An Eye-Tracking Study of Attention Biases in Children at High Familial Risk for Depression and Their Parents with Depression.

Attention biases (AB) are a core component of cognitive models of depression yet it is unclear what role they play in the transgenerational transmission of depression. 44 children (9-14 years) with a high familial risk of depression (HR) were compared on multiple measures of AB with 36 children with a low familial risk of depression (LR). Their parents: 44 adults with a history of depression (HD) and 36 adults with no history of psychiatric disorder (ND) were also compared. There was no evidence of group differences in AB; neither between the HR and LR children, nor between HD and ND parents. There was no evidence of a correlation between parent and child AB. The internal consistency of the tasks varied greatly. The Dot-Probe Task showed unacceptable reliability whereas the behavioral index of the Visual-Search Task and an eye-tracking index of the Passive-Viewing Task showed better reliability. There was little correlation between the AB tasks and the tasks showed minimal convergence with symptoms of depression or anxiety. The null-findings of the current study contradict our expectations and much of the previous literature. They may be due to the poor psychometric properties associated with some of the AB indices, the unreliability of AB in general, or the relatively modest sample size. The poor reliability of the tasks in our sample suggest caution should be taken when interpreting the positive findings of previous studies which have used similar methods and populations.

Effects of a modified clinoptilolite zeolite on growth performance, health status and detoxification of aflatoxin B1 and ochratoxin A in male broiler chickens.

1. The aim of the present study was to test the ability of an in-feed modified clinoptilolite zeolite-based mycotoxin binding substance (Minazel® Plus, Patent Co, Misicevo, Serbia; MP) to prevent gastrointestinal absorption of aflatoxin B1 (AFB1) and ochratoxin A (OTA) and its effects on health status and performance parameters of broilers.2. A total of 375, 1 d old male broiler chickens (Cobb 500) were used for a total trial period of 42 d (from hatch to 42 d of age). Animals were randomly allocated to five treatment groups (T1-T5), in 25 pens (15 male broilers per pen, five pens per treatment). T1 was the control maize-based diet without the addition of mycotoxins, or the test product. T2 and T3 groups received contaminated maize in the diet containing 0.02 mg AFB1/kg feed and 0.1 mg OTA/kg feed, whereas T4 and T5 groups received 0.05 mg AFB1/kg feed and 0.5 mg OTA/kg feed. The MP was added to T3 (1 g/kg feed), and T5 (2 g/kg feed) groups.3. Results showed that exposure to AFB1 and OTA at low or moderate levels, as used in this study, did not markedly affect growth performance, blood profile or organ weights. Improvements in feed conversion ratio (FCR) were observed in birds receiving MP, whereby FCR of T3 group was improved in comparison with T2 group, although there was no significant difference between T5 and T4 groups. However, average body weight gain (ABWG) was improved in the T5 group compared to T4, but not in the T3 versus T2 group comparison.4. For serum biochemical parameters, glutamate-dehydrogenase (GLDH) was significantly improved in T5 birds in comparison with T4. The addition of MP significantly decreased residue levels of AFB1 in liver and OTA in the spleen of the treated groups.5. The improvements in productive performance and reduction of mycotoxin residue levels in tissues demonstrated a beneficial effect of MP in cases of concurrent AFB1 and OTA ingestion by broilers.

Preterm neonates display altered plasmacytoid dendritic cell function and morphology.

Bacterial and viral infections cause high rates of morbidity and mortality in premature newborns. In the setting of viral infection, pDCs play a key role as strong producers of IFN-α upon TLR9 activation. We analyzed pDC frequency, phenotype, morphology, and function in CB of preterm and term newborns in comparison with adults. Whereas all age groups show similar pDC numbers, BDCA-2, CD123, and TLR9 levels, the expression of BDCA-4 and capacity to produce IFN-α upon TLR9 challenge were decreased significantly in preterm neonates. Furthermore, we show by means of electron microscopy that pDCs from preterm newborns exhibit a distinct, "immature" morphology. Taken together, these findings suggest decreased functionality of pDCs in the premature newborn. The reduced capacity to produce IFN-α is likely to render such infants more susceptible to viral infections.

Photooxidation technology for correlated light and electron microscopy.

The combination of the capabilities of light microscopical techniques with the power of resolution of electron microscopy along with technical advances has led to a gradual decline of the gap between classical light and electron microscopy. Among the correlative techniques using the synergistic opportunities, photooxidation methods have been established as valuable tools for visualizing cell structures at both light and electron microscopic level. Fluorescent dyes are used to oxidize the substrate diaminobenzidine, which in its oxidized state forms fine granular precipitates. Stained with osmium, the diaminobenzidine precipitates are well discernible in the electron microscope, thus labelling and defining the cellular structures, which at light microscopy level are recorded by fluorescent probes. The underlying photooxidation reaction is based on the excitation of free oxygen radicals that form upon illumination of fluorochromes; this is a central step in the procedure, which mainly influences the success of the method. This article summarizes basic steps of the technology and progresses, shows efforts and elaborated pathways, and focuses on methodical solutions as to the applicability of different fluorochromes, as well as conditions for fine structural localizations of the reaction products.

The cannabinoid receptor-1 antagonist rimonabant inhibits platelet activation and reduces pro-inflammatory chemokines and leukocytes in Zucker rats.

BACKGROUND AND PURPOSE: We investigated the effect of rimonabant on inflammation and enhanced platelet reactivity in type 2 diabetic Zucker rats, an experimental model of impaired glucose tolerance and the metabolic syndrome. EXPERIMENTAL APPROACH: Rimonabant (10 mg kg(-1) by gavage) was fed for 2 weeks to 3-month-old male obese Zucker rats as an impaired glucose tolerance model and for 10 weeks to 6-month-old male obese Zucker rats as a model of the metabolic syndrome. RANTES (Regulated upon Activation, Normal T cell Expressed, and Secreted) and MCP-1 (monocyte chemotactic protein-1) serum levels were determined by ELISA. Leukocyte populations were quantitatively assessed using a veterinary differential blood cell counter. Platelet activation was assessed by flow-cytometry, platelet aggregation, and adhesion of isolated platelets to immobilized fibrinogen. KEY RESULTS: RANTES and MCP-1 serum levels were increased in obese vs lean Zucker rats and significantly reduced by long-term treatment with rimonabant, which slowed weight gain in rats with the metabolic syndrome. Neutrophils and monocytes were significantly increased in young and old obese vs lean Zucker rats and lowered by rimonabant. Platelet-bound fibrinogen was significantly enhanced in obese vs lean Zucker rats of both age, and was reduced by rimonabant. Platelets from obese rats were more sensitive to thrombin-induced aggregation and adhesion to fibrinogen, which were both attenuated by rimonabant therapy. CONCLUSIONS AND IMPLICATIONS: We demonstrate positive modulation of circulating neutrophil and monocyte numbers, reduced platelet activation and lower RANTES and MCP-1 levels by rimonabant in Zucker rats. This may potentially contribute to a reduction of cardiovascular risk.

Platelet content and growth factor release in platelet-rich plasma: a comparison of four different systems.

BACKGROUND: Different systems for preparation of platelet-rich plasma are commercially available, but data for comparison of these systems have not been published so far. MATERIALS AND METHODS: We investigated the performance of Vivostat PRF Preparation Kit, PCCS Platelet Concentrate Collection System, Harvest SmartPReP 2 APC 60 Process, and Fibrinet Autologous Fibrin & Platelet System. The preparations provided by these systems are platelet concentrates with high numbers of platelets in a small volume of plasma and PDGF-AB is released continuously during the 5 days after preparation. RESULTS: Vivostat PRF Preparation Kit, PCCS Platelet Concentrate Collection System, Harvest SmartPReP 2 APC 60 Process are comparable in platelet yield and total amount of released PDGF-AB after 120 h while with Fibrinet the lowest platelet yield and PDGF-AB content of supernatant was achieved. The ability of growth factor release was equal in all four systems. CONCLUSION: In conclusion, all four systems for preparation of platelet-rich plasma investigated result in considerable growth factor release. In what extent the total content of PDGF-AB as a consequence of platelet yield has an impact on wound healing has to be further investigated.

