Beáta Dérfalvi

Improved flow cytometric assessment reveals distinct microvesicle (cell-derived microparticle) signatures in joint diseases.

Introduction Microvesicles (MVs), earlier referred to as microparticles, represent a major type of extracellular vesicles currently considered as novel biomarkers in various clinical settings such as autoimmune disorders. However, the analysis of MVs in body fluids has not been fully standardized yet, and there are numerous pitfalls that hinder the correct assessment of these structures. Methods In this study, we analyzed synovial fluid (SF) samples of patients with osteoarthritis (OA), rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA). To assess factors that may confound MV detection in joint diseases, we used electron microscopy (EM), Nanoparticle Tracking Analysis (NTA) and mass spectrometry (MS). For flow cytometry, a method commonly used for phenotyping and enumeration of MVs, we combined recent advances in the field, and used a novel approach of differential detergent lysis for the exclusion of MV-mimicking non-vesicular signals. Results EM and NTA showed that substantial amounts of particles other than MVs were present in SF samples. Beyond known MV-associated proteins, MS analysis also revealed abundant plasma- and immune complex-related proteins in MV preparations. Applying improved flow cytometric analysis, we demonstrate for the first time that CD3+ and CD8+ T-cell derived SF MVs are highly elevated in patients with RA compared to OA patients (p = 0.027 and p = 0.009, respectively, after Bonferroni corrections). In JIA, we identified reduced numbers of B cell-derived MVs (p = 0.009, after Bonferroni correction). Conclusions Our results suggest that improved flow cytometric assessment of MVs facilitates the detection of previously unrecognized disease-associated vesicular signatures.

Novel and recurrent STAT3 mutations in hyper-IgE syndrome patients from different ethnic groups

We performed clinical, immunological and genetic studies of 12 hyper-IgE syndrome (HIES) patients from 4 Hungarian, 2 Lebanese, one Russian, one Polish, and one Swedish families with autosomal dominant (AD) or sporadic forms of the disease to reveal cross-ethnicity of recurrent and novel mutations in the signal transducer and activator of transcription-3 gene (STAT3). Four patients from 3 Hungarian families, and one Russian, and one Swedish patient carried the heterozygous R382W germline mutation at the DNA-binding site of STAT3. The recurrent V637M mutation affecting the SRC homology 2 (SH2) domain was detected in one Lebanese and one Polish family, and the V463del deletion located in the DNA-binding domain was unveiled in another Lebanese family. A novel H332Y mutation affecting the DNA-binding site of STAT3 in three Hungarian patients from a Gypsy family was also found. The segregation of this mutation with HIES, restriction fragment length polymorphism analysis of STAT3 from patients and controls and the negligible production upon IL-6 stimulation of monocyte chemotactic protein-1 by the patients blood mononuclear cells suggested that the H332Y mutation was disease-causing. These data suggest, that dominant negative mutations of the DNA-binding and SH2 domains of STAT3 cause AD and sporadic cases of HIES in different ethnic groups with R382W as the predominant mutation found in 5 of the 9 families. Functional and genetic data support that the novel H332Y mutation may result in the loss of function of STAT3 and leads to the HIES phenotype.

National registry of patients with juvenile idiopathic inflammatory myopathies in Hungary—Clinical characteristics and disease course of 44 patients with juvenile dermatomyositis

Idiopathic inflammatory myopathies (IIMs) are systemic autoimmune diseases characterized by chronic muscle inflammation resulting in progressive weakness and frequent involvement of internal organs, mainly the pulmonary, gastrointestinal and cardiac systems which considerably contribute to the morbidity and mortality of the IIMs. Aim of this study was to present clinical characteristics, disease course, frequency of relapses and survival in patients with juvenile dermatomyositis (DM). A national registry of patients with juvenile IIMs was elaborated by the authors in Hungary. We have summarized data of the register according to signs and symptoms, disease course, frequency of relapses and survival of patients with juvenile IIM. Analysis was performed using data of 44 patients with juvenile DM diagnosed between 1976 and 2004 according to Bohan and Peters criteria. Survival probability was calculated by Kaplan–Meier method. Data of patients with juvenile DM were compared with data of 66 patients with adult DM. The most frequent cutaneous features were facial erythema and heliotrope rash. Extramuscular and extraskeletal manifestations of the disease were more frequent in adult patients. The most common extramuscular feature was arthralgia in both groups of patients with juvenile or adult DM. Cardiac manifestation of the disease was not observed in juvenile patients. Respiratory muscle involvement and interstitial lung disease (ILD) were more frequent among adult DM patients than cardiac manifestation of the myositis. In view of the disease course, the authors found that frequency of polycyclic and monophasic subtypes of the disease were mainly similar. The hazard of relapse was found higher during the first year after the remission. None of the juvenile patients died. Among adult patients four disease-specific deaths occurred. There was no correlation between relapse free survival and initial therapeutic regimen. Many of our patients had polycyclic or chronic disease. As relapses can occur after a prolonged disease-free interval, patients should be followed up for at least 2 years. Although we found favourable survival probability, further investigations are needed to assess functional outcome.

