Four-Parameter FluoroSpot Assay Reveals That the Varicella Zoster Virus Elicits a Robust Memory T Cell IL-10 Response throughout Childhood.

During childhood, the composition and function of the T cell compartment undergoes significant changes. In healthy individuals, primary infection with herpesviruses is followed by latency, and occasional subclinical reactivation ensures transmission and contributes to an emerging pool of memory T cells. In immunocompromised individuals, herpesviruses can be life threatening. However, knowledge about the spectrum of virus-specific cytokine responses is limited. Here, we investigated peripheral blood mononuclear cells (PBMCs) from children with differential carrier statuses for cytomegalovirus (CMV), Epstein-Barr virus (EBV), and varicella zoster virus (VZV) (n = 32, age 1 to 17 years). We examined memory T cell subsets as well as IFN-γ-, IL-10-, IL-17A-, and IL-22-producing T cells after polyclonal activation or stimulation with viral peptides using flow cytometry and a 4-parameter FluoroSpot assay. Age and herpesvirus carriage influenced the size of the memory T cell subsets. A positive association between age and the number of IFN-γ-, IL-17A- and IL-22-producing T cells was found following polyclonal activation. For CMV, age was positively associated with IL-17A spot-forming cells (SFC), while for VZV, age was negatively associated with IL-22 and positively associated with IFN-γ SFC. Upon activation with CMV, VZV, and EBV peptides, IFN-γ SFCs dominated. Notably, VZV responses were characterized by a higher IL-10 SFC population compared to both CMV and EBV. Our findings suggest that cytokine responses vary across herpesvirus-type-specific memory T cells and may more adequately reflect their composition. An observed deviation between polyclonal and herpesvirus-specific T cell cytokine responses in children needs to be considered when interpreting the associations between herpesvirus carrier status and bulk T cell reactivity. In summary, these findings may have implications for the treatment of immunocompromised patients. IMPORTANCE Infection with herpesviruses accounts for 35 to 40 billion human cases worldwide. Despite this, little is known about how herpesviruses shape the immune system in the asymptomatic carrier. Particularly in children, primary infection is connected to no or mild symptoms ahead of latency for life. Most research on cellular responses against herpesviruses focuses on inflammatory cytokines associated with antiproliferative and antitumor mechanisms and not the spectrum of cytokine responses in healthy humans. This study investigated four divergent cytokine-producing T cell responses to herpesviruses, reflecting different immunological functions. Three common childhood herpesviruses were selected: Epstein-Barr virus, cytomegalovirus, and varicella-zoster virus. Curiously, not all viruses induced the same pattern of cytokines. Varicella-zoster responses were characterized by IL-10, which is considered regulatory. Besides broadening understanding of responses to herpesviruses, our results raise the possibility that reactivation of varicella-zoster may be counterproductive in cancer treatment through the action of IL-10-producing T-cells.

The facial nerve palsy and cortisone evaluation (FACE) study in children: protocol for a randomized, placebo-controlled, multicenter trial, in a Borrelia burgdorferi endemic area.

BACKGROUND: Children with acute peripheral facial nerve palsy cannot yet be recommended corticosteroid treatment based on evidence. Adults with idiopathic facial nerve palsy are treated with corticosteroids, according to guidelines resulting from a meta-analysis comprising two major randomized placebo-controlled trials. Corresponding trials in children are lacking. Furthermore, acute facial nerve palsy in childhood is frequently associated with Lyme neuroborreliosis, caused by the spirochete Borrelia burgdorferi. The efficacy and safety of corticosteroid treatment of acute facial nerve palsy associated with Lyme neuroborreliosis, has not yet been determined in prospective trials in children, nor in adults. METHOD: This randomized double-blind, placebo-controlled study will include a total of 500 Swedish children aged 1-17 years, presenting with acute facial nerve palsy of either idiopathic etiology or associated with Lyme neuroborreliosis. Inclusion is ongoing at 12 pediatric departments, all situated in Borrelia burgdorferi endemic areas. Participants are randomized into active treatment with prednisolone 1 mg/kg/day (maximum 50 mg/day) or placebo for oral intake once daily during 10 days without taper. Cases associated with Lyme neuroborreliosis are treated with antibiotics in addition to the study treatment. The House-Brackmann grading scale and the Sunnybrook facial grading system are used for physician-assessed evaluation of facial impairment at baseline, and at the 1- and 12-month follow-ups. Primary outcome is complete recovery, measured by House-Brackmann grading scale, at the 12-month follow-up. Child/parent-assessed questionnaires are used for evaluation of disease-specific quality of life and facial disability and its correlation to physician-assessed facial impairment will be evaluated. Furthermore, the study will evaluate factors of importance for predicting recovery, as well as the safety profile for short-term prednisolone treatment in children with acute facial nerve palsy. DISCUSSION: This article presents the rationale, design and content of a protocol for a study that will determine the efficacy of corticosteroid treatment in children with acute facial nerve palsy of idiopathic etiology, or associated with Lyme neuroborreliosis. Future results will attribute to evidence-based treatment guidelines applicable also in Borrelia burgdorferi endemic areas. TRIAL REGISTRATION: The study protocol was approved by the Swedish Medical Product Agency (EudraCT nr 2017-004187-35) and published at ClinicalTrials.gov ( NCT03781700 , initial release 12/14/2018).

