LFRET, a novel rapid assay for anti-tissue transglutaminase antibody detection

The diagnosis of celiac disease (CD) is currently based on serology and intestinal biopsy, with detection of anti-tissue transglutaminase (tTG) IgA antibodies recommended as the first-line test. Emphasizing the increasing importance of serological testing, new guidelines and evidence suggest basing the diagnosis solely on serology without confirmatory biopsy. Enzyme immunoassays (EIAs) are the established approach for anti-tTG antibody detection, with the existing point-of-care (POC) tests lacking sensitivity and/or specificity. Improved POC methods could help reduce the underdiagnosis and diagnostic delay of CD. We have previously developed rapid homogenous immunoassays based on time-resolved Förster resonance energy transfer (TR-FRET), and demonstrated their suitability in serodiagnostics with hanta- and Zika virus infections as models. In this study, we set out to establish a protein L -based TR-FRET assay (LFRET) for the detection of anti-tTG antibodies. We studied 74 patients with biopsy-confirmed CD and 70 healthy controls, with 1) the new tTG-LFRET assay, and for reference 2) a well-established EIA and 3) an existing commercial POC test. IgG depletion was employed to differentiate between anti-tTG IgA and IgG positivity. The sensitivity and specificity of the first-generation tTG-LFRET POC assay in detection of CD were 87.8% and 94.3%, respectively, in line with those of the reference POC test. The sensitivity and specificity of EIA were 95.9% and 91.9%, respectively. This study demonstrates the applicability of LFRET to serological diagnosis of autoimmune diseases in general and of CD in particular.Peer reviewe

Pre-and postdiagnosis growth failure, adult short stature, and untreated growth hormone deficiency in radiotherapy-treated long-term survivors of childhood brain tumor

Peer reviewe

Novel NPHS2 variant in patients with familial steroid-resistant nephrotic syndrome with early onset, slow progression and dominant inheritance pattern

Background Steroid-resistant nephrotic syndrome (SRNS) is a common cause of end-stage renal disease in children but also occurs as an adult-onset condition. In a subset of SRNS patients, pathogenic variants are found in genes coding for podocyte foot process proteins. The aim of this study was to define the role of pathogenic variants in Finnish patients with familial and sporadic SRNS. Methods We analyzed SRNS-related genes NPHS1, NPHS2, NEPH1, ACTN4, TRPC6, INF2, WT1, CD2AP, LAMB2, and PLCE1 for disease-causing variants using direct sequencing of exons and intron/exon boundaries in all members of a family with dominant SRNS with early onset and slow progression to end-stage renal disease. We carried out a whole genome sequencing in two affected and two healthy family members. The function of found podocin variant was studied using co-immunoprecipitation and immunohistochemistry. Podocyte gene sequences were analyzed in a cohort of Finnish non-familial SRNS patients. Results A heterozygous de novo deletion, c. 988_989delCT in NPHS2, was found in all affected family members and in none of their healthy relatives, non-familial patients or controls. No other SRNS-related gene variant, coding or non-coding co-segregated with the disease phenotype in the family. While the truncated podocin remained able to bind nephrin, the expression of nephrin was fragmented and podocin expression reduced. The gene analysis of the non-familial SRNS patients revealed few variants. Conclusion The role of podocin variants in nephrotic syndrome may be more varied than previously thought.Peer reviewe

FOXP3(+) T cells are present in kidney biopsy samples in children with tubulointerstitial nephritis and uveitis syndrome

Tubulointerstitial nephritis (TIN) is an inflammatory disease of unknown pathogenesis. To evaluate a possible role of regulatory T cells (Tregs) in the pathophysiology of TIN with (TINU) and without uveitis, we investigated the presence and quantity of FOXP3(+) T regulatory lymphocytes in diagnostic kidney biopsies from pediatric patients. A total of 33 patients (14 TIN and 19 TINU) were enrolled. The quantity of CD4(+), FOXP3(+) and double-positive T cells in formalin-fixed kidney biopsies was determined using double label immunohistochemistry with anti-human CD4 and FOXP3 antibodies. FOXP3 staining was successful in all 33 patients. In patients with chronic uveitis, the density of FOXP3(+) cells was significantly lower (p = 0.046) than in TIN patients without uveitis or with uveitis lasting <3 months. CD4(+) staining was successful in 23 patients. The density of all lymphocytes (CD4(+), CD4(+)FOXP3(+) and FOXP3(+) cells) was significantly lower (p = 0.023) in patients with chronic uveitis than in other patients. FOXP3(+) T cells are present in kidney biopsy samples from TIN and TINU patients. In patients with chronic uveitis, the density of FOXP3(+) T cells is significantly lower than in other patients, suggesting a different pathomechanism for these clinical conditions.Peer reviewe

Long-term outcome of biopsy-proven idiopathic tubulointersitial nephritis with or without uveitis in children-a nationwide follow-up study