Genetic and demographic features of X-linked agammaglobulinemia in Eastern and Central Europe: A cohort study

Primary immunodeficiency disorders are a recognized public health problem worldwide. The prototype of these conditions is X-linked agammaglobulinemia (XLA) or Brutons disease. XLA is caused by mutations in Brutons tyrosine kinase gene (BTK), preventing B cell development and resulting in the almost total absence of serum immunoglobulins. The genetic profile and prevalence of XLA have not previously been studied in Eastern and Central European (ECE) countries. We studied the genetic and demographic features of XLA in Belarus, Croatia Hungary, Poland, Republic of Macedonia, Romania, Russia, Serbia, Slovenia, and Ukraine. We collected clinical, immunological, and genetic information for 122 patients from 109 families. The BTK gene was sequenced from the genomic DNA of patients with a high susceptibility to infection, almost no CD19(+) peripheral blood B cells, and low or undetectable levels of serum immunoglobulins M, G, and A, compatible with a clinical and immunological diagnosis of XLA. BTK sequence analysis revealed 98 different mutations, 46 of which are reported for the first time here. The mutations included single nucleotide changes in the coding exons (35 missense and 17 nonsense), 23 splicing defects, 13 small deletions, 7 large deletions, and 3 insertions. The mutations were scattered throughout the BTK gene and most frequently concerned the SH1 domain; no missense mutation was detected in the SH3 domain. The prevalence of XLA in ECE countries (total population 145,530,870) was found to be 1 per 1,399,000 individuals. This report provides the first comprehensive overview of the molecular genetic and demographic features of XLA in Eastern and Central Europe.

Interleukin-6-induced production of type II acute phase proteins and expression of junB gene are downregulated by human recombinant growth hormone in vitro.

Growth hormone (GH), given therapeutically in many human diseases, is able to modulate the maturation and function of many cells of immune system. The present study demonstrates the effect of human recombinant GH on the production of acute phase proteins (APP) as well as on the gene expression of junB proto‐oncogene on human hepatoma cell line, HepG2. When applied alone GH resulted in an increase in the transcription of junB proto‐oncogene within 30min. The production of α2‐macroglobulin, haptoglobin and fibrinogen was also enhanced by rhGH treatment. However, both IL‐6‐stimulatedjunB gene expression (junB mRNA) and biosynthesis of type II APP (α2‐macroglobulin, fibrinogen, haptoglobin) were strongly inhibited by the GH. The results indicate that GH has a modulatory role in regulating inflammation both in the absence and presence of IL‐6. These findings call for further in vivo studies to determine the potential anti‐inflammatory actions of GH therapy.

In vitro effect of human recombinant growth hormone on lymphocyte and granulocyte function of healthy and uremic children

The recombinant human growth hormone (rhGH), currently used in supraphysiological doses to promote growth acceleration in chronic renal failure children (CRF), also has the ability to influence their impaired immune functions. The effect of human growth hormone on the lymphoproliferative response in vitro was analyzed in the peripheral blood lymphocytes of 25 healthy and 11 uremic children. In 72% of the uremic cases and in 60% of the healthy individual children the hormone increased the lymphoproliferation alone, and/or when used in combination with phytohaemagglutinine. The range of the effective hormone concentrations differed individually. Using semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) a great variation in the gene expression of growth hormone- (GH)-receptor in peripheral lymphocytes was detected. The respiratory burst activity of peripheral polymorphonuclear leukocytes (PMN) in vitro, in response to GH alone and when combined with suboptimal dose of phorbolester (PMA), was assessed by measuring luminol enhanced chemiluminescence in ten uremic and 18 healthy children. In six out of the ten of the CRF patients and in eight out of 18 of the healthy children the GH enhanced the oxidative burst activity of granulocytes provoked by a suboptimal dose of PMA. However, the effective doses (10, 50 and 300 ng/ml) and incubation times (0, 45 and 90 min) showed individual variations. Our data suggest that rhGH treatment in uremic children could be advantageous considering this populations enhanced susceptibility to bacterial, viral and fungal infections.