Peripheral facial nerve palsy in children in a Borrelia high-endemic area, a retrospective follow-up study.

AIM: To identify the incidence, aetiology and prognosis of acute peripheral facial nerve palsy (FNP) in children in the Borrelia high-endemic region of Stockholm. METHODS: The present study identified children from 0 to 18 years of age who visited a paediatric emergency department for acute peripheral FNP during a 1-year period from 2014 to 2015. Data were collected retrospectively. The Sunnybrook and House-Brackmann facial grading systems were used to measure clinical outcome. RESULTS: A total of 77 children were identified with FNP, an estimated incidence of 30 per 100 000 children/year. Forty-five children (58%) were diagnosed with neuroborreliosis, 28 (36%) with idiopathic FNP and four (6%) with other rarer causes. Neuroborreliosis was common from June to November and mainly seen in children below 10 years of age. Six patients (8%) had remaining symptoms at least 3 months after onset; three had idiopathic facial palsy (IFP) and were all older than 10 years, one had neuroborreliosis and two had other causes. CONCLUSION: Neuroborreliosis and IFP were the major causes of FNP during the study period. Neuroborreliosis-associated facial palsy had a seasonal variation and dominated in younger ages.

Kidney volume, kidney function, and ambulatory blood pressure in children born extremely preterm with and without nephrocalcinosis.

BACKGROUND: Reduced kidney volume (KV) following prematurity is a proxy for reduced nephron number and is associated with the development of hypertension and end-stage renal disease in adults. We investigated whether extreme prematurity affects KV, function, and blood pressure in school-aged children and if nephrocalcinosis (NC) developed during the neonatal period had additional effects. METHODS: We investigated 60 children at a mean age of 7.7 years: 20 born extremely preterm (EPT < 28 weeks gestational age with NC (NC+)), 20 born EPT without NC (NC-), and 19 born as full-term infants (control). We measured KV by ultrasound, collected blood and urine samples to evaluate renal function, and measured office and 24-h ambulatory blood pressure (ABPM). RESULTS: Children born EPT had significantly smaller kidneys (EPT (NC+ NC-) vs control (estimated difference, 11.8 (CI - 21.51 to - 2.09 ml), p = 0.018) and lower but normal cystatin C-based glomerular filtration rate compared with control (estimated difference, - 10.11 (CI - 0.69 to - 19.5), p = 0.035). KV and function were not different between NC+ and NC- groups. Change in KV in relation to BSA (KV/BSA) from the neonatal period to school age showed significantly more EPT children with neonatal NC having a negative evolution of KV (p = 0.01). Blood pressure was normal and not different between the 3 groups. Fifty percent of EPT had a less than 10% day-to-night decline in ABPM. CONCLUSIONS: Kidney growth and volume is affected by EPT birth with NC being a potential aggravating factor. Circadian blood pressure regulation seems abnormal in EPT-born children.

Altered proportions of circulating CXCR5+ helper T cells do not dampen influenza vaccine responses in children with rheumatic disease.

Biological therapy options for the treatment of rheumatic disease target molecules that can affect the cross-talk between innate and adaptive immune responses upon vaccination. Influenza vaccination in children with rheumatic disease has been recommended, but there are only sparse data on the quality of vaccine responses from pediatric patients treated with biological therapy. We conducted an influenza vaccine study over 3 consecutive seasons where the antibody response to TIV was evaluated in children with PRD (n = 78), including both non-treated (n = 17) and treated (with methotrexate, TNF-inhibitors with or without methotrexate, or IL-inhibitors, n = 61) children as well as healthy age-matched controls (n = 24). Peripheral B cells, T and NK cell populations, as well as CXCR5+ (follicular) helper T cells (TFH) and chemokines involved in antibody responses were assessed prior to immunization in the same cohort. Data on disease duration, therapy and data on previous influenza vaccinations were retrieved. The proportion of circulating TFH cells were significantly lower in non-treated children with PRD compared to treated patients and healthy controls. The significantly lower proportion of TFH cells was mirrored by a marked significant increase in CXCL13 serum level, the ligand for CXCR5, with higher levels in non-treated children with PRD compared to treated patients and healthy controls. However, the proportion of TFH cells or CXCL13 level at the time of vaccination was not a predictor of the antibody response to TIV in this cohort of children. Children with PRD had an overall similar response to TIV as healthy children. Although not significant, children treated with TNF-inhibitors differed as a few children remained seronegative towards H3N2- and influenza B viruses after immunization. Our data show that children with PRD respond to TIV as healthy children. Furthermore, plasma CXCL13 levels did not correlate to the proportion of TFH cells in blood prior to immunisation, or to antibody responses following immunization.