Structure of synovial lymphatic capillaries in rheumatoid arthritis and juvenile idiopathic arthritis.

The structure of lymphatic capillaries (LC) of the synovial membrane (SM) from patients with rheumatoid arthritis and juvenile idiopathic arthritis obtained by synovectomy was investigated by transmission electron microscopy. This method allows comparison of the structure of the same vessel under light and electron microscope and clear differentiation between lymphatic and blood capillaries and venules. Synovial LC were localized in the subintimal connective tissue of the SM in the vicinity of venules. The shape of some LC was irregular, suggesting edema of the interstitium. Lymphatic endothelium has extremely attenuated cytoplasm with the exception of the perinuclear region. Many nuclei of endothelial cells had distinct nucleoli. The basal lamina was discontinuous. The walls of LC showed close connection with the interstitium represented by anchoring filaments that were attached to the endothelial cells and to the surrounding connective tissue. In some LC connective tissue appeared to be disconnected from endothelium and gaps between their walls and the interstitium were seen. Mononuclear cells were accumulated adjacent to some LC. Specialized interendothelial junctions (endothelial microvalves) were observed in the LC walls. Their structure and function in the migration of cells and debris from synovial interstitium into LC lumina in rheumatoid arthritic synovium deserves further investigation. In the lumina of some of the LC lymphocytes, monocytes, macrophages, cell debris and enlarged endothelium were observed. Accumulation of such material may cause obstruction of tiny LC. We suggest that reported alterations of the fine synovial lymphatic vessels can contribute to the progression of the inflammatory process to chronicity.

Removal of T and B lymphocytes by in-line filtration: evaluation of the efficiency of a polyester filter type (Pall WBF-2) by flow cytometric counting.

BACKGROUND AND OBJECTIVES: The aim of this study was to investigate whether in-line filtration, using a polyester filter for the preparation of red cell concentrates (RCC) and plasma (PL), leads to an altered proportion of T and B lymphocytes in the fraction of residual white blood cells (WBC). MATERIALS AND METHODS: The capacity of Pall WBF-2 in-line filters to reduce the numbers of T and B lymphocytes from red blood cell concentrates (RCC) and plasma (PL) of 22 donations was investigated by three-colour flow cytometry (FC) using the Tritest-Trucount kit. T and B lymphocytes were identified using monoclonal antibodies (mAbs) against CD3, CD19 and CD45, conjugated with fluorescein isothiocyanate, phycoerythrin or peridinin chlorophyll protein-A, respectively. As the number of B cells was below the detection limit of the FC method, WBC of the respective blood components of healthy donors were concentrated 25-fold by Percoll density-gradient centrifugation. In this fraction the absolute numbers of T and B cells, as well as their ratio, were determined using the Attractor software, which provides a discrimination of rare cell counts from FC in relation to debris. RESULTS: The mean numbers, as well as minima and maxima of T and B lymphocytes per unit, were as follows. T cells in RCC: 4.51 x 10(3) (1.68 x 10(2)-4.09 x 10(4)) and in PL: 1.35 x 10(3) (2.21-1.78 x 10(4)); B cells in RCC: 2.33 x 10(3) (7.10 x 10(1)-9.15 x 10(3)) and in PL: 2.33 x 102 (7.5 x 10(2)-2.8 x 10(3)). T cells were retained, on average, at a higher level than B cells: 3.01 times higher in RCC and 1.01 times higher in PL. CONCLUSION: After filtration, the ratio of T and B lymphocytes changed in RCC (1.95 : 1) compared with unfiltered blood, where it was 5.83 : 1. In PL the ratio did not change notably compared to unfiltered blood. The results of this research show that cell concentration (using gradient centrifugation) in combination with an appropriate FC acquisition and analysis procedure, allows both residual T and B lymphocytes (being under the detection limit without cell concentration) to be determined.