GROWTH HORMONE RECEPTOR GENE EXPRESSION ON HUMAN LYMPHOCYTIC AND MONOCYTIC CELL LINES

The potential effect of growth hormone (GH) in tumorigenesis, particularly in acute leukemia is controversial. Human growth hormone has the ability to influence certain immune functions; the majority of immune cells express growth hormone receptor (GHR) on plasma membranes. We determined GHR gene expression on different human lymphocyte (JURKAT, CESS) and monocyte (U937, THP1) cell lines by reverse transcriptase polymerase chain reaction analysis of GHR mRNA after stimulating the cells with phytohaemagglutinin or phorbolester, human growth hormone and with a combination of these. The receptor gene expression showed differences; in the U937 and CESS cell lines only the stimulants were able to induce GHR mRNA expression; in the case of JURKAT cells even the hormone alone had the ability to express its own receptor gene. Both the increased TNF‐α production of U937 (but not that of THP1 cells), and the decreased proliferation of JURKAT cells in response to GH stimuli also prove the presence of biologically active GHR on the cell surface. Our data suggest asymmetric interaction between GH or phorbolester‐induced signal pathways in U937 cells sharply depending on the temporal sequence of treatments. THP1 monocytes showed no gene expression in response to any of the stimulants. The phenomenon that certain human lymphoid and monocytoid cell lines at different levels of cell differentiation are able to express the GH receptor gene could have importance in the rhGH therapy.

[When does an autoimmune disease begin? Importance of the early diagnosis].

In autoimmune diseases, such as type I diabetes mellitus, systemic autoimmune diseases and the early phase of rheumatoid arthritis, before the development of a definitive disease, clinical and laboratory alterations can be observed. Being aware of these symptoms is crucial both for family practitioners and specialists, handling autoimmune and early arthritis patients. The early recognition and prognosticating of the disease and sending the patient immediately to a specialist will lead to the exponential improvement of the patients life expectancies and will also help to avoid complications. The need for special diagnostics, care and treatment made the development of national immunological and rheumatological centers imperative, where sufficient experience and professional knowledge helps the proper medical attendance.

A novel flow cytometric approach reveals abundant CD8+ T cell derived microvesicles in rheumatoid arthritis synovial fluid samples

Backgroundand objectives Microvesicle (MV) secretion represents an evolutionally conserved feature of all living cells. The assessment of MVs may give insight into the pathomechanism of various disorders. Furthermore, it may serve as potential novel biomarker of disease. However, the characterisation of MVs in body fluids has not been fully standardised yet, and there are numerous pitfalls that hinder the correct assessment of MVs. Previously, the authors developed a ‘differential detergent lysis’ method to exclude MV-mimicking protein aggregates and immune complexes during flow cytometry. Using our novel method here the authors analysed synovial fluid (SF) derived MVs. Materials and methods The authors tested plasma and SF samples of patients with osteoarthritis (OA), rheumatoid arthritis (RA) and juvenile idiopathic arthritis. The authors used electron microscopy and nanoparticle tracking analysis (NTA) to determine the particle size distributions in SF samples. The authors also applied mass spectrometry to determine the MV protein composition in SFs. To immune phenotype SF MVs, the authors applied flow cytometry using ‘differential detergent lysis’ method. Results The different techniques gave concordant results regarding the size distribution of MVs in SF samples (80–400 nm). However, NTA analysis and MS revealed that most of the events were related to protein aggregates rather than cell-derived vesicles. Using our novel flow cytometric approach, the authors demonstrate for the first time that CD3+ and CD8+ T cell derived SF MVs are highly elevated in SFs of patients with RA compared to OA patients (p=0.027 and p=0.009, respectively after Bonferroni corrections). In contrast, T cell derived MVs were undetectable in the blood plasma of patients with OA or RA. B cell and T cell derived MV counts strongly correlated with rheumatoid factor positivity in RA (r=0.912, p=0.002 and r=0.956, p=0.001, respectively). Conclusions The correct assessment of MVs in RA suggests local CD8+ T cell activation in the joints and may shed light on hidden immunpathological processes of this disease.

The Hungarian version of the Juvenile Arthritis Multidimensional Assessment Report (JAMAR)

The Juvenile Arthritis Multidimensional Assessment Report (JAMAR) is a new parent/patient-reported outcome measure that enables a thorough assessment of the disease status in children with juvenile idiopathic arthritis (JIA). We report the results of the cross-cultural adaptation and validation of the parent and patient versions of the JAMAR in the Hungarian language. The reading comprehension of the questionnaire was tested in 10 JIA parents and patients. Each participating centre was asked to collect demographic, clinical data and the JAMAR in 100 consecutive JIA patients or all consecutive patients seen in a 6-month period and to administer the JAMAR to 100 healthy children and their parents. The statistical validation phase explored descriptive statistics and the psychometric issues of the JAMAR: the three Likert assumptions, floor/ceiling effects, internal consistency, Cronbach’s alpha, interscale correlations, test–retest reliability, and construct validity (convergent and discriminant validity). A total of 206 JIA patients (3.9% systemic, 41.3% oligoarticular, 28.2% RF-negative polyarthritis, 26.6% other categories) and 90 healthy children, were enrolled in two centres. The JAMAR components discriminated healthy subjects from JIA patients. All JAMAR components revealed good psychometric performances. In conclusion, the Hungarian version of the JAMAR is a valid tool for the assessment of children with JIA and is suitable for use both in routine clinical practice and clinical research.