Carbon-based nanomaterials accelerate arteriolar thrombus formation in the murine microcirculation independently of their shape.

Although carbon-based nanomaterials (CBNs) have been shown to exert prothrombotic effects in microvessels, it is poorly understood whether CBNs also have the potential to interfere with the process of leukocyte-endothelial cell interactions and whether the shape of CBNs plays a role in these processes. Thus, the aim of this study was to compare the acute effects of two differently shaped CBNs, fiber-shaped single-walled carbon nanotubes (SWCNT) and spherical ultrafine carbon black (CB), on thrombus formation as well as on leukocyte-endothelial cell interactions and leukocyte transmigration in the murine microcirculation upon systemic administration in vivo. Systemic administration of both SWCNT and CB accelerated arteriolar thrombus formation at a dose of 1 mg kg(-1) body weight, whereas SWCNT exerted a prothrombotic effect also at a lower dose (0.1 mg kg(-1) body weight). In vitro, both CBNs induced P-selectin expression on human platelets and formation of platelet-granulocyte complexes. In contrast, injection of fiber-shaped SWCNT or of spherical CB did not induce leukocyte-endothelial cell interactions or leukocyte transmigration. In vitro, both CBNs slightly increased the expression of activation markers on human monocytes and granulocytes. These findings suggest that systemic administration of CBNs accelerates arteriolar thrombus formation independently of the CBNs' shape, but does not induce leukocyte-endothelial cell interactions or leukocyte transmigration.

A summary of characteristics of the first wave of new type coronavirus epidemic and potential association with tuberculosis vaccination / Az új koronavírus okozta járvány elso hullámának jellemzoi és esetleges kapcsolata a tuberkulózis elleni oltottsággal.

Összefoglaló. Bevezetés: A COVID-19-járvány az egész világon elterjedt. A járvány Európában való elso megjelenése során megfigyelheto volt, hogy a terjedés mértéke kisebb azokban az országokban, ahol a tuberkulózis elleni védekezésül kiterjedt BCG-vakcinációt végeznek. Célkituzés: A jelen munkában olyan összefüggéseket igyekeztünk feltárni, amelyek befolyásolták a járványterjedés paramétereit, különös figyelemmel a BCG-vakcinációs gyakorlatra. Módszerek: A világ összes olyan országára vonatkozóan, ahol megfelelo minoségu statisztikai adatok álltak rendelkezésünkre, vizsgáltuk a járvány terjedésének elso hullámát. A mozgóátlagolt járványgörbéken elemeztük a járvány idotartamát, a tetozés mértékét, a fertozöttek és a halálesetek egymillió lakosra vetített számát. Figyelembe vettük az országok gazdasági mutatóit (GDP, légi forgalom, a tengeri hajózás mértéke). Statisztikai analízis: A vizsgált paraméterek nem mutattak normális eloszlást, így nemparaméteres próbákkal (rangkorreláció, Kruskal-Wallis ANOVA) statisztikai kapcsolatot kerestünk a járványterjedés mértéke, a BCG-vakcináció és más paraméterek között. Eredmények: A járvány gyorsan elterjedt a világon, de mégis, február elso három hetében a terjedésben egy szünet volt megfigyelheto. A járványhullám Európában nagyjából egyszerre ért véget. A járvány által leginkább azok az országok érintettek, ahol nem alkalmaztak rendszeres BCG-vakcinációt, bár a képet bonyolítja, hogy ezek az országok gazdaságilag többnyire fejlettek. A halálozási rátában nem mutatkozott ilyen különbség. Következtetés: Statisztikailag igazolható tény, hogy a vakcinációt végzo országokból az elso hullám alatt kevesebb fertozöttet jelentettek; az ok-okozati összefüggés bizonytalan, hiszen az országok múltja, szokásai, társadalmi berendezkedése, gazdasági fejlettsége nem azonos. Eredményeink alátámasztják az összehasonlító kontaktkutatás fontosságát annak tisztázására, hogy a BCG-oltás hogyan befolyásolja az emberek vírussal szembeni érzékenységét, valamint a vírus terjesztésének, továbbadásának képességét. Orv Hetil. 2021; 162(4): 123-134. INTRODUCTION: The new type of coronavirus (SARS-CoV-2) epidemic is widespread throughout the world. During the outbreak of the pandemic in Europe it was revealed that the rate of spread was lower in countries where extensive BCG vaccination is used to protect against tuberculosis. OBJECTIVE: In the present work, we sought to explore relationships that influenced epidemic spreading parameters, with particular reference to BCG vaccination practice. METHODS: We examined the first wave of the spread of the epidemic for all countries in the world where adequate quality statistics were available. We analyzed the duration of the epidemic, the extent of the peak, the number of infected people, and the number of deaths per million inhabitants with the moving average of epidemic curves. We took into account the economic indicators of the countries (GDP, air traffic and extent of maritime shipping). STATISTICAL ANALYSIS: The examined parameters did not show a normal distribution, so we looked for a statistical relationship with non-parametric tests (rank correlation, Kruskal-Wallis ANOVA) between the extents of epidemic spread, BCG vaccination and other parameters. RESULTS: The epidemic spread rapidly around the world, but still, in the first three weeks of February, there was a pause in the spread. The first wave of epidemics ended roughly at the same time in Europe. Those countries are the most affected by the epidemic where regular BCG vaccination has not been used, although the picture is complicated by the fact that these countries are mostly economically developed. There was no such difference observable in the mortality rate. CONCLUSION: Although this work clearly demonstrates that during the first wave of the pandemic, fewer infections were reported worldwide in countries where BCG vaccination is obligatory, however, the causal relationship is uncertain, as the countries' past, customs, social organization and economic development are different. Our results support the necessity of comparative contact tracing to clarify how BCG vaccination affects people's susceptibility to this new type of coronavirus as well as their ability to spread and transmit the virus. Orv Hetil. 2021; 162(4): 123-134.

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.

In vitro platelet activation, aggregation and platelet-granulocyte complex formation induced by surface modified single-walled carbon nanotubes.

Surface modification of single-walled carbon nanotubes (SWCNTs) such as carboxylation, amidation, hydroxylation and pegylation is used to reduce the nanotube toxicity and render them more suitable for biomedical applications than their pristine counterparts. Toxicity can be manifested in platelet activation as it has been shown for SWCNTs. However, the effect of various surface modifications on the platelet activating potential of SWCNTs has not been tested yet. In vitro platelet activation (CD62P) as well as the platelet-granulocyte complex formation (CD15/CD41 double positivity) in human whole blood were measured by flow cytometry in the presence of 0.1mg/ml of pristine or various surface modified SWCNTs. The effect of various SWCNTs was tested by whole blood impedance aggregometry, too. All tested SWCNTs but the hydroxylated ones activate platelets and promote platelet-granulocyte complex formation in vitro. Carboxylated, pegylated and pristine SWCNTs induce whole blood aggregation as well. Although pegylation is preferred from biomedical point of view, among the samples tested by us pegylated SWCNTs induced far the most prominent activation and a well detectable aggregation of platelets in whole blood.

[Right prefrontal cerebral hemispheric activation by symmetrical carotid sinus baroreceptor stimulation]. / Jobb agyféltekei praefrontalis aktiváció szimmetrikus carotis sinus baroreceptor-ingerlés hatására.

This study was performed to test the hypothesis of greater right hemispheric involvement in the processing of signals related to baroreceptor stimuli. Carotis sinus baroreceptors were stimulated by rhythmically decreasing air pressure in a neck chamber, and as a control the thorax was stimulated in a similar manner. Changes in regional cerebral blood flow (rCBF) were measured by positron emission tomography (PET). Baroreceptor stimulation resulted in rCBF increase in the right anterior-inferior prefrontal cortex (Brodmann areas [BA] 10/44/47) and bilaterally in BA 6/8. The authors conclude that, at least in some stages of baroreceptor information processing, the right hemisphere plays a greater role than the left one.

[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.

Single-walled carbon nanotubes activate platelets and accelerate thrombus formation in the microcirculation.

OBJECTIVES: Although ambient nanoparticles have been shown to exert prothrombotic effects, manufactured nanoparticles are in this aspect less well investigated. Thus, the aim of this study was to characterize the effects of diesel, titanium dioxide rutile, and single-walled carbon nanotube nanoparticles on (i) platelet activation in vitro and (ii) on macro- and microcirculatory thrombus formation in vivo. METHODS: Platelet P-selectin expression was measured by flow cytometry after incubation of whole blood with diesel (0.1mg/mL), titanium dioxide (0.1mg/mL) or single-walled nanotubes (0.001-0.1mg/mL). Platelet-granulocyte complexