Jan Vavrinec

Anti-cyclic citrullinated peptide antibodies in patients with juvenile idiopathic arthritis.

We analysed the presence of anti-cyclic citrullinated peptide (anti-CCP) and anti-keratin (AKA) antibodies of the IgG class in sera of patients with defined juvenile idiopathic arthritis (JIA) of various subgroups with more than one year duration of the disease. Enzyme-linked immunosorbent assay (Immunoscan RA, Eurodiagnostica, The Netherlands) and an indirect immunofluorescence (IIF) test on rat oesophagus substrate (ImmuGloTM, Immco Diagnostics, Buffalo, USA) were used for the detection and quantification of anti-CCP and AKA antibodies in 140 patients with JIA (64 male and 76 female) aged 2-47 years (median 16.5 years). Overall, anti-CCP were found in 7/140 (5.0%) patients including 3/52 RF negative polyarthritis, 2/18 RF positive polyarthritis, 1/15 enthesitis related arthritis and 1/5 unclassifiable arthritis. AKA were detected in 40/140 patients (28.6%, p =0.04) including 2/11 systemic arthritis, 2/32 oligoarthritis, 18/52 patients with RF negative polyarthritis (34.6%, p =0.01), 14/18 RF positive polyarthritis (77.8%, p =0.000002), 2/15 enthesitis related arthritis and 2/3 psoriatic arthritis. While simultaneous negativity for AKA and anti-CCP occurred in most (97/140; 69.3%) studied cases, simultaneous antibody positivity was found only in few (4/140; 2.9%) studied samples. We conclude that while AKA measured using IIF on rat esophagus can be detected approximately in one third of patients with definite JIA with more than 1 year duration of the disease, only rare occurrence of anti-CCP was observed. We conclude that AKA seem to be partly useful to confirm JIA diagnosis, however, useless to follow-up severity or activity in JIA patients. Anti-CCP do not have any additional value in JIA cohort in comparison to RA where their diagnostic and prognostic importance was reported.

Type 1 diabetes mellitus in Czech children diagnosed in 1990–1997: a significant increase in incidence and male predominance in the age group 0–4 years

Aims To overview total, age‐and sex‐specific incidence rates of type 1 diabetes mellitus and their trends in Czech children 0–14 years of age in the period of 1990–1997.

HLA class II genetic association of type 1 diabetes mellitus in Czech children

Abstract:  To examine human leukocyte antigen (HLA) class II association of type 1 diabetes mellitus (DM) in Czech children, we performed a case–control study of 261 patients diagnosed before the age of 15 and 289 non‐diabetic control children. Complete HLA‐DQA1, DQB1 genotyping and DRB1*04 subtyping were carried out by polymerase chain reactions with sequence‐specific primers. The effect of the DRB1*04 subtypes was studied in DRB1*04 alleles carried on DQB1*0302‐DQA1*03 haplotypes. The risk was statistically evaluated by testing 2 × 2 tables, considering corrected p‐values < 0.05 significant. The DQB1*0302 (odds ratio, OR = 9.0), DQB1*0201 (OR = 3.4) and DQA1*03 (OR = 7.5) alleles were significantly associated with diabetes risk, while the DQB1*0602 (OR = 0.02), DQB1*0301 (OR = 0.08), DQB1*0503 (OR = 0.13), DQB1*0603 (OR = 0.20), DQA1*01 (OR = 0.28) and DQA1*02 (OR = 0.26) alleles were significantly protective. Of the DQA1‐DQB1 genotypes, we point out the extremely high risk of OR = 116 conferred by HLA‐DQA1*05‐DQB1*0201/DQA1*03‐DQB1*0302. Among DRB1*04 subtypes, DRB1*0403 was significantly protective (OR = 0.05, CI 95% 0.01–0.45). Since none of the remaining DRB1*04 subtypes was associated with type 1 DM, our study may present another piece of evidence that the DRB1*0401 and DRB1*0404 alleles do not modify type 1 diabetes risk generally in European populations.

Use of a skin explant model for predicting GVHD in HLA-matched bone marrow transplants - effect of GVHD prophylaxis.

Over the last decade we have successfully evaluated the use of a human skin explant assay for predicting acute GVHD in HLA-matched sibling transplants. In the present study, we modified GVHD prophylaxis on an individual patient basis depending on the GVHD outcome predicted by the skin explant model. We have summarised our previous data describing how the skin explant assay results correctly predict GVHD occurrence and severity in 45/56 patients (80%); P< 0.0001, χ2 19.97, df = 1. In a further cohort of 19 patients, all were predicted to develop grade II or above GVHD. These patients were given increased GVHD prophylaxis with the addition of methotrexate and a significant reduction in the expected incidence of GVHD was observed (P = 0.02; χ2 7.7, df = 1; Fisher exact test P = 0.04). The results from these studies suggest that modifying GVHD prophylaxis, based on skin explant assay results, may reduce the expected incidence and severity of GVHD. We suggest that the technique might be used for selective GVHD prophylaxis in T cell non-depleted HLA matched sibling transplants.