Background Only a few studies reporting the long-term outcome of children with idiopathic tubulointerstitial nephritis (TIN) and uveitis syndrome (TINU) are available. We studied the long-term kidney and ocular outcome in a nationwide cohort of children with TIN or TINU.Methods All patients followed up for a minimum of 1 year by a paediatrician and an ophthalmologist were enrolled. The data on plasma creatinine (P-Cr), estimated glomerular filtration rate (eGFR), proteinuria, hypertension and uveitis were collected retrospectively.Results Fifty-two patients were studied. Median age at time of diagnosis was 13.1 (1.8-16.9) years and median follow-up time was 5.7 (1.1-21.2) years. Forty-five (87%) patients were initially treated with glucocorticoids. The median of the maximum P-Cr was 162 mu mol/l (47-1,016) and that of eGFR 47 ml/min/1.73m(2) (8-124). Uveitis was diagnosed in 33 patients (63%) and 21 (40%) patients developed chronic uveitis. P-Cr normalised in a median of 2 months. Eleven (21%) patients had nephritis recurrence during or after discontinuation of glucocorticoids. At the latest follow-up, 13 (25%) patients had eGFR Conclusions Our study questions the previously reported good long-term kidney and ocular outcome of patients with TIN/TINU. Decreased kidney function and/or ocular co-morbidities may persist for several years; thus, both kidney and ocular follow-up for at least 1 year is warranted.</div

Development of gliadin-specific immune responses in children with HLA-associated genetic risk for celiac disease

Objective. The development of gliadin-specific antibody and T-cell responses were longitudinally monitored in young children with genetic risk for celiac disease (CD). Material and methods. 291 newborn children positive for HLA-DQB1*02 and -DQA1*05 alleles were followed until 3-4 years of age by screening for tissue transglutaminase autoantibodies (tTGA) by using a commercial ELISA-based kit and antibodies to deamidated gliadin peptide (anti-DGP) by an immunofluorometric assay. Eighty-five of the children were also followed for peripheral blood gliadin-specific CD4(+) T-cell responses by using a carboxyfluorescein diacetate succinimidyl ester-based in vitro proliferation assay. Results. The cumulative incidence of tTGA seropositivity during the follow-up was 6.5%. CD was diagnosed in nine of the tTGA-positive children (3.1%) by duodenal biopsy at a median 3.5 years of age. All of the children with confirmed CD were both IgA and IgG anti-DGP positive at the time of tTGA seroconversion and in over half of the cases IgG anti-DGP positivity even preceded tTGA seroconversion. Peripheral blood T-cell responses to deamidated and native gliadin were detected in 40.5% and 22.2% of the children at the age of 9 months and these frequencies decreased during the follow-up to the levels of 22.2% and 8.9%, respectively. Conclusions. Anti-DGP antibodies may precede tTGA seroconversion and thus frequent monitoring of both tTGA and anti-DGP antibodies may allow earlier detection of CD in genetically susceptible children. Peripheral blood gliadin-specific T-cell responses are relatively common in HLA-DQ2-positive children and are not directly associated with the development of CD.Peer reviewe

Impaired cardiorespiratory and neuromuscular fitness in children and adolescents with juvenile idiopathic arthritis : a cross-sectional case–control study in the era of biologic drug therapies

Background: In recent years, biologic drug therapies have altered the course of juvenile idiopathic arthritis (JIA) possibly also improving the patients’ physical fitness. However, studies measuring both cardiorespiratory and muscular fitness in children with JIA are sparse and have failed to show consistent results. Our aim was to assess both cardiorespiratory and neuromuscular fitness and contributing factors in children and adolescents with JIA in the era of biologic drug therapies. Methods: This cross-sectional study consisted of 73 JIA patients (25 boys, 48 girls) aged 6.8- 17.5 years and 73 healthy age- and sex-matched controls, investigated in 2017–2019. Cardiorespiratory fitness was assessed by maximal ergospirometry and neuromuscular fitness by speed, agility, balance, and muscle strength tests. Results: Means (± SD) of maximal workload (Wmax/kg) and peak oxygen uptake (VO2peak/kg,) were lower in JIA patients than in controls (Wmax/kg: 2.80 ± 0.54 vs. 3.14 ± 0.50 Watts, p < 0.01; VO2peak/kg: 38.7 ± 7.53 vs. 45.8 ± 6.59 ml/min/kg, p < 0.01). Shuttle-run, sit-up and standing long jump test results were lower in JIA patients than in controls (p < 0.01). Mean (± SD) daily activity was lower (89.0 ± 44.7 vs. 112.7 ± 62.1 min/day, p < 0.05), and sedentary time was higher (427 ± 213 vs. 343 ± 211 min/day, p < 0.05) in JIA patients compared to controls. Physical activity and cardiorespiratory or neuromuscular fitness were not associated with disease activity. Conclusions: JIA patients were physically less active and had lower cardiorespiratory and neuromuscular fitness than their same aged controls with no JIA. Therefore, JIA patients should be encouraged to engage in physical activities as a part of their multidisciplinary treatment protocols to prevent adverse health risks of low physical activity and fitness.publishedVersionPeer reviewe

Pre- And postdiagnosis growth failure, adult short stature, and untreated growth hormone deficiency in radiotherapy-treated long-term survivors of childhood brain tumor