[The child immunisation programme--effective but insufficient. Experiences from the Astrid Lindgren pediatric hospital, 2008­2013]. / Barnvaccinationsprogrammet ­ effektivt men otillräckligt - Erfarenheter från Astrid Lindgrens barnsjukhus, 2008­2013.

The national Swedish immunisation programme includes vaccine against diphtheria, tetanus, polio, pertussis, Haemophilus influenzae type B, Pneumococcus as well as measles, mumps and rubella. Data were collected on hospital admissions for children 0- 17 years of age of vaccine-preventable diseases during 2008-2013 at Astrid Lindgren Children's Hospital, Stockholm. Patients were identified by discharge diagnosis codes as well as from the clinical microbiology laboratory. There were rare cases of measles and mumps, and only a few cases of invasive bacterial diseases. The dominating pathogens were influenza A and B, rotavirus and varicella zoster virus. Our national programme is effective, but vaccine preventable infections still cause significant morbidity in young children. An extended vaccine programme might significantly reduce the need of hospitalisations.

Diverse effects on vaccine-specific serum IgG titres and memory B cells upon methotrexate and anti-TNF-α therapy in children with rheumatic diseases: A cross-sectional study.

OBJECTIVES: We aimed at a comprehensive evaluation of how anti-TNF-α therapy and methotrexate treatment interferes with B cell memory in children with Paediatric Rheumatic Disease (PRD), by evaluating existing B cell phenotypes, and preserved vaccine-specific memory B cells and IgG titres generated prior to disease and treatment. METHODS: In a cross-sectional study on children with PRD on various treatments, we measured titre levels and avidity strength of serum IgG specific against measles, rubella and tetanus. We also quantified transitional B cells and resting, atypical, and activated memory B cells with flow cytometry, and enumerated antigen-specific memory B cells with ELISpot. RESULTS: For children who had received a tetanus booster, patients treated with any disease-modifying anti-rheumatic drug (DMARD) had lower tetanus serum IgG compared to healthy controls and NSAID-treated patients. Patients without a measles booster had lower levels of measles-specific memory B cells, but all vaccine-specific memory B cells were preserved in patients with booster. We furthermore found that the mature B cell compartment was phenotypically similar between patients and healthy controls. CONCLUSIONS: We concluded that the general and vaccine-specific memory B cell compartment is well preserved in children with PRD and DMARD treatment, but that they might have lower serum tetanus IgG. We emphasize the importance for these children to follow the full vaccination schedule, and suggest to re-measure tetanus titres as they reach adulthood.

HMGB1 levels are increased in patients with juvenile idiopathic arthritis, correlate with early onset of disease, and are independent of disease duration.

OBJECTIVE: High mobility group box chromosomal protein 1 (HMGB1) has been implicated as a mediator of inflammation in rheumatoid arthritis (RA), while its role in juvenile idiopathic arthritis (JIA) has not been described. To evaluate the role of HMGB1 in the inflammatory process in JIA and its potential as a therapeutic target, we investigated whether extracellular HMGB1 is detectable in JIA and if so, to correlate the levels with established inflammatory markers and clinical measures. METHODS: Matching samples of blood and synovial fluid (SF) were collected from 23 patients with JIA. Levels of HMGB1, soluble receptor for advanced glycation endproducts, S100A12, myeloid-related protein 8/14, and other inflammatory mediators were analyzed. RESULTS: Significantly increased HMGB1 levels were recorded in SF compared to blood samples from patients with JIA. The amount of HMGB1 was highest in patients with early disease onset irrespective of disease duration. In contrast, the proinflammatory S100 protein and interleukin 8 were highest in patients in early phases of disease. Matrix metalloproteinase-3, a marker of cartilage destruction, was higher in patients with late disease onset, indicating similarities with RA in that patient subgroup. CONCLUSION: Levels of extracellular HMGB1 are increased in the inflamed joints of patients with JIA. This warrants further studies of HMGB1 as a mediator of JIA pathogenesis as well as a biomarker for inflammatory activity and as a target for therapy. The variation in levels of HMGB1 and S100 proteins in relation to disease onset indicates a difference in inflammatory phenotype during disease progression.

