Michael W. Beresford

Gain-of-function mutations in IFIH1 cause a spectrum of human disease phenotypes associated with upregulated type I interferon signaling

The type I interferon system is integral to human antiviral immunity. However, inappropriate stimulation or defective negative regulation of this system can lead to inflammatory disease. We sought to determine the molecular basis of genetically uncharacterized cases of the type I interferonopathy Aicardi-Goutières syndrome and of other undefined neurological and immunological phenotypes also demonstrating an upregulated type I interferon response. We found that heterozygous mutations in the cytosolic double-stranded RNA receptor gene IFIH1 (also called MDA5) cause a spectrum of neuroimmunological features consistently associated with an enhanced interferon state. Cellular and biochemical assays indicate that these mutations confer gain of function such that mutant IFIH1 binds RNA more avidly, leading to increased baseline and ligand-induced interferon signaling. Our results demonstrate that aberrant sensing of nucleic acids can cause immune upregulation.

Tartrate-resistant acid phosphatase deficiency causes a bone dysplasia with autoimmunity and a type I interferon expression signature

We studied ten individuals from eight families showing features consistent with the immuno-osseous dysplasia spondyloenchondrodysplasia. Of particular note was the diverse spectrum of autoimmune phenotypes observed in these individuals (cases), including systemic lupus erythematosus, Sjögrens syndrome, hemolytic anemia, thrombocytopenia, hypothyroidism, inflammatory myositis, Raynauds disease and vitiligo. Haplotype data indicated the disease gene to be on chromosome 19p13, and linkage analysis yielded a combined multipoint log10 odds (LOD) score of 3.6. Sequencing of ACP5, encoding tartrate-resistant acid phosphatase, identified biallelic mutations in each of the cases studied, and in vivo testing confirmed a loss of expressed protein. All eight cases assayed showed elevated serum interferon alpha activity, and gene expression profiling in whole blood defined a type I interferon signature. Our findings reveal a previously unrecognized link between tartrate-resistant acid phosphatase activity and interferon metabolism and highlight the importance of type I interferon in the genesis of autoimmunity.

Disease activity, severity, and damage in the UK juvenile-onset systemic lupus erythematosus cohort

OBJECTIVE The UK Juvenile-Onset Systemic Lupus Erythematosus (JSLE) Cohort Study is a multicenter collaborative network established with the aim of improving the understanding of juvenile SLE. The present study was undertaken to describe the clinical manifestations and disease course in patients with juvenile SLE from this large, national inception cohort. METHODS Detailed data on clinical phenotype were collected at baseline and at regular clinic reviews and annual followup assessments in 232 patients from 14 centers across the UK over 4.5 years. Patients with SLE were identified according to the American College of Rheumatology (ACR) SLE classification criteria. The present cohort comprised children with juvenile SLE (n=198) whose diagnosis fulfilled ≥4 of the ACR criteria for SLE. RESULTS Among patients with juvenile SLE, the female:male sex distribution was 5.6:1 and the median age at diagnosis was 12.6 years (interquartile range 10.4-14.5 years). Male patients were younger than female patients (P<0.01). Standardized ethnicity data demonstrated a greater risk of juvenile SLE in non-Caucasian UK patients (P<0.05). Scores on the pediatric adaptation of the 2004 British Isles Lupus Assessment Group disease activity index demonstrated significantly increased frequencies of musculoskeletal (82%), renal (80%), hematologic (91%), immunologic (54%), and neurologic (26%) involvement among the patients over time. A large proportion of the patients (93%) were taking steroids and 24% of the patients required treatment with cyclophosphamide. Disease damage was common, with 28% of the patients having a Systemic Lupus International Collaborating Clinics/ACR damage score of ≥1. CONCLUSION The data on these patients from the UK JSLE Cohort Study, comprising one of the largest national inception cohorts of patients with juvenile SLE to date, indicate that severe organ involvement and significant disease activity are primary characteristics in children with juvenile SLE. In addition, accumulation of disease-associated damage could be seen.

Protein kinase Cδ deficiency causes mendelian systemic lupus erythematosus with B cell-defective apoptosis and hyperproliferation

OBJECTIVE Systemic lupus erythematosus (SLE) is a prototype autoimmune disease that is assumed to occur via a complex interplay of environmental and genetic factors. Rare causes of monogenic SLE have been described, providing unique insights into fundamental mechanisms of immune tolerance. The aim of this study was to identify the cause of an autosomal-recessive form of SLE. METHODS We studied 3 siblings with juvenile-onset SLE from 1 consanguineous kindred and used next-generation sequencing to identify mutations in the disease-associated gene. We performed extensive biochemical, immunologic, and functional assays to assess the impact of the identified mutations on B cell biology. RESULTS We identified a homozygous missense mutation in PRKCD, encoding protein kinase δ (PKCδ), in all 3 affected siblings. Mutation of PRKCD resulted in reduced expression and activity of the encoded protein PKCδ (involved in the deletion of autoreactive B cells), leading to resistance to B cell receptor- and calcium-dependent apoptosis and increased B cell proliferation. Thus, as for mice deficient in PKCδ, which exhibit an SLE phenotype and B cell expansion, we observed an increased number of immature B cells in the affected family members and a developmental shift toward naive B cells with an immature phenotype. CONCLUSION Our findings indicate that PKCδ is crucial in regulating B cell tolerance and preventing self-reactivity in humans, and that PKCδ deficiency represents a novel genetic defect of apoptosis leading to SLE.

Respiratory syncytial virus infection in high risk infants and the potential impact of prophylaxis in a United Kingdom cohort

BACKGROUND Bronchiolitis caused by respiratory syncytial virus (RSV) is an important cause of morbidity in ex-premature infants. In a randomised placebo controlled trial monoclonal antibody prophylaxis showed a 55% reduction in relative risk of hospital admission for these high risk infants, against a background incidence of 10.6 admissions per 100 high risk infants. AIMS To follow a cohort of high risk infants in order to assess hospitalisation rate from RSV and the potential impact of prophylaxis for these patients in a UK local health authority. METHODS A cohort of high risk infants from a local health authority were followed over the 1998/99 and 1999/2000 RSV seasons. The high risk population was defined as infants who, at the beginning of the seasons studied, were: (1) under 6 months old and born prior to 36 weeks gestation with no domiciliary oxygen requirement; or (2) under 24 months of age and discharged home in supplemental oxygen. All admissions with bronchiolitis during the season were identified. RESULTS A total of 370 high risk infants were identified for the 1998/99 season and 286 for the following year. Over the two years there were 68 admissions. Significantly more admissions occurred from group 2 infants. RSV was identified in 27 cases (four admissions per hundred high risk infants). Prophylaxis may have saved up to £195 134 in hospital costs over the two years, but would have cost £1.1 million in drug acquisition costs. CONCLUSIONS Careful consideration of risk factors is needed when selecting infants for RSV prophylaxis.

Validity of a three-variable Juvenile Arthritis Disease Activity Score in children with new-onset juvenile idiopathic arthritis

Objectives To investigate the validity and feasibility of the Juvenile Arthritis Disease Activity Score (JADAS) in the routine clinical setting for all juvenile idiopathic arthritis (JIA) disease categories and explore whether exclusion of the erythrocyte sedimentation rate (ESR) from JADAS (the ‘JADAS3’) influences correlation with single markers of disease activity. Methods JADAS-71, JADAS-27 and JADAS-10 were determined at baseline for an inception cohort of children with JIA in the Childhood Arthritis Prospective Study. JADAS3-71, JADAS3-27 and JADAS3-10 were determined using an identical formula but with exclusion of ESR. Correlation of JADAS with JADAS3 and single measures of disease activity/severity were determined by category. Results Of 956 eligible children, sufficient data were available to calculate JADAS-71, JADAS-27 and JADAS-10 at baseline in 352 (37%) and JADAS3 in 551 (58%). The median (IQR) JADAS-71, JADAS-27 and JADAS-10 for all 352 children was 11 (5.9–18), 10.4 (5.7–17) and 11 (5.9–17.3), respectively. Median JADAS and JADAS3 varied significantly with the category (Kruskal–Wallis p=0.0001), with the highest values in children with polyarticular disease patterns. Correlation of JADAS and JADAS3 across all categories was excellent. Correlation of JADAS71 with single markers of disease activity/severity was good to moderate, with some variation across the categories. With the exception of ESR, correlation of JADAS3-71 was similar to correlation of JADAS-71 with the same indices. Conclusions This study is the first to apply JADAS to all categories of JIA in a routine clinical setting in the UK, adding further information about the feasibility and construct validity of JADAS. For the majority of categories, clinical applicability would be improved by exclusion of the ESR.

Does sustained lung inflation at resuscitation reduce lung injury in the preterm infant

Background: Bronchopulmonary dysplasia (BPD) is a common outcome of preterm birth. Experimental animal work has shown that initial ventilation strategies injure the immature lung and may lead to BPD. Studies with asphyxiated babies have shown that, if tidal ventilation at birth is preceded by sustained lung inflation, larger inflation volumes can be achieved, which is thought to lead to clearance of lung fluid and formation of the functional residual capacity (FRC). Objective: To see if sustained lung inflation at initial resuscitation of preterm babies would facilitate the removal of lung fluid, establish the FRC, and allow an even distribution of alveolar opening, permitting less aggressive ventilation, leading to a reduction in pulmonary inflammation and subsequent BPD. Method: The outcomes of 52 babies of less than 31 weeks gestation, resuscitated at birth using either a sustained lung inflation of five seconds or a conventional lung inflation of two seconds for the first assisted breath of resuscitation, were examined. Evidence of pulmonary inflammation was determined by quantification of interleukins 6, 10, and 1β and tumour necrosis factor α in bronchoalveolar lavage fluid by enzyme linked immunosorbent assay. Results: There were no significant differences in any of the cytokines. Death occurred in 3/26 babies in the conventional group and 6/26 babies in the sustained lung inflation group. Survival without BPD occurred in 13/26 and 14/26 respectively. Conclusion: The use of sustained lung inflation at resuscitation did not reduce lung injury, as measured by inflammatory markers.

Detectable IL-8 and IL-10 in bronchoalveolar lavage fluid from preterm infants ventilated for respiratory distress syndrome.

Pro-inflammatory cytokines such as IL-8 play an important role in the inflammatory response to neonatal airway injury. Difficulty in detecting counter-regulatory cytokines such as IL-10 in lavage fluid from preterm infants led to the suggestion that its deficit may be a factor in the etiology of chronic lung disease of prematurity (CLD). The aim of the study was to determine IL-8 and IL-10 concentrations in lavage fluid from preterm infants ventilated for respiratory distress syndrome. Fifty infants <30 wk gestation were studied who had been randomized to receive a natural or synthetic surfactant. Lavage samples were collected daily for the first week and twice weekly thereafter. Samples were immediately centrifuged and stored at −70°C. Cytokine concentrations were quantified in duplicate using commercially available sandwich ELISA kits. Lavage IL-10 concentration, at a minimum initially, rose significant over the first five postnatal days (p = 0.009). In the same samples, lavage IL-8 concentrations rose significantly over the first postnatal week (p < 0.001), the rise preceding that of IL-10. Infants dying or developing CLD had a significant early rise in both cytokine concentrations. Compared with infants developing CLD, lavage IL-10 concentrations were significantly higher on d 1 among those not developing CLD but significantly lower on d 4 (p < 0.05). To conclude, IL-10 is detectable in lavage fluid from ventilated preterm infants and its concentrations rise significantly over the first five postnatal days. In the same samples, IL-8 concentration also rises and this increase precedes the rise in IL-10.

Randomised controlled trial of patient triggered and conventional fast rate ventilation in neonatal respiratory distress syndrome

AIM To compare patient triggered, with conventional fast rate, ventilation in a randomised controlled trial using the incidence of chronic lung disease as the primary outcome measure. METHODS Three hundred and eighty six preterm infants with birthweights from 1000 to 2000 g, and requiring ventilation for respiratory distress syndrome within 24 hours of birth, were randomised to receive either conventional or trigger ventilation with the SLE 2000 ventilator. RESULTS There were no significant differences in the incidence of chronic lung disease (28 day and 36 week definitions), death, pneumothorax, intraventricular haemorrhage, number of ventilator days, or length of oxygen dependency between groups. CONCLUSIONS Patient triggered ventilation in preterm infants with respiratory distress syndrome is feasible. No significant differences, when compared with conventional fast rate ventilation in important medium and longer term outcome measures, were evident.

The role of neutrophil apoptosis in juvenile-onset systemic lupus erythematosus.

OBJECTIVE Accumulation of apoptotic cells may lead to the development of systemic lupus erythematosus (SLE) through a breakdown in immune tolerance. Altered neutrophil apoptosis may contribute to nuclear autoantigen exposure, ultimately leading to autoantibody generation. This study aimed to determine whether neutrophil apoptosis is altered in patients with juvenile-onset SLE as compared with controls. METHODS Apoptosis was measured in neutrophils from patients with juvenile-onset SLE (n=12), adult-onset SLE (n=6), and pediatric patients with inflammatory (n=12) and noninflammatory (n=12) conditions. Annexin V staining and flow cytometry were used to determine neutrophil apoptosis. Proapoptotic and antiapoptotic proteins were measured in sera and in neutrophil cell lysates. RESULTS Neutrophil apoptosis was significantly increased in patients with juvenile-onset SLE as compared with the noninflammatory controls at time 0. Incubation of neutrophils with sera from patients with juvenile-onset SLE further increased neutrophil apoptosis as compared with incubation with sera from pediatric controls. Concentrations of TRAIL and FasL were significantly increased in sera from patients with juvenile-onset SLE, whereas interleukin-6, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were significantly decreased. Addition of GM-CSF to sera from patients with juvenile-onset SLE significantly decreased neutrophil apoptosis as compared with juvenile-onset SLE sera alone. The expression of proapoptotic proteins (caspase 3, Fas, and FADD) was elevated in juvenile-onset SLE neutrophils, whereas the expression of antiapoptotic proteins (cellular inhibitor of apoptosis 1 and 2 and X-linked inhibitor of apoptosis) was decreased. Neutrophil apoptosis correlated with biomarkers of disease activity (erythrocyte sedimentation rate and double-stranded DNA concentration) and the British Isles Lupus Assessment Group disease activity score. CONCLUSION Our data demonstrate an imbalance in proapoptotic and antiapoptotic factors in both neutrophils and sera from patients with juvenile-onset SLE. This imbalance results in increased neutrophil apoptosis in these patients. Correlations with markers of disease activity indicate that altered neutrophil apoptosis in juvenile-onset SLE patients may play a pathogenic role in this condition.