High prevalence of coeliac disease in siblings of children with type 1 diabetes.

Coeliac disease has been shown to occur more frequently among first-degree relatives of diabetic patients than in the general population. Our objective was to assess the prevalence of endomysium antibodies (EMA) in non-diabetic siblings of Czech diabetic children and to evaluate the effects of HLA-DQ polymorphisms in determining the genetic susceptibility to coeliac disease (CD) in these subjects. We investigated 240 siblings of diabetic children from 213 families (125 males and 115 females, aged 12.6±4.9 years, mean ± SD). All subjects were tested for the total IgA level to exclude IgA deficiency, and for endomysium IgA to disclose CD. In five IgA-deficient subjects, anti-gliadin IgG was used instead. Small bowel biopsy was offered to subjects with confirmed positive EMA. The HLA-DQA1, -DQB1 genotypes were determined using PCR-SSP. Positive EMA were found in 9/240 (3.8%) subjects (three males, six females). The biopsy confirmed CD in six children, two had a normal mucosal finding and one refused the biopsy. The HLA-DQ2 polymorphism was more frequent among siblings with EMA (seven of nine) than in siblings without EMA (33%), corrected P =0.031. Conclusion:The 3.8% frequency of coeliac disease found in siblings of diabetic children is close to the 4.3% found previously in Czech children with type 1 diabetes mellitus and is substantially higher than the rate in the healthy children population.

Anti-GAD65 reactive peripheral blood mononuclear cells in the pathogenesis of cystic fibrosis related diabetes mellitus

Objective: A role of autoreactive T cells for type 1 diabetes pathogenesis is considered crucial. In our pilot study we addressed if autoreactive mononuclear cells are present also in peripheral blood of patients with other specific forms of diabetes as cystic fibrosis related diabetes (CFRD). Methods: Cellular immune responses to a known β-cell autoantigen (GAD65 and GAD65 derived peptides) were analysed by ELISPOT (IFN-γ) and by protein microarray analysis in four patients suffering from CFRD, in four cystic fibrosis (CF) patients without diabetes, in eight type 1 diabetes patients (without CF) and in four healthy controls. Results: Response to the autoantigen GAD65 (protein and peptides) was observed in 7/8 patients suffering from CF and in all type 1 diabetes patients. Post-stimulation production of Th1 cytokines (IFN-γ, TNF-β) was observed in 2/4 CFRD, 1/4 CF patients and in 7/8 type 1 diabetes patients. All these patients carry prodiabetogenic HLA-DQ genotype. Th2- and Th3 type of cytokine pattern was observed in 2/4 CF patients. Production of IL-8 was observed in the third CFRD as well as in the third CF patient and in 1/8 type 1 diabetes patient and borderline production of this chemokine was also observed in 2/4 healthy controls. No reaction was observed in the other 2/4 healthy controls and in the fourth CFRD patient who carried a strongly protective genotype and did not produce autoantibodies. The most potent peptide of GAD65 was amino acids 509–528. Conclusions: We consider our observations as a sign of a reaction directed against the self-antigen GAD65 that are closely connected to type 1 diabetes. In CF patients who do not develop diabetes autoreactive mechanisms are very probably efficiently suppressed by immune self-tolerance mechanisms. CFRD patients are a heterogenic group. To disclose those who may display features of autoimmune diabetes could have an impact for their therapy and prognosis.

An in vitro skin explant assay as a predictive assay for graft-versus-host disease in a cohort of pediatric transplants.