Developmental aspects of Escherichia coli-induced innate responses in rat renal epithelial cells.

Renal scarring after pyelonephritis is common in infancy. In this experimental study performed on tissue from 10-d-old infant and 40-d-old pubertal rats, several aspects of the renal innate immune response to a pyelonephritogenic strain of alpha-hemolysin-expressing Escherichia coli were compared. The kidney typically responds to urinary tract infection with release of proinflammatory cytokines, e.g. IL-6. Basal production of IL-6 from 10-d-old renal cortical tissue was approximately 20% of that from 40-d-old tissue. Six-hour incubation in the presence of supernatant from the E. coli culture caused an approximately 15-fold increase of IL-6 release in 10-d-old tissue and a 5-fold increase in 40-d-old tissue. The absolute level of IL-6 release in stimulated tissue was, however, significantly lower at 10 d than at 40 d. Lipopolysaccharide, the most immunogenic component of E. coli, signals via Toll-like receptor 4. Reverse transcriptase PCR performed on outer renal cortex indicated that expression of Toll-like receptor 4 mRNA was similar in both ages. Microdissection studies revealed that Toll-like receptor 4 mRNA was expressed in proximal tubules but not in glomeruli. The exotoxin alpha-hemolysin, expressed by a majority of uropathogenic E. coli isolates, stimulates IL-6 release via an alternative pathway that signals via intracellular calcium oscillations. We conclude that the higher susceptibility to pyelonephritic scarring is unlikely related to immaturity of innate immune system, as measured by cellular release of IL-6. Instead, the underlying mechanisms for pyelonephritic scarring are most likely multifactorial and may be mainly attributed to anatomic immaturity of the urinary tract.

Dual effects of Escherichia coli alpha-hemolysin on rat renal proximal tubule cells.

BACKGROUND: alpha-Hemolysin (HlyA) producing Escherichia coli is a common cause of pyelonephritis and subsequent renal scarring. Recent studies have suggested that toxin secreted from HlyA E. coli may not only have a lytic effect, but also may activate a calcium signaling pathway in renal tubule cells. A dose dependent study was performed on the interaction between HlyA E. coli secretions and rat renal proximal tubule (PT) cells with regards to calcium signaling and cell morphology. The site of interaction between HlyA secretion and PT cells was examined by using an antagonist to a common binding motif in bacterial proteins. METHODS: Supernatant from an overnight culture of HlyA was freshly prepared for each experiment. Renal PT cells from infant rats were cultured for three days and exposed for 30 minutes to four hours to supernatant or purified HlyA. Effects on cell morphology were studied semiquantitatively with light microscopy. Intracellular calcium was measured ratiometrically in the presence or absence of drugs. RESULTS: Renal PT cells incubated with low doses of HlyA supernatant responded within five minutes with calcium oscillations. Morphology appeared unchanged after four hours of incubation. In contrast, high doses of HlyA caused a sustained increase in intracellular calcium and majority of cells were lysed within four hours. Calcium oscillations caused by lower doses of HlyA supernatant were highly regular and slow in the 10 to 12 minute range. Oscillations were abolished by 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX), indicating that HlyA is interacting with a QPB/LAOBP-motif. CONCLUSION: HlyA secreted from uropathogenic E. coli exerts a dual action on renal PT cells. Sublytical concentrations induce a response that may serve as a host defense, while high concentrations cause irreversible cell damage. The data emphasize the importance of high diuresis in urinary tract infection.

Toxin-induced calcium oscillations: a novel strategy to affect gene regulation in target cells.

Although the mucosal linings continuously are exposed to microbes, the microbes rarely induce disease. This is because mucosal surfaces are protected by a first line of host defence termed the innate immunity system. The innate immune response is an outcome of the complex interplay between microbes and host target cells, and leads to the activation of inflammatory processes. Although inflammation is essential for clearing out infectious agents, it can also be harmful to the host and is therefore subjected to control at multiple levels. We recently discovered that alpha-haemolysin, a toxin secreted by uropathogenic E. coli induces constant, low-frequency Ca2+ oscillations in renal epithelial cells (Uhlén et al., Nature 405, 694-696 (2000)). Ca2+ oscillation at a specific periodicity of 12 min was found to affect gene expression in target epithelial cells, as the proinflammatory cytokine IL-6 and chemokine IL-8 were specifically induced by alpha-haemolysin-induced Ca2+ oscillations. This demonstrates a novel feature of bacterial toxin effects on host target cells: as inducers of second messenger responses which fine-tune gene expression in target epithelial cells into pathways leading to e. g. a pro-inflammatory response.