variant in autocrine motility factor receptor (AMFR), encoding a ubiquitin ligase involved in innate cytosolic DNA sensing and interferon (IFN) production through the cyclic GMP-AMP synthase-stimulator of IFN genes (cGAS-STING) pathway. Peripheral blood mononuclear cells (PBMCs) from the patient exhibited impaired signaling downstream of STING in response dsDNA and 2'3'-cGAMP, agonists of cGAS and STING, respectively, and fibroblasts from the patient showed impaired type I IFN responses and significantly increased VZV replication. Overexpression of the variant AMFR R594C resulted in decreased K27-linked STING ubiquitination compared to WT AMFR. Moreover, ImageStream technology revealed reduced STING trafficking from ER to Golgi in cells expressing the patient AMFR R594C variant. This was supported by a dose-dependent dominant negative effect of expression of the patient AMFR variant as measured by IFN-ß reporter gene assay. Finally, lentiviral transduction with WT AMFR partially reconstituted 2'3'-cGAMP-induced STING-mediated signaling and ISG expression in patient PBMCs. This work links defective AMFR-STING signaling to severe VZV disease and hyperinflammation and suggests a direct role for cGAS-STING in the control of viral infections in humans. In conclusion, we describe a novel genetic etiology of severe VZV disease in childhood, also representing the first inborn error of immunity related to a defect in the cGAS-STING pathway.

STK4 Deficiency Impairs Innate Immunity and Interferon Production Through Negative Regulation of TBK1-IRF3 Signaling.

BACKGROUND: STK4 deficiency due to homozygous mutations in the STK4 gene encoding the STK4/MST1 kinase was first described in 2012. STK4/MST1 kinase regulates cell proliferation, survival, differentiation, and immune responses through canonical and non-canonical Hippo signaling pathways. OBJECTIVE: We describe an 11-year-old girl with a clinical presentation consisting of severe recurrent herpes zoster, chronic warts, and recurrent pneumonias, as well as a somatic phenotype with hypothyroidism and low stature. Whole exome sequencing revealed STK4 deficiency due to homozygosity for a novel frameshift variant in STK4, c.523dupA, p.(L174fsTer45), resulting in a premature stop codon within the kinase domain. METHODS: We performed a thorough investigation of the genetics and innate and adaptive immunological abnormalities in STK4 deficiency. RESULTS: We show significantly impaired type I, II, and III interferon (IFN) responses and partly reduced proinflammatory cytokine responses to ligands of Toll-like receptor (TLR)3, TLR9, and the cytosolic RNA and DNA sensors as well as to microorganisms. Impaired IFN responses could be attributed to reduced phosphorylation of TBK1 and IRF3. Moreover, virus infection induced enhanced cell death by apoptosis. Importantly, autophagy pathways were slightly disturbed, with enhanced LC3B-Ito LCB3-II conversion at the single cell level but normal overall formation of LCB3 punctae. Finally, the patient displayed some indicators of impaired adaptive immunity in the form of insufficient vaccination responses, T cell lymphopenia, and reduced Treg fractions, although with largely normal T cell proliferation and normal IFNg production. CONCLUSION: Here, we demonstrate disturbances in various immune cell populations and pathways involved in innate immune responses, cell death, autophagy, and adaptive immunity in a patient homozygous for a novel STK4 frameshift mutation.

Sinogenic intracranial complications: is adalimumab a culprit?

We present two 11-year-old girls with chronic recurrent multifocal osteomyelitis, treated with adalimumab. Both developed severe intracranial complications to sinusitis. Patient 1 had been treated with adalimumab for 15 months when she developed acute sinusitis complicated by an orbital abscess, forehead swelling, a subdural empyema and osteomyelitis of the frontal bone. She was treated with a rhinosurgical and neurosurgical approach with intravenous antibiotics.Patient 2 had been in adalimumab treatment for 10 weeks. Adalimumab was discontinued 8 weeks prior to developing subdural empyema and subcortical abscesses in combination with sinusitis. She was treated with endoscopic sinus surgery and intravenous antibiotics. Both patients had developed psoriasis and episodes of infection during treatment. They were non-septic and had low fever on presentation. None of the patients suffered any long-term neurological sequelae. The immunosuppressive treatment with adalimumab is considered to be the cause of the sinogenic intracranial complications in our cases.