Purpose Growth failure is common in radiotherapy-treated long-term survivors of pediatric brain tumors, but studies on longitudinal growth in this patient group are lacking. Here, the aim was to assess the changes in growth patterns before and after brain tumor diagnosis, the adult height, and the risk factors for compromised growth. The incidence and treatment practices of growth hormone deficiency were analyzed. Methods A cohort of 73 survivors of childhood brain tumor (median age 27.2 years, range 16.2 to 43.8 years) was studied after a median follow-up period of 20.4 years from diagnosis (IQR 14.9 to 22.9 years). Patients were treated in five university hospitals in Finland between 1970 and 2008. Growth curves, final height, and patient- and disease-related risk factors for compromised growth during different growth periods were analyzed. Laboratory analyses for IGF-1 and IGFBP-3 were performed at the follow-up. Results Growth failure was evident at diagnosis, with a mean height decline of -0.6 SDS (standard deviation score) from birth (95% CI -1.15 to -0.05). Mean height SDS decline after the diagnosis was -1.09 SDS (95%CI -1.51 to -0.66). At follow-up, 37% of the study subjects (27/73) had true short stature (height < -2 SDS). The mean height deficit corrected for target height was -1.9 SDS (95% CI -1.45 to -2.40). Growth failure was associated with the age at diagnosis, corticosteroid dose, radiotherapy modality and mean dose of irradiation in the thalamic area. Low IGF-1 level (below -2.0 SDS) was found in 32% (23/72), and untreated growth hormone deficiency in 40% (29/72) of the subjects. Conclusion Longitudinal growth impairment was common in radiotherapy-treated survivors of childhood brain tumor, resulting in compromised adult height. Loss of growth potential was evident already at diagnosis and further accelerated by the treatments. At young adulthood, unrecognized growth hormone deficiency was common.publishedVersionPeer reviewe

Health-related quality of life in long-term survivors of childhood brain tumors : a population-based cohort study

Purpose Survivors of childhood brain tumors (BT) are at high risk for long-term physical and psychological sequelae. Still, knowledge about health-related quality of life (HRQL) and associated factors in this population is sparse. This study investigated HRQL and its predictors in long-term survivors of childhood BT. Methods Survivors of childhood BT (mean age = 28.1 years, SD = 6.8, n = 60) underwent clinical examination and neurocognitive examination, and completed self-rating questionnaires assessing HRQL (RAND-36) and depressive symptoms (Beck Depression Inventory-II). Socio-demographic information was gathered via a questionnaire. Tumor- and treatment-related information was collected from medical records. Control group data were collected from age-matched controls (n = 146) without a history of cancer, randomly selected from the local population registry. Multiple linear regression models were used to investigate predictors of HRQL; separate models were fitted for each domain of the RAND-36. Results Male survivors (mean age = 27.0, SD = 6.0, n = 39) reported significantly lower HRQL than male controls in the domains of physical functioning, general health, vitality, social functioning, and role limitations-emotional. Female survivors (mean age = 30.2 years, SD = 7.6, n = 21) reported comparable levels as female controls in all domains except physical functioning. A higher burden of late effects, not working/studying, being diagnosed with BT during adolescence, and reporting current depressive symptoms were significant predictors of lower HRQL. Conclusion Our results highlight that male survivors of childhood BT are at particular risk of impaired HRQL. Also, results point to the close relation between symptoms of depression and impaired HRQL in survivors of childhood BT which should be acknowledged by long-term follow-up care.Peer reviewe

LFRET, a novel rapid assay for anti-tissue transglutaminase antibody detection

The diagnosis of celiac disease (CD) is currently based on serology and intestinal biopsy, with detection of anti-tissue transglutaminase (tTG) IgA antibodies recommended as the first-line test. Emphasizing the increasing importance of serological testing, new guidelines and evidence suggest basing the diagnosis solely on serology without confirmatory biopsy. Enzyme immunoassays (EIAs) are the established approach for anti-tTG antibody detection, with the existing point-of-care (POC) tests lacking sensitivity and/or specificity. Improved POC methods could help reduce the underdiagnosis and diagnostic delay of CD. We have previously developed rapid homogenous immunoassays based on time-resolved Förster resonance energy transfer (TR-FRET), and demonstrated their suitability in serodiagnostics with hanta- and Zika virus infections as models. In this study, we set out to establish a protein L -based TR-FRET assay (LFRET) for the detection of anti-tTG antibodies. We studied 74 patients with biopsy-confirmed CD and 70 healthy controls, with 1) the new tTG-LFRET assay, and for reference 2) a well-established EIA and 3) an existing commercial POC test. IgG depletion was employed to differentiate between anti-tTG IgA and IgG positivity. The sensitivity and specificity of the first-generation tTG-LFRET POC assay in detection of CD were 87.8% and 94.3%, respectively, in line with those of the reference POC test. The sensitivity and specificity of EIA were 95.9% and 91.9%, respectively. This study demonstrates the applicability of LFRET to serological diagnosis of autoimmune diseases in general and of CD in particular