Microparticles expressing myeloperoxidase as potential biomarkers in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV).

To investigate presence of circulating myeloperoxidase-positive microparticles (MPO+MPs) in relation to disease activity in patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Forty-six patients with AAV and 23 age- and sex-matched healthy controls were included. Vasculitis disease activity was assessed using the Birmingham Vasculitis Activity Score (BVAS). MPs were analyzed in citrate plasma by flow cytometry and phenotyped based on MPO expression and co-expression of pentraxin-3 (PTX3), high mobility group box 1 protein (HMGB1), and tumor necrosis factor-like weak inducer of apoptosis (TWEAK). Serum levels of PTX3, sTWEAK, and HMGB1 were also determined. Twenty-three patients had active vasculitis (BVAS ≥ 1). Concentrations of MPO+MPs expressing PTX3, HMGB1, and TWEAK were significantly higher in patients compared to healthy controls (p < 0.001, p < 0.01, p < 0.001, respectively), while concentrations of PTX3+ and HMGB1+MPO+MPs were significantly higher in active AAV compared to patients in remission. MPO+MPs expressing either PTX3 or HMGB1 were associated with BVAS (r = 0.5, p < 0.001; r = 0.3, p = 0.04, respectively). Significantly higher serum PTX3 levels were found in active- than in inactive AAV (p < 0.001), correlating strongly with BVAS (r = 0.7, p < 0.001). Serum levels of sTWEAK and HMGB1 did not differ between patients and controls. Concentration of MPO+MPs is increased in plasma from AAV patients compared to healthy individuals. PTX3 in serum as well as PTX3 and HMGB1 expressed on MPO+MPs were associated with disease activity in the investigated patients. KEY MESSAGES: Myeloperoxidase-positive microparticles (MPO+MPs) are increased in plasma from patients with ANCA-associated vasculitis. Concentrations of MPO+MPs expressing PTX3, HMGB1, and TWEAK were significantly higher in patients compared to healthy controls. MPO+MPs expressing PTX3 and HMGB1 are associated with disease activity in ANCA-associated vasculitis.

Amendment of the OMERACT ultrasound definitions of joints' features in healthy children when using the DOPPLER technique.

BACKGROUND: Recently preliminary ultrasonography (US) definitions, in B mode, for normal components of pediatric joints have been developed by the OMERACT US group. The aim of the current study was to include Doppler findings in the evaluation and definition of normal joint features that can be visualized in healthy children at different age groups. METHODS: A multistep approach was used. Firstly, new additional definitions of joint components were proposed during an expert meeting. In the second step, these definitions, along with the preliminary B-mode-US definitions, were tested for feasibility in an exercise in healthy children at different age groups. In the last step, a larger panel of US experts were invited to join a web-based consensus process in order to approve the developed definitions using the Delphi methodology. A Likert scale of 1-5 was used to assess agreement. RESULTS: Physiological vascularity and fat pad tissue were identified and tested as two additional joint components in healthy children. Since physiological vascularity changes over the time in the growing skeleton, the final definition of Doppler findings comprised separate statements instead of a single full definition. A total of seven statements was developed and included in a written Delphi questionnaire to define and validate the new components. The final definitions for fat pad and physiological vascularity agreed by the group of experts reached 92.9% and 100% agreement respectively in a web survey. CONCLUSION: The inclusion of these two additional joints components which are linked to detection of Doppler signal in pediatric healthy joints will improve the identification of abnormalities in children with joint pathologies.

Educational recommendations for the conduct, content and format of EULAR musculoskeletal ultrasound Teaching the Teachers Courses.

OBJECTIVE: To produce educational guidelines for the conduct, content and format of theoretical and practical teaching at EULAR musculoskeletal ultrasound (MSUS) Teaching the Teachers (TTT) Courses. METHODS: A Delphi-based procedure with 24 recommendations covering five main areas (Duration and place of the course; Faculty members; Content of the course; Evaluation of the teaching skills; TTT competency assessment) was distributed among a group of experts involved in MSUS teaching, in addition to an advisory educational expert being present. Consensus for each recommendation was considered achieved when the percentage of agreement was >75%. RESULTS: 21 of 24 invited participants responded to the first Delphi questionnaire (88% response rate). All 21 participants also responded to the second round. Agreement on 19 statements was obtained after two rounds. CONCLUSIONS: This project has led to the development of guidelines for the conduct, content and format of teaching at the EULAR MSUS TTT Courses that are organised annually, with the aim of training future teachers of EULAR MSUS Courses, EULAR Endorsed MSUS Courses, as well as national and local MSUS Courses. The presented work gives indications on how to homogenise the teaching at the MSUS TTT Courses, thus resolving current discrepancies in the field.

EULAR-PReS points to consider for the use of imaging in the diagnosis and management of juvenile idiopathic arthritis in clinical practice.

To develop evidence based points to consider the use of imaging in the diagnosis and management of juvenile idiopathic arthritis (JIA) in clinical practice. The task force comprised a group of paediatric rheumatologists, rheumatologists experienced in imaging, radiologists, methodologists and patients from nine countries. Eleven questions on imaging in JIA were generated using a process of discussion and consensus. Research evidence was searched systematically for each question using MEDLINE, EMBASE and Cochrane CENTRAL. Imaging modalities included were conventional radiography, ultrasound, MRI, CT, scintigraphy and positron emission tomography. The experts used the evidence obtained from the relevant studies to develop a set of points to consider. The level of agreement with each point to consider was assessed using a numerical rating scale. A total of 13 277 references were identified from the search process, from which 204 studies were included in the systematic review. Nine points to consider were produced, taking into account the heterogeneity of JIA, the lack of normative data and consequent difficulty identifying pathology. These encompassed the role of imaging in making a diagnosis of JIA, detecting and monitoring inflammation and damage, predicting outcome and response to treatment, use of guided therapies, progression and remission. Level of agreement for each proposition varied according to the research evidence and expert opinion. Nine points to consider and a related research agenda for the role of imaging in the management of JIA were developed using published evidence and expert opinion.

Increased activity of lymph node cells in experimental thermal injury: changes in accessory cells in injured area-draining lymph nodes.

Accessory cell content and some of their functional characteristics were determined in regional lymph nodes which drain burn injury (DLN) in rats. Increase in percentages of non-specific esterase-positive cells and NBT+ macrophages and in numbers of dendritic cells were noted in cytospin preparations of draining lymph node cells (DLC) 24 and 72 h following thermal injury. An accumulation of B cells was also noted in the DLN paracortex region at these time points. Enrichment of ED1+ (rat macrophage marker) cells was noted in the adherent DLC population. Increased activity of interleukin-1 (IL-1) in conditioned medium from adherent DLC population and the increased stimulatory capacity of whole DLC or dendritic cell enriched-DLC fraction were noted in functional assays. Enrichment in accessory cells and an increase in their functional activity could contribute to the endogenous activity of regional lymph nodes which drain burned areas.

Disease modifying and immunomodulatory effects of high dose 1 alpha (OH) D3 in rheumatoid arthritis patients.

OBJECTIVE: Vitamin D analogues such as 1 alpha (OH) D3 (alphacalcidiol) have a possible physiological paracrine effect on cell proliferation and differentiation. Experimentally established possibilities to prevent autoimmune diseases suggest that alphacalcidiol may have therapeutic value as an immunomodulatory agent in patients with rheumatoid arthritis. METHODS: We organized a 3-month open-label trial on 19 patients being treated with standard DMARD therapy for acute RA. They were divided into 2 subgroups, those with highly active RA and those with moderately active RA. Their regular drug regimen was maintained during the trial and oral alphacalcidiol 2 micrograms/day was added. Therapy results were evaluated by ESR, CRP, morning stiffness, the Richie index, and the Lee index. Immunomodulatory effects were investigated by measuring lymphocyte proliferation and apoptosis both in the patients and in vitro in 10 nM alphacalcidiol-supplemented culture medium. RESULTS: After 3 months, high dose oral alphacalcidiol therapy showed a positive effect on disease activity in 89% of the patients (45% or 9 pts. with complete remission and 44% or 8 pts. with a satisfactory effect). Only two patients (11%) showed no improvement, but no new symptoms occurred. No side effects were observed. CONCLUSION: These results suggest that alphacalcidiol is a powerful immunomodulatory agent with fairly low hypercalcemic activity. Clinical improvement was strongly correlated with the immunomodulating potential of this agent. We noticed dual effects on lymphocyte proliferation and apoptosis according to the prior cell activation state. Alphacalcidiol could therefore possibly be used as an adjunct therapy with DMARDs in patients with rheumatoid arthritis.

[Bone marrow stromal damage mediated by immune response activity]. / Ostecenje strome kostne srzi u uslovima aktivnosti imunskog odgovora.

The aim of this work was to estimate influence of activated immune response on hematopoiesis in vitro, using the experimental model of BCG immunized BALB/c mice and in patients with chronic immunoactivation: long-lasting infections, autoimmunity or malignancy. We correlated changes in long term bone marrow cultures (Dexter) and NBT reduction with appearance of anemia in patients and experimental model of immunization by BCG. Increased spontaneous NBT reduction pointed out role of macrophage activation in bone marrow stroma damage. Long-term bone marrow cultures showed reduced number of hematopoietic cells, with predomination of fibroblasts and loss of fat cells. This results correlated with anemia and leucocytosis with stimulated myelopoiesis in peripheral blood. Activation of immune response, or acting of any agent that directly changes extracellular matrix and cellularity of bone marrow, may result in microenviroment bone marrow damage that modify hematopoiesis.

[Importance of immunoregulation and erythropoiesis estimation in disorders with activated immune system]. / Znacaj ispitivanja imunoregulacije i eritropoeze u oboljenjima s aktivisanim imunskim sistemom.

Complicated immunoregulatory events in different disorders depend on the pathologic process itself and the phase of disease. Immune parameters aberrations in advanced phase of disease, compared to initial ones, may be significant, making hard the understanding of basic immunoregulatory process of the disease. Intending to explain immune mechanisms in different disorders with activation of the immune system as basic pathogenic event (infections, autoimmune diseases), particularly in advanced phase of the disease, on a parallel with standard laboratory investigations of erythropoiesis (hemoglobin, red blood cell count, hematocrit) the following examinations of the immune system were done: white blood cell count, immunophenotyping of lymphocytes, ability of the phagocytes to reduce NBT, chemiluminescent response after stimulation with opsonized and nonopsonized particles. Analysis of erythropoietic parameters in order to see the consequences of the regulatory mechanisms of the immune system (cytokines, interleukins) on hemopoiesis may enable us to understand the mechanisms of disease with the initial activation of the immune system. Finding anemia might be useful in defining the immunoregulatory mechanisms involved in particular pathologic process important for therapeutic approach.

[Effect of exanguinotransfusion on the immune system of neonate]. / Uticaj eksangvinotransfuzije na imunski sistem novoredjenceta.

Exanguinotransfusion is commonly used during therapeutic procedure in neonates with hyperbillirubinemia risking to cause brain injury of the neonate. This therapy without alternative may couse anemia, hyper or hypotonus, and behavior problems. Those disturbances are not clearly understood yet, but it appears that this process is mediated by the immune system. In order to confirm this hypothesis parallely with clinical examination, we performed the following laboratory investigations: red blood cell count, hemoglobin, hematocrite, white blood cells, natural killer cells, T lymphocyte subsets, ability of the peripheral blood phagocytes to reduce NBT, the phagocytic ability and hemiluminescent response. In the clinical examination the most common finding was hypertonus or hypotonus of the muscules. The values of hemoglobin and hematocrite were reduced, red blood cell count was similar, while the white blood cell particularly the monocyte count was increased compared to the control. The absolute number of T lymphocytes defined by CD2 and CD3 surface marker expression was depleted, while the number of DR positive cells as well as the number of NK cells was increased in group treated with exanguinotransfusion. We have also found an increase of NBT positive cells in the same group. There were no differences in the number of NBT positive cells, when they were prestimulated by PMA. Phagocytic ability was lower compared to the control, while hemiluminescent response was faster remaining on high level for a long time. All those differences remained for several months after exanguinotransfusion.

[Immunophenotyping in diagnostic and classification of leukemia]. / Imunofenotipizacija u dijagnostici i klasifikaciji leukemija.

Markers of differentiation on cell membrane are recognized by monoclonal antibodies, capable to define more precisely the level of cell maturation. The immunofluorescent method, used in this work, is based on the discoveration of surface molecules by means of fluorescein labeled monoclonal antibodies. 74 patients with leukemia, 11 children and 63 adults persons, have been examined. The results show that malignant cells express certain markers characteristic for some forms of leukemias. The most common marker expression in pre-B ALL was: CD34, CD10, CD22, CD24, CD19 and DR. Expression of CD10 marker was characteristic for CALLA + pre-B ALL cells. T leukemias expressed CD38, CD2, CD3, CD4 and CD8 markers. The expression of CD36, CD13, CD14, CD15, CD11b and DR markers was seen in AML. Surface markers may determine type of leikemia. Immunophenotyping of leukemias has enabled us to make a precise diagnostic, determine its prognosis, and select appropriate therapeutic approach.

Periodontal cell migration into the apical pulp during the repair process after pulpectomy in immature teeth: an autoradiographic study.