Interaction of monocytes from patients with psoriatic arthritis with cultured microvascular endothelial cells.

The aim of this study was to investigate the interaction of monocytes of the peripheral blood of patients with psoriatic arthritis with cultured human dermal microvascular endothelial cells (HDMEC) compared to monocytes from control persons. The surface expression of adhesion molecules (ADM) and other cell surface molecules in psoriatic arthritis and control monocytes was investigated by quantitative flow cytometry. The receptor densities of these molecules were determined in terms of monoclonal antibody (mAb) binding sites. Cocultivation experiments including peripheral blood mononuclear cells and HDMEC were performed to determine the adhesion to and transmigration through activated or resting endothelial cell monolayers. In order to achieve optimal responses of cellular functions, activation for adhesion experiments was induced by lipopolysaccharide (LPS), while in transmigration experiments the endothelial cells were activated by TNF-alpha. For transendothelial migration studies HDMEC cultivated on collagen gels were used. In the supernatants of cocultivated cells the cytokines IL-6 and IL-8 were determined by ELISA. A significantly reduced expression of CD11b in nonactivated psoriatic arthritis peripheral blood monocytes compared to control monocytes was verified (mean number of adhesion molecules/cell: 33,756 +/- 10,138 vs 61,023 +/- 6925). In agreement with these findings, adhesion to, as well as transendothelial migration through, activated HDMEC was found to be significantly reduced in psoriatic arthritis monocytes. Transendothelial migration engendered an enrichment of monocytes in the migrated cell fraction for both control and psoriatic arthritis peripheral blood mononuclear cells. The activation of HDMEC by LPS induced a highly significantly enhanced cytokine release for IL-6 and IL-8, irrespective of the origin of monocytes (psoriatic arthritis vs. controls). However, IL-8 production in the supernatants of nonactivated monocytes/HDMEC cocultures was significantly reduced in the case of monocytes from psoriatic arthritis patients (6650 +/- 2489.32 pg/ml) vs 9280.00 +/- 3209.51 pg/ml in control patients. Impaired adhesion as well as transendothelial migration of monocytes derived from peripheral blood of psoriatic arthritis patients can be explained by the reduced expression of adhesion molecules MAC-1 (CD11b/CD18) at the surface of monocytes. The reduced IL-8 production also corresponds to a diminished cellular interaction under nonflow conditions. These results support the view that there are systemic immunological alterations in psoriatic arthritis patients.

Interactions of lymphocytes from patients with psoriatic arthritis or healthy controls and cultured endothelial cells.

