Elevation of monocyte-platelet aggregates is an early marker of type 2 diabetes.

BACKGROUND: Diabetes has been shown to be an accelerating factor in the progression of atherosclerosis. The metabolic changes in diabetes contribute to modified platelet function and enhanced leukocyte-platelet aggregate formation. The attachment of activated platelets leads to the activation of leukocytes causing enhanced cytokine production and upregulation of surface adhesion molecules. Therefore, platelet-leukocyte aggregates may be of great importance in the development of cardiovascular complications. MATERIALS AND METHODS: Monocyte-platelet aggregates and monocyte Mac-1 expression were measured by flow cytometry to obtain differences between type 2 diabetic and healthy subjects. Inflammatory mediators were evaluated to assess the presence of inflammation. RESULTS: We found no signs of inflammation in type 2 diabetes; however, we observed enhanced aggregation level of monocytes and platelets. The expression of Mac-1 did not differ between diabetic and control subjects, but it was significantly higher on monocytes bearing platelets in both groups. CONCLUSIONS: Elevation of monocyte-platelet aggregates is an early marker of diabetes, which precedes the signs of inflammation. Enhanced Mac-1 expression can be observed on monocytes bearing platelets, independent from the presence of diabetes.

Extramedullary hematopoiesis is dysregulated in histamine-free histidine decarboxylase knockout (HDC-/-) mice.

OBJECTIVE AND DESIGN: In this study we investigated the role of histamine on the extramedullary hematopoiesis. METHODS: Male histidine decarboxylase knockout (HDC(-/-)) mice and wild-type mice were used (n = 5/group). Groups of mice received sublethal total-body gamma irradiation at a single dose of 4 Gy. Spleen cells were studied at different time points post-irradiation by flow cytometry, colony forming unit (CFU) assay, and real-time PCR. For statistical analysis Student's t test, ANOVA, and Holm-Sidak post-hoc test were used. RESULTS: By day 14 after irradiation, spleen cell counts increased almost eightfold in wild-type and not even fourfold in HDC(-/-) mice (P < 0.01). The proliferative capacity and interleukin-3 signaling of stem cells were impaired in HDC(-/-) mice. STAT5 mRNA expression was decreased in granulocyte-myeloid colonies by 72.9 +/- 8.6% (P < 0.001), compared to the wild-type. CONCLUSIONS: The absence of histamine adversely affects splenic hematopoiesis via direct and indirect mechanisms.

An easy-to-use practical method to measure coincidence in the flow cytometer--the case of platelet-granulocyte complex determination.

Cell complexes composed of two different cells labeled with different fluorophores can be detected as double positive events in the flow cytometer. Double positivity can originate not only from real complexes but from non-interacting coinciding cells as well. Coincidence has a high impact on the determination of the amount of platelet-granulocyte complexes since platelet concentration is in the orders of magnitude higher than that of the granulocytes. Mixtures of non-interacting fluorescent beads as well as EDTA anticoagulated or citrated blood samples were analyzed in the flow cytometer in the presence and absence of fluorescent beads at various dilutions. Experimental data were evaluated by mathematical means. The bead or platelet concentration dependence of double positivity was converted into linear functions using Poisson distribution. This linearised form contains information on the detection volume as well as on the presence/absence of dilution independent complexes. The presence of appropriate fluorescent beads in the blood sample makes possible to estimate the fraction of double positivity originating from coincidence if data collection is triggered by the granulocytes or by the fluorescent beads, alternatively. Mixing fluorescent beads into a blood sample is a simple experimental method to distinguish double positivity originating from real cell-cell complexes from the coincidence of cells in a flow cytometer, thus providing a tool for the determination of the real amount of cell-cell complexes.

Impact of coincidence on granulocyte-platelet complex determination by flow cytometry is evaluated by a novel computer simulation model of coincidence.

Cell complexes composed of two different cells labeled with different fluorophores can be detected as double positive events in the flow cytometer. Double positivity can originate not only from real complexes but from non-interacting coinciding cells as well. Coincidence has a high impact on the determination of the amount of platelet-granulocyte complexes since platelet concentration is in the orders of magnitude higher than that of the granulocytes. A computer model has been developed to simulate coincidence in the flow cytometer to reveal the contribution of coincidence to the overestimation of the total amount of platelet-granulocyte complexes. Mixtures of non-interacting fluorescent beads as well as EDTA-anticoagulated blood samples were analyzed in the flow cytometer. An excellent fit was found between computer simulated and measured data pairs. Bead mixture in the flow cytometer and simulation of that resulted in 37.3+/-1.3 and 35.7+/-0.6% double positivity, respectively. 30.2+/-4.3% double positivity was measured for EDTA-anticoagulated blood samples while simulation of that resulted in 28.3+/-0.6%. Double positivity attributed to platelet-granulocyte complexes in slightly diluted blood samples might originate in coincidence and not from true complexes.

The effects of platelet receptor GPIIb/IIIa polymorphism (Leu Pro33) on the receptor expression and platelet aggregation in patients with ischaemic stroke.

Platelet hyperaggregation in ischaemic stroke patients is a proven finding, and associated with increased expression of the platelet surface GPIIb/IIIa receptor. The polymorphism occurs at nucleotide position 1565 of the GPIIIa gene resulting a 33Leu-Pro change. Data are conflicting regarding the abnormal function of the PlA1/A2 receptor in stroke. The aim of the study was to address the difference of platelet receptor function in ischemic stroke patients with the wild PlA1/A1 and heterozygous PlA1/A2 genotype. A total of 51 patients with PA1/A1 and 54 patients with PlA1/A2 genotypes were enrolled. Polymerase chain reaction was used for genotyping of platelets. Platelet aggregation was measured in whole blood and in platelet rich plasma (PRP). Flow cytometry was used for measuring surface molecule expression (CD42b, CD41a, CD61, CD62P) and fibrinogen binding capacity of cells with phosphate buffer solution (PBS) in comparison with activation by ristocetin in whole blood as well as by adenosine diphosphate (ADP) in PRP. In comparison with wild types, platelets carrying the PlA1/A2 genotypes showed hyperaggregation measured in whole blood and induced by ristocetin (p< 0.05). Using whole blood flow cytometry with ristocetin induction, the CD62P+/FIB- (P selectin) and the CD62P+/FIB+ were more expressed in heterozygous platelets as compared to wild types (p< 0.01 and p< 0.05), respectively. According to mean fluorescence intensity with ADP induction, an increased expression of CD61+, CD61+/CD41+ and CD62P+ in PlA1/A2 platelets were detected as compared to the group carrying the wild type (p< 0.0001, p= 0.006, p= 0.0001), respectively. These findings support the possibility that in ischaemic stroke patients, platelets carrying PlA1/A2 genotypes can be activated by different inductors in a way, which leads to permanent hyperfunction of platelet surface receptor GPIIIa.

Prolonged intestinal mucosal acidosis is associated with multiple organ failure in human acute pancreatitis: gastric tonometry revisited.

AIM: To evaluate whether multiple determinations of intramucosal pH (pHi) in acute pancreatitis (AP) patients could provide additional information of the disease severity during early hospitalization. METHODS: Twenty-one patients suffering from acute pancreatitis were monitored by gastric tonometry in the first 72 h after hospital admission. RESULTS: In the survivor group (n = 15) the initially low pHi values returned to normal level (pHi > or = 7.32) within 48 h (median pHi: d 1: 7.21; d 2: 7.32; d 3: 7.33). In contrast, pHi values in the non-survivor group n = 6) were persistently either below or in the low normal range (median pHi 7.12; 7.12; 7.07 respectively), but pHi differences between the two groups reached significance only after 24 h (P<0.01). Mucosal acidosis detected at any time during the monitored period was associated with the emergence of single or multiple organ dysfunction (P<0.01). CONCLUSION: Prolonged gastric mucosal acidosis was associated with remote organ dysfunction and failure in Acute Pancreatitis, however, correlation with the fatal outcome became significant only 24 h after admission. Due to its non-invasive nature gastric tonometry may supplement the pro-inflammatory markers to achieve a multi-faceted monitoring of the disease.

Self-regulation of neutrophils during phagocytosis is modified after severe tissue injury.

Neutrophil (PMNL) function is influenced by factors released by other immune cells during the course of the immune response. We investigated the effect of neutrophil cell density and the effect of supernatant of the phagocytosis assay on the phagocytosis activity of PMNLs. The measurements were carried out with naive (control) PMNLs of healthy donors and with PMNLs obtained from patients with severe tissue injury. Phagocytosis index (FI) of PMNLs was determined at cell densities of 7.5 x 10(5)/ml and 15 x 10(5)/ml. E. coli phagocytosis of heparinized whole blood from healthy donors and patients with severe tissue injury was measured and evaluated at three different cell densities (normal, half, and double densities) by flow cytometry. Supernatants of phagocytosis assays of either control or trauma (ISS >18) patient PMNLs were added to the assay suspensions of control and trauma PMNLs. An increase in cell density of healthy donor PMNLs increased yeast phagocytic activity. In cases of tissue injury, PMNLs showed increased phagocytic activity at lower cell densities. E. coli phagocytosis was increased with the increase of cell density, and tissue injury PMNLs were more active at each cell concentration compared to naive cells. Polytrauma supernatants in most cases inhibited, while healthy supernatants mostly increased the yeast phagocytosis of healthy and trauma PMNLs. These results reinforce the idea that primed PMNLs in the presence of microbial agents produce factor(s) which inhibit some of the cell's antimicrobial functions contributing to immune-dysfunction, while unprimed PMNLs produce factor(s) which facilitate antimicrobial countermeasure. These results also demonstrate that reduced phagocytosis of tissue injury primed PMNLs is not due to cytoskeletal changes but to the humoral environment.

The prevalence and incidence of Helicobacter pylori infections among young recruits during service in the Hungarian Army.

BACKGROUND: A vast number of data indicate that the prevalence of Helicobacter pylori infections is positively correlated with age and is different in various countries. Although our knowledge of transmission of H. pylori is very limited, it is reasonable to assume that it could be much more contagious in closed communities, for example in garrisons, than in normal populations. METHODS: Young male recruits (aged 19-23 years) in the Hungarian Army were tested for seropositivity at the beginning and at the end of their military service. RESULTS: The prevalence of H. pylori seropositivity was found to be 23% (*CI95%: 21-24%) among the young male recruits. Seroconversion among the formerly seronegative persons after completing either their 9-month or 6-month military service was 30% (CI95%: 25-35%) and 23% (CI95%: 8-45%), respectively. In those groups, where either the H. pylori infection was eradicated by antibiotics or hygienic countermeasures were introduced, the infection rate was reduced from 23% to 11% (CI95%: 3-25%) and to 0% (CI95%: 0-6%); p > .2 and p < .002, respectively. CONCLUSION: Our data suggest that, although H. pylori has a very high contagiosity in closed communities, its spread can be reduced or even prevented by medication of the infected persons and/or by improving the hygienic conditions and introducing anti-infective sanitary regulations.

[First attempts of detecting fetal cells in the maternal circulation]. / Elso lépéseink a magzati sejtek anyai keringésbol történo kimutatásában.

INTRODUCTION: In prenatal diagnosis there is great interest for noninvasive diagnostic methods. Authors report their first results in detecting fetal cells in the maternal circulation during pregnancy. OBJECTIVE: The aim of the study was to detect fetal gender from maternal peripheral blood samples during pregnancy. METHOD: Authors have analysed fetal nucleated red blood cells. In 12 cases after a double density Percoll gradient separation they labelled the surface antigens of the cells with anti-glycophorin-A and anti-CD45 fluorescent antibodies, did an intracellular staining of the epsilon haemoglobin chain, and analysed the cells with flow cytometry. The CD45 negative/glycophorin-A positive/epsilon-haemoglobin chain positive cells were considered as fetal cells. Having the results, in another 13 cases magnetic activated cell sorting with CD71 antibody were used as an enrichment step. Authors made an intracellular staining of the epsilon haemoglobin chain, the positive cells were isolated by micromanipulation, and analysed by single cell fluorescent polymerase chain reaction. Primers for the amelogenin gene were used to detect fetal gender. RESULTS: Only the Percoll enrichment step itself is not enough for using the samples for diagnostic molecular-biologic examinations, a following enrichment step is needed. For this the authors used magnetic activated cell sorting with CD71 antibody. With the help of this enrichment step, after the intracellular staining of the epsilon haemoglobin chain the direct micromanipulator isolation of the epsilon haemoglobin chain positive cells could be done. After analysing single cells by fluorescent polymerase chain reaction, in 8 out of the 11 comparable cases the results were similar to those, what was found during the genetic amniocentesis. In 2 cases from this 8, genetic amniocentesis proved Klinefelter syndrome, which they could also confirm with the examination of fetal cells in the maternal circulation. CONCLUSION: The results of the study suggest that the method described above can be useful in prenatal genetic diagnosis, and improving it could be useful to detect other genetic abnormalities (chromosomal abnormalities, single gene disorders) as well.

[Therapy resistant leg ulcer caused by multiple myeloma]. / Myeloma multiplex szokásos terápiával nem befolyásolható lábszárfekély hátterében.

The authors review an elderly woman suffering from leg ulcer with bad curability which was a consequence of a malignant haematologic disease. Multiple relapse of this ulcer was observed and did not react to usual conservative therapy. The only sign of multiple myeloma was the extremely high level of iron measured in the blood serum bound to a monoclonal paraprotein. Sternum aspiration was made and the sample showed presence of plasmoblasts supporting diagnosis of multiple myeloma. The poor therapeutical results were caused by hyperviscosity syndrome in consequence of the high level of the monoclonal component in the blood serum. The ulcer was cured within eight weeks by suppression of the monocloclonal component and thus, elimination of hyperviscosity. This case is a special one from several points of view. Leg ulcers not reacting to usual therapy may be caused by haematologic disease thus the physician should consider this and extend examinations as well and necessarily hospitalize patient. Appearance of multiple myeloma is unusual in this case. Hystology made from the skin excised from the periphery of the wound has not showed signs of pyoderma gangrenosum, which is known mostly being associated with multiple myeloma.

Luminol-dependent chemiluminescence is related to the extracellularly released reactive oxygen intermediates in the case of rat neutrophils activated by formyl-methionyl-leucyl-phenylalanine.

Luminol is a non-radical-specific amplifying molecule which produces light upon interaction with various reactive oxygen intermediates (ROIs). ROI production of rat peritoneal polymorphonuclear leukocytes (PMNLs) elicited by 2.3 microM formyl-methionyl-leucyl-phenylalanine (fMLP) results in a biphasic luminol-dependent chemiluminescence (LDCL) signal. Whereas ROIs are also produced intracellularly, as judged by flow cytometry, addition of non-membrane-permeable catalase reduces the first and second phases of the LDCL signal to around 3% and less than 3%, respectively. This suggests that in the case of fMLP-stimulated rat PMNLs, the LDCL signal is related to the ROIs in the extracellular medium and hydrogen peroxide has a key role in the formation of the LDCL signal. In the presence of the non-specific myeloperoxidase inhibitor Na-azide, the first phase of the LDCL signal decreases slightly (87+/-8%), while the second phase almost disappears (< 3%), indicating the myeloperoxidase dependence of the second phase. The hydroxyl radical scavenger histidine results in an 84+/-4% and a 71+/-4% decrease in the intensity of the first and second phases, respectively. Based on these data, it is concluded that hydrogen peroxide might be the source of hydroxyl radicals directly oxidizing luminol in the first phase of the LDCL signal, while in the second phase it serves as a substrate of myeloperoxidase in the peroxidation reaction of the luminol.