The migration of dental papilla cells into the periodontium during the process of root development may occur as part of the process involved in the formation of the periodontal tissues. The question posed is whether such cells under pathological conditions could retromigrate from periodontium into dental pulp and together with other apical pulp cells of immature teeth, take part in the production of additional dental tissue, e.g. 1) the tertiary/dentine under deep carious lesion where odontoblasts had been destroyed 2) the dentine bridge on an amputation wound and 3) calcified tissue which closes an apex during the apexification process in immature teeth. The migration of periodontal cells locally marked by H3 thymidine immediately after partial pulpectomy in immature dog's teeth was analysed at observation periods of 2, 24 and 50 h and also without H3 thymidine labelling of periodontal cells 8 weeks after pulpectomy. The marked cells were found in the early observation periods after pulpectomy just in the places where the hard tissues were formed in the later observation period of 8 weeks. They were found in large numbers just around the coagulated necrotic foci. The finding supports the assumption that firm necrotic masses are a very important stimulative factor in the reparation process in pulp and periodontium. The experiment also corroborated the existence of periodontal cell retromigration into apical dental papilla of immature teeth. Future research should assess the possible role of the pathological condition in the determination of undifferentiated odontogenic ectomesenchymal periodontal cells into odontoblasts.

[Fissure sealants as primary preventive measures. Four-year evaluation in Stara Pazova]. / Fissurenversiegelungen als primäre Vorbeugungsmassnahme. Eine vierjährige Bewertungsstudie in Stara Pazova.

Worldwide studies have shown that fissure caries prophylaxis is very difficult using conventional preventive methods. The scientific council of the prophylaxis programme for the protection of mouth and teeth in Vojvodina has begun sealing fissures in Stara Pazova and Novi Sad. The molars of all 6-year-old children were sealed completely. A 77.8% reduction in fissure caries was found at the end of the four-year study. Losses of sealing material came to 16% after four years. Cost-effectiveness analysis: The sealings are three times cheaper than an amalgam filling. Application time: 8-9 minutes.

Histochemical localization at the electron microscopic level of sulfated glycosaminoglycans in the rat gingiva.

Using the high iron diamine thiocarbohydrazide silver proteinate (HID-TCH-SP) staining technique, we investigated ultrastructural localization of sulfated glycosaminoglycans (GAGs) in the rat gingiva shortly after eruption, especially those associated with internal and external basal laminae. In the apical portion of the internal basal lamina, HID-TCH-SP stain deposits were distributed mainly in the region of the lamina lucida located between the lamina densa and the distal surface membrane of the junctional epithelium and inside the depression of the distal surface membrane adjacent to the basal lamina. Stain deposits were also detected on the surface membrane of the cytoplasmic protrusion. Interestingly, the density of HID-TCH-SP stain deposits in the internal basal lamina was highest in the apical portion of the junctional epithelium and decreased in the coronal direction, finally tending to disappear completely. On the other hand, in the external basal lamina the deposits were localized in the whole region of the basal lamina or at both sites of the lamina densa. HID-TCH-SP stain deposits were also detected external to the lamina densa in the basement membrane associated with capillaries and in the connective tissue where they were distributed in close relation to collagen fibrils. Testicular hyaluronidase digested most HID-TCH-SP stain deposits in the connective tissue, whereas those in the region of basement membranes resisted this enzymatic digestion.

[The influence of the optimal fluoride concentration upon the gingival health during the period of mixed dentition]. / Uticaj fluorida u vodi za pice na gingivu u vreme mesovite denticije.

The paper is presenting analyzes of the condition of marginal periodontium in children age 7 to 14 from Novi Sad (F level 0.2 ppm) and Sombor (F level 1.2 ppm). The gingival health was determined by methodology WHO-TRS 621 for every tooth which had reached occlusal plane. As statistical unit side, tooth and person (with 80-100% of healthy sides) were used. With a side as a unit the health condition was higher in Sombor for every age. In the 8, 10 and 12 year health condition was lower for both locality. Correlation coefficient for both distribution was r = 0.94. In the case of tooth as a unit no difference was found in 8 year of age. Correlation coefficient was lower (r = 0.74). Concerning person there were no difference in the ages of 8, 10 and 12. The results pointed to the necessity to use person as experimental unit together as side. Observed difference between two localities is not enough to exclude other primary preventive measures for periodontal diseases. The higher incidence of the inflamed gingival units in ages of 8, 10 and 12 deserves more detailed investigation of possible causes.

[Experimental testing of effects of organic fluorides on prevention of gingival inflammation]. / Eksperimentalno ispitivanje moguceg mehanizma dejstva organskih fluorida u prevenciji inflamacije gingive.

The possible mechanismus of Aminfluoride action in the inhibition of plaque maturation is presented in the paper. Experiment was performed on dogs during the eruption of permanent molars. Experimental side was brushed with Aminfluride gel and control without any paste during 5 weeks. The groove in the enamel representing a border between supra and subgingival surface was prepared. The enamel was investigated by means of electron probe for elements Ca, P and F. The presence of fluoride in the surface layers was determined on the experimental side supragingivali and subgingivali till the depth of 500 in the sulcus. A release of fluorides from these deposits could be responsible for detoriation of plaque maturation and gingivitis prevention.