Psoriatic arthritis (PA) is an inflammatory rheumatic disease that can concomitantly occur in patients with psoriasis vulgaris. Psoriatic synovitis shows alterations of the synovial microvasculature. Inflammatory cells adhere to endothelial cells (EC) and migrate through the vascular wall of postcapillary venules located in the subintimal layer of the synovial membrane. The aim of our study was to investigate, first, the phenotype of lymphocytes (LC) of PA patients using flow cytometry (FC) with regard to activation antigens and adhesion molecules; second, the adhesion of LC of PA patients on cultivated resting or activated (with thrombin, LPS, IFN-gamma, or TNF-alpha) human umbilical vein endothelial cells (HUVEC) by counting the Feulgen-stained nuclei of both adherent LC and HUVEC using image analysis; and third, the synthesis of IL-6 and IL-8 in both LC and HUVEC 24 hr after cell contact. These cytokines were determined qualitatively by immunofluorescence and quantitatively at the single-cell level by FC as well as in the supernatants of the cultures using commercial cytokine ELISAs. Fourth, we investigated whether or not the LC adhesion on HUVEC as well as the cytokine production could be inhibited by monoclonal antibodies against LC- or EC-specific adhesion molecules. In contrast to controls PA patients showed an increased surface expression of CD11a, b, and c as well as of CD44 but a reduced surface expression of CD49d/CD29, and CD49e/CD29, and cell-bound fibronectin on CD3+ LC. The activation markers CD25 and HLA-DR were found to be slightly enhanced in PA. The cell adhesion was generally enhanced in PA patients vs controls. It could be reduced with monoclonal antibodies (MoAbs) against CD11a and CD18 on IFN-gamma- or TNF-alpha-activated HUVEC but was generally enhanced after treatment of HUVEC with MoAbs against CD54, CD62E, or CD106. Due to LC adhesion on HUVEC IL-6 and IL-8 were produced in significantly higher amounts in PA patients compared to controls. This effect occurred already in resting but was enhanced in activated HUVEC. While IL-6 is mainly produced by HUVEC but also in smaller quantities by LC, IL-8 is synthesized only by HUVEC and could be modified by preincubation with MoAbs against LC- or EC-specific adhesion molecules in parallel to the cell adhesion. The experiments show that the main adhesion pathway in LC homing of PA patients is the interaction of the LC adhesion molecule CD11a/CD18 with CD54 on EC followed by an enhanced synthesis of proinflammatory and chemotactic cytokines. These results favor the hypothesis that the pathological alterations of the microvasculature in PA patients are generated by altered homing processes.

Effects of ciprofloxacin and ofloxacin on adult human cartilage in vitro.

Chondrocyte toxicity and necrosis were seen with electron microscopy after incubation of human adult cartilage biopsy specimens in ciprofloxacin or ofloxacin. In vitro exposure of chondrocytes to fluoroquinolones did not affect apoptosis as determined by flow cytometry. While the immediate clinical significance of this finding remains unclear, the possibility of long-term cartilage damage after fluoroquinolone treatment cannot be excluded.

Effects of aminoguanidine on adhesion molecule expression of human endothelial cells.

The effect of aminoguanidine (AG) on the expression of adhesion molecules on nonactivated human umbilical vein endothelial cells (HUVEC) was investigated in vitro. Nonactivated HUVEC cultivated on long-term glycated fibronectin (FN) as compared to native FN showed a significant upregulation of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and CD31 which could be further promoted by long-term glycated bovine serum albumin. AG, at a concentration of 0.01 mol/l, caused an upregulation of ICAM-1 of 48 +/- 17.4% in HUVEC cultivated on gelatin. In contrast, VCAM-1 and E-selectin remained unaffected. At this concentration, formation of advanced glycation end products (AGE) was inhibited by 57%, as determined immunologically, and by 50%, as verified by AGE-specific fluorescence. A hypothesis concerning the upregulation of ICAM-1 by AG as compared to VCAM-1 is proposed relating to its relative redox insensitivity. Our results demonstrate that the beneficial effect of AG in reducing the risk of accelerated development of atherosclerosis in diabetic patients by inhibiting formation of AGE on matrix proteins such as FN might be hampered by its tendency to upregulate ICAM-1 on endothelial cells.

Demonstration by flow cytometry of the numbers of residual white blood cells and platelets in filtered red blood cell concentrates and plasma preparations.

BACKGROUND AND OBJECTIVES: New-generation polyester filters provide significant depletion of white blood cells (WBC) and platelets (PLT) in filtered red blood cell concentrates (FRCC) and in filtered plasma preparations (FP). The aim of this study was to elaborate a sensitive flow cytometric method for monitoring residual WBC and PLT in FRCC and FP. MATERIALS AND METHODS: We determined the number of WBC in 500 microliters FRCC of FP using 50 microliters of a combination of monoclonal antibodies (MAB) against CD45 (FITC labeled) and CD19 (PE labeled). After lysis of red blood cells, we mixed a specific number of reference beads with the remaining WBC. The number of residual WBC related to the acquisition volume was defined by the acquired reference beads. Using this method, the detection limit (DL) was 3 WBC/microliter. Alternative methods used MAB against CD45 (FITC and PerCP labeled) and CD14 (PE labeled) or lymphocyte subsets such as CD3 (FITC labeled) and CD19, CD4, CD8, CD16 and CD56 (PE labeled) in combination with CD45 (PerCP labeled). The DL values were 10 WBC/microliter for the CD45/CD14 staining and 0.1 WBC/microliter for the determination of both CD3+ and CD19+ lymphocytes. For residual PLT in FRCC or FP, we used an FITC-conjugated MAB against CD41, with reference beads to determine the acquisition volume. PLT were demonstrated in a green-fluorescence (FL1) single histogram after gating in the forward light scatter x 90 degrees light scatter signal dot plot. PLT counting was as described for WBC. The DL value was about 2 PLT/microliter. RESULTS: Filtration with Pall WBF-1 filters reduces WBC by 4 log and PLT by 3-4 log, resulting in cell counts which are below the critical limit for causing adverse transfusion reactions. CONCLUSIONS: Flow cytometry techniques provide a reproducible and objective tool for counting residual WBC and PLT in blood preparations compared with the Nageotte hemocytometer. Absolute numbers of leukocyte and lymphocyte subpopulations are obtainable.

Evaluation of four monoclonal antibodies against HLA-B27 for their reliability in HLA-B27 typing with flow cytometry (FC): comparison with the classic microlymphocytotoxic test (MLCT).

Typing for HLA-B27 by serological methods is routinely performed using the microlymphocytotoxic test (MLCT). Since monoclonal antibodies (MAB) against HLA-B27 are available, flow cytometry (FC), which requires less time than the MLCT has been developed as an alternative technique. The aim of the present study was to check the accuracy and reliability of this method using different MAB against HLA-B27 in comparison to MLCT (using polyclonal antibodies against HLA-B27 and cross-reacting specificities [CRS]). FC was performed in 144 patients with HLA-B27-related rheumatic disorders (seronegative spondarthritides) using a special software package which requires corresponding calibration beads in order to achieve a standardized setup of the flow cytometer. MAB from the following producers were used: Becton & Dickinson (BD), Behringwerke (BE), One Lambda (OL), and Immunotech (IT). In addition to the critical limit of fluorescence intensity (FI) which indicates positivity if exceeded, provided by the software (but valid only for the MAB from BD), empirically twice the value of the STD calculated from the mean of the FI values of HLA-B27 positive patients was regarded a good cut off for the HLA-B27 positivity in FC measurements with the MAB used. Using a standard protocol including an incubation of whole EDTA-anticoagulated blood for 15 min with MAB against HLA-B27 (FITC-conjugated) and CD3 (PE-conjugated) and a lysis of erythrocytes, good discrimination between HLA-B27 positive and negative patients was obtained. Cross reactions with HLA-B27 positive patients occurred except when the MAB from OL was used. One false-negative result was found with OL's MAB (out of 22) and false-positive results occurred in HLA-B7+ patients when MAB from BD, BE, and IT were used. Unfortunately also 1 false-positive result (out of 57) was obtained in HLA-B7-, B27- patients with IT's MAB. Errors in the interpretation of the FC analysis might be avoided if more than one MAB (including those not cross reacting with HLA-B7) are used.

HLA-B27 determination using serological methods. A comparison of enzyme immunoassay and a microlymphocytotoxic test with flow cytometry and a molecular biological assay.

Typing for HLA-B27 is routinely performed in patients with seronegative spondarthritides. Besides the microlymphocytotoxic test (MLCT), other serological techniques have been developed such as enzyme immunoassays (EIA) using serum or plasma as a source for the determination of soluble HLA-B27 (sHLA-B27) and flow cytometric (FC) methods. The aim of the present study was to check the accuracy and reliability of the EIA for sHLA-B27 in comparison to the MLCT using antibodies against HLA-B27 and cross-reacting specificities (CRS), as well as an FC method and a molecular biological method. Any discrepant results should be typed with the MLCT using a complete panel of anti-HLA-class I antibodies, with FC and with a molecular biological technique. The EIA should also be repeated in those patients, using serum and plasma from a new venipuncture. In 81 patients with rheumatic disorders, the EIA and the MLCT using antibodies against HLA-B27 and CRS were performed. Based on the MLCT with a complete panel of anti-HLA-class I antibodies as a standard, discrepant test results were obtained for 9 out of 81 patients with the MLCT using antibodies against HLA-B27 and CRS and with the EIA. The following wrong results occurred: in the MLCT with anti-HLA-B27 and CRS, there were two false-negative results; in the EIA there were four false-negative and one false-positive results; one sample was undeterminable. In comparison with the MLCT, including the complete panel of HLA-class I antibodies, as well as with a molecular biological technique, typing with FC showed a complete concordance. Our investigations demonstrated that for routine typing for HLA-B27 the MLCT cannot be replaced by EIA because of a significant number of mistypings. The MLCT performed only with antibodies against HLA-B27 and CRS may also lead to typing errors. No errors were detected using flow cytometry. If only serological methods can be performed in a laboratory a combination of flow cytometry and MLCT could therefore enhance the safety of HLA-B27 typing.

Prevalence of HLA-DR3 and autoantibodies to connective tissue components in Dupuytren's contracture.

Forty-six patients with Dupuytren's contracture (DC) and 55 control persons were HLA-typed for class I and HLA-DR class II antigens and were investigated for the presence of autoantibodies against elastin (ELAB) and collagen types I-IV (ACA I-IV). Using the chi 2 test, obtained from 2 x 2 contingency tables, a significant association was found between DC and HLA-DR3 and autoantibodies to types I-IV collagen. ELAB and ACA I and III were significantly correlated with HLA-DR3 in the whole group of patients plus controls. In analogy to other diseases with autoimmunologic features the presence of HLA-DR3 therefore seems to indicate a higher risk for the formation of connective tissue autoantibodies. The remodeling processes during the course of fibrosis in DC might be responsible for this autoantibody formation.

Comparative in vitro investigations of the influence of mofebutazone, phenylbutazone and diclofenac on phagocytosis and respiratory burst of human peripheral blood leucocytes.

The phagocytosis and the release of oxidative products generated by the respiratory burst have been studied under the influence of the non-steroidal anti-inflammatories (NSAID) phenylbutazone (PB), mofebutazone (monophenylbutazone, MPB) and diclofenac (DF) using phagocytes of the peripheral blood from healthy human donors and patients with soft tissue rheumatism. The measurement of phagocytosis by flow cytometry (FC) was carried out in order to investigate the uptake of FITC-labelled bacteria (E. coli), separately by monocytes (MON) and polymorphonuclear leucocytes (PMN). In addition the luminol-dependent chemiluminescence (CL) was measured using opsonized Zymosan on leucocytes of the peripheral blood that were purified by lysis of erythrocytes. In FC, PMNs and MONs could be identified by gating in the whole blood in which erythrocytes had been lysed. The NSAID were added to the in vitro tests for 30 min in concentrations of 10(-3) mol/l, 10(-4) mol/l, 10(-5) mol/l, 10(-6) mol/l, and 10(-8) mol/l. Using the FC phagocytosis test it was found that PB and MPB decreased the percentage of phagocytosing PMNs as well as MONs while DF increased it slightly in contrast to the controls. However, the fluorescence intensity of the phagocytes, which indicates the amount of ingested bacteria, was found to be unchanged. PB effected a significant concentration-dependent inhibition of CL in all of the concentrations used, with the exception of 10(-8) mol/l. MPB resulted in a borderline inhibition at 10(-3) mol/l although the measurement of every individual proband showed a concentration dependency. DF inhibited the luminol-dependent CL significantly only at a concentration of 10(-3) mol/l.