Successful management of transfusion-dependent congenital dyserythropoietic anemia type 1b with interferon alfa-2a.

The congenital dyserythropoietic anemias (CDAs) are a group of rare inherited blood disorders characterized by ineffective erythropoiesis as the principal cause of anemia. We present a child with CDA 1b-the rarest and least well-described type-due to a mutation in the C15orf41 gene. The patient presented with severe in utero and neonatal manifestations, typical peripheral limb anomalies as well as rarely reported cardiac manifestations, visual impairment, short stature, and hip dysplasia. Anemia was complicated by iron overload and pronounced extra medullary erythropoiesis leading to skull deformities. The patient responded to treatment with pegylated interferon alfa-2a.

Infantile hemophagocytic lymphohistiocytosis in a case of chediak-higashi syndrome caused by a mutation in the LYST/CHS1 gene presenting with delayed umbilical cord detachment and diarrhea.

A 2-month-old female infant, born to consanguineous parents, presented with infections in skin and upper respiratory tract. She was notable for delayed umbilical cord detachment, partial albinism, and neurological irritability. Giant granules were present in white blood cells. The intracellular perforin content in CD8 T cells seems to correlate to the immune activation state of the patient with 82% and 8% perforin-containing CD8 T cells at active and nonactive hemophagocytic lymphohistiocytosis (HLH) disease, respectively. HLH was confirmed by hemophagocytosis in bone marrow and absent natural killer cell activity. The patient carried a homozygous G>A mutation in the 3' splice site of intron 24 of the LYST/CHS1 gene, leading to the use of an alternative YAG splice site located in exon 25, introducing a premature STOP codon (L2355fsX2370; NP_000072.2). The early-onset accelerated phase in this severe phenotype of Chediak-Higashi syndrome was probably induced by rotaviral infection. Interestingly, the intracellular perforin content in CD8 T cells seems to correlate to the immune activation state of the patient. Late separation of the umbilical cord in concordance with clinical symptoms should lead to evaluation of a possible neutrophil dysfunction including Chediak-Higashi syndrome before onset of HLH.

Caesarean section and hospitalization for respiratory syncytial virus infection: a population-based study.

BACKGROUND AND OBJECTIVE: Hospitalization for respiratory syncytial virus (RSV) infection and asthma share common determinants, and meta-analyses indicate that children delivered by caesarean section (CS) are at increased risk of asthma. We aimed to investigate whether birth by CS is associated with an increased risk of hospitalization for RSV illness. METHODS: This was a population-based national register-based cohort study, conducted between January, 1997 and June, 2003, which included all children born in Denmark and all hospitalizations for RSV disease in them from 0 to 23 months of age. We used Cox regression with adjustment for prematurity, asphyxia, birthweight, multiple births, single parenthood, maternal smoking during pregnancy, older siblings and asthma diagnoses up to 2 weeks before hospitalization for RSV infection, to compare the effects of acute or elective CS versus vaginal delivery, on subsequent hospitalization for RSV disease. A test for homogeneity was used to assess for effect over time. RESULTS: 399,175 children with 10,758 hospitalizations for RSV illness were included; 31,715 were born by acute CS and 30,965 by elective CS. Adjusted hazard ratios for hospitalization for RSV infection in children born by acute CS and by elective CS were 1.09 (1.01-1.17) and 1.27 (1.19-1.36), respectively. The effect of elective CS remained unchanged throughout the first 2 years of life (P = 0.53), whereas the effect of acute CS was only present in the second year of life (P = 0.001). CONCLUSION: Delivery by caesarian section is associated with an increased risk of hospitalization for RSV infection. This effect continues at least throughout the first 2 years of life.

The burden of rotavirus disease in Denmark 2009-2010.

BACKGROUND: This study sought to determine the incidence and the burden of severe diarrheal disease in Denmark with emphasis on rotavirus (RV) disease. METHODS: This study was designed as a national prospective disease surveillance of children <5 years of age hospitalized for acute gastroenteritis in Denmark during March 2009 to April 2010, using rapid RV and adenovirus antigen detection. RESULTS: A total of 3100 hospitalizations annually among Danish children <5 years of age can be attributed to acute gastroenteritis and 1210 (39%) of these to RV disease. The majority of RV-associated hospitalizations occur among children ≤ 24 months of age (RV-associated hospitalization rate: 7.7/1000 children ≤ 24 months of age and 3.8/1000 children <5 years of age). Although the well-known seasonal pattern of RV was evident with a peak during the spring months of March through April, our active surveillance demonstrated RV-associated hospitalizations throughout the year. Genotyping of a subset of RV-samples demonstrated high frequency of G1 (39%) and G4 (32%). Adenovirus was detected in 350 acute gastroenteritis-associated hospitalizations (11.2%). CONCLUSION: In conclusion, we present national disease burden data on severe cases of gastroenteritis and specifically RV-associated disease and demonstrate that RV is indeed ubiquitous in the population and can be considered a major health burden among young Danish children.

Sphingosine kinase 1 is a relevant molecular target in gastric cancer.

Sphingosine kinase 1 (Sphk1), a lipid kinase implicated in cell transformation and tumor growth, is overexpressed in gastric cancer and is linked with a poor prognosis. The biological relevance of Sphk1 expression in gastric cancer is unclear. Here, we studied the functional significance of Sphk1 as a novel molecular target for gastric cancer by using an antisense oligonucleotide approach in vitro and in vivo. Gastric cancer cell lines (MKN28 and N87) were treated with Sphk1 with locked nucleic acid-antisense oligonucleotides (LNA-ASO). Sphk1 target regulation, cell growth, and apoptosis were assessed for single-agent Sphk1 LNA-ASO and for combinations with doxorubicin. Athymic nude mice xenografted with gastric cancer cells were treated with Sphk1 LNA and assessed for tumor growth and Sphk1 target regulation, in vivo. In vitro, nanomolar concentrations of Sphk1 LNA-ASO induced an approximately two-fold reduction in Sphk1 mRNA in both the cell lines. This resulted in a 1.6-fold increase in apoptosis and inhibited the growth of gastric cancer cells by more than 50% (P < 0.05). The combination of Sphk1 LNA-ASO with doxorubicin resulted in significant chemosensitization. In vivo, Sphk1 LNA-ASO displayed neither mRNA target regulation in xenografts nor antitumor activity in two independent nude mouse xenograft models. In conclusion, the potent single-agent activity and the synergistic effect of Sphk1 LNA-ASO in combination with chemotherapy in vitro highlight Sphk1 as a biologically relevant molecular target for gastric cancer. Further studies are warranted to overcome the challenge of delivering Sphk1-targeting RNA-therapeutics to solid tumors in vivo.

Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates.

The potency and specificity of locked nucleic acid (LNA) antisense oligonucleotides was investigated as a function of length and affinity. The oligonucleotides were designed to target apolipoprotein B (apoB) and were investigated both in vitro and in vivo. The high affinity of LNA enabled the design of short antisense oligonucleotides (12- to 13-mers) that possessed high affinity and increased potency both in vitro and in vivo compared to longer oligonucleotides. The short LNA oligonucleotides were more target specific, and they exhibited the same biodistribution and tissue half-life as longer oligonucleotides. Pharmacology studies in both mice and non-human primates were conducted with a 13-mer LNA oligonucleotide against apoB, and the data showed that repeated dosing of the 13-mer at 1-2 mg/kg/week was sufficient to provide a significant and long lasting lowering of non-high-density lipoprotein (non-HDL) cholesterol without increasing serum liver toxicity markers. The data presented here show that oligonucleotide length as a parameter needs to be considered in the design of antisense oligonucleotide and that potent short oligonucleotides with sufficient target affinity can be generated using the LNA chemistry. Conclusively, we present a 13-mer LNA oligonucleotide with therapeutic potential that produce beneficial cholesterol lowering effect in non-human primates.

A locked nucleic acid antisense oligonucleotide (LNA) silences PCSK9 and enhances LDLR expression in vitro and in vivo.

BACKGROUND: The proprotein convertase subtilisin/kexin type 9 (PCSK9) is an important factor in the etiology of familial hypercholesterolemia (FH) and is also an attractive therapeutic target to reduce low density lipoprotein (LDL) cholesterol. PCSK9 accelerates the degradation of hepatic low density lipoprotein receptor (LDLR) and low levels of hepatic PCSK9 activity are associated with reduced levels of circulating LDL-cholesterol. METHODOLOGY/PRINCIPAL FINDINGS: The present study presents the first evidence for the efficacy of a locked nucleic acid (LNA) antisense oligonucleotide (LNA ASO) that targets both human and mouse PCSK9. We employed human hepatocytes derived cell lines HepG2 and HuH7 and a pancreatic mouse beta-TC3 cell line known to express high endogenous levels of PCSK9. LNA ASO efficiently reduced the mRNA and protein levels of PCSK9 with a concomitant increase in LDLR protein levels after transfection in these cells. In vivo efficacy of LNA ASO was further investigated in mice by tail vein intravenous administration of LNA ASO in saline solution. The level of PCSK9 mRNA was reduced by approximately 60%, an effect lasting more than 16 days. Hepatic LDLR protein levels were significantly up-regulated by 2.5-3 folds for at least 8 days and approximately 2 fold for 16 days. Finally, measurement of liver alanine aminotransferase (ALT) levels revealed that long term LNA ASO treatment (7 weeks) does not cause hepatotoxicity. CONCLUSION/SIGNIFICANCE: LNA-mediated PCSK9 mRNA inhibition displayed potent reduction of PCSK9 in cell lines and mouse liver. Our data clearly revealed the efficacy and safety of LNA ASO in reducing PCSK9 levels, an approach that is now ready for testing in primates. The major significance and take home message of this work is the development of a novel and promising approach for human therapeutic intervention of the PCSK9 pathway and hence for reducing some of the cardiovascular risk factors associated with the metabolic syndrome.

SPC3042: a proapoptotic survivin inhibitor.

The ability to regulate the cellular homeostasis of a higher organism through tight control of apoptosis and cell division is crucial for life. Dysregulation of these mechanisms is often associated with cancerous phenotypes in cells. Optimal cancer therapy is a fine balance between effective cancer cell killing and at the same time minimizing, or avoiding, damage to the surrounding healthy tissue. To obtain this, it is necessary to identify and inhibit molecular targets on which the cancer cells are strongly dependent. Survivin represents such a target, and it has been published previously that peptide vaccines, the small-molecule YM155, and the antisense molecule LY2181308/ISIS23722, via different mechanisms, have been used as survivin inhibitors. In this article, a new potent antisense inhibitor of survivin, SPC3042, is presented, and the properties of SPC3042 are compared with the previously published antisense drug, LY2181308/ISIS23722. SPC3042 is a 16-mer locked nucleic acid (LNA) oligonucleotide and designed as a fully phosphorothiolated gapmer containing 7 LNA nucleotides in the flanks. The LNA nucleotides in SPC3042 provide nuclease stability and higher potency for survivin mRNA inhibition compared with earlier generations of antisense reagents. It is shown that the down-regulation of survivin with SPC3042 leads to cell cycle arrest, pronounced cellular apoptosis, and down-regulation of Bcl-2. It is also shown that SPC3042 is a sensitizer of prostate cancer cells to Taxol treatment in vitro and in vivo.

Survivin mRNA antagonists using locked nucleic acid, potential for molecular cancer therapy.

We have investigated the effects of different locked nucleic acid modified antisense mRNA antagonists against Survivin in a prostate cancer model. These mRNA antagonists were found to be potent inhibitors of Survivin expression at low nanomolar concentrations. Additionally there was a pronounced synergistic effect when combining the mRNA antagonists against Survivin with the chemotherapeutic Taxol. This effect was demonstrated at concentrations of antagonists far lower than any previously demonstrated, indicating the high potential of locked nucleic acid for therapeutic use. Further characterisations in vivo are ongoing.