Abstract: Severe acute graft‐versus‐host disease (GvHD) remains a serious complication of allogeneic stem cell transplantation. An in vitro skin explant assay was used to predict the occurence and severity of acute GvHD in a cohort of 30 pediatric patients undergoing human leucocyte antigen (HLA)‐matched sibling transplants (20 patients) and matched or one antigen mismatched unrelated donor transplants (10 patients). In the cohort of HLA‐matched sibling transplants, the result appeared to reflect the degree of GvHD prophylaxis. The skin explant assay correlated with GvHD outcome in 12 of 20 children, but this did not reach statistical significance (chi‐square 0.95, d.f.=1, p=0.32). These results support previous observations. In this present cohort, patients were treated either with cyclosporin A (CsA) monotherapy (n=7) or with CsA plus additional methotrexate (MTX) (n=13). We have previously demonstrated that the skin explant assay was not as predictive in patients receiving CsA plus additional MTX compared to cohorts treated with CsA alone. In the group of patients treated with CsA alone, four of five patients (80%) predicted to develop GvHD developed acute GvHD of grade II or above; of two patients predicted to develop only grade 0–I GvHD, one patient developed no GvHD and the other grade II GvHD. In the CsA plus MTX group, nine patients were predicted to develop GvHD. Five of nine (55%) developed acute GvHD of grade II or above, while three of four with grade 0 or I skin explant assay results developed only grade 0–I GvHD. In a cohort of 10 patients who received unrelated donor transplants, the skin explant assay correlated with GvHD outcome in all 10 patients (Fishers exact test p=0.008). Hence, the skin explant assay is a pretransplant in vitro GvHD predictive test that predicts the occurence and severity of acute GvHD in pediatric unrelated donor transplants and to varying degrees, depending on the GvHD prophylaxis protocols, in HLA‐matched sibling cohorts.

Continuing increase in incidence of childhood-onset type 1 diabetes in the Czech Republic 1990-2001

Using a prospective (1990–2001) population-based registration of new cases of type 1 diabetes among children under the age of 15 years, we demonstrate a continuing increase in incidence, markedly shown in the lowest ageat-onset category. Incidence rates of type 1 diabetes are known to rise significantly in most European populations. While this trend is modest or absent in populations of Scandinavia (Denmark, Norway, Sweden), it is markedly expressed in the countries of Eastern and Central Europe [3, 4, 5]. Here we report the type 1 diabetes incidence rates and their trends for the Czech Republic, 1990–2001. Cases of childhood-onset type 1 diabetes (0–14 years of age) were registered over the period 1990–2001 by the population-based Czech Childhood Diabetes Register according to EURODIAB criteria [3]. Ascertainment rates were calculated by a capture-recapture method using the Association of Parents and Friends of Diabetic Children as the secondary data source. The ascertainment rate was calculated to be 97% for the primary data source, 76% for the secondary, and 99% for both data sources combined. Population data were taken from annual reports of the Czech Statistics Bureau. Apart from calculating crude incidence rates, the rates were also standardised on a virtual population with equal numbers of individuals in the age groups 0–4, 5–9 and 10–14 years. Trends in incidence were estimated using Poisson regression, sex differences were tested using heterogeneity tests, and cyclic trends of seasonality of onset were investigated according to Edwards [2]. A total of 2,644 cases were registered by the Czech Childhood Diabetes Register throughout the period 1990–2001. The average standardised incidence rate was 11.4/100,000/year. The rates did not significantly differ between sexes. The crude and adjusted incidence rates are shown in Table 1. The incidence rates in the age-atonset groups were 7.6/100,000/year in 0–4 completed years at diagnosis, 12.0/100,000/year in 5–9, and 14.6/ 100,000/year in 10–14. Seasonality of onset was most apparent in children with diabetes onset at 10–14 years of age, with high numbers of new cases in September to November, and a trough in July and August (P<10). A significant increase in incidence was seen in all categories of age at onset. The yearly increase in standardised incidence was 6.3% (P<10); the yearly agespecific increases were 10.3% (P<10), 6.0% (P<10), and 4.4% (P=0.009) for the categories 0–4, 5–9, and 10–14, respectively. The absolute average yearly increase in incidence was, however, very similar in all age-at-onset categories (+0.73, +0.70, +0.79/ 100,000/year for the three categories). Consequently, the relative difference in incidence between the three age-atonset bands has weakened over time: this is demonstrated in Fig. 1 where age-specific incidence rates are compared between the first and the second half of the observation period, i.e. between 1990–1995 and 1996– 2001. A significant increase in type 1 diabetes incidence in Czech children has been already demonstrated for the time period 1990–1997 [1]. The present data show that the incidence rate continued to rise for further 4 years, with a similar absolute increase in all three age-at-onset categories. The variations in diabetes incidence between populations are partly attributable to differences in genetic background of the population [8], and are linked to several indicators of national prosperity [7] as proxy measures of yet unknown factors. The explanations for the observed increase in incidence still remain rather speculative. The increase may be, for instance, partly attributed to the rise in the body mass index of Czech children [6], according to the ‘accelerator hypothesis’ which deems insulin resistance resulting from weight gain to be one of the leading causes of rising incidence of Eur J Pediatr (2003) 162: 428–429 DOI 10.1007/s00431-003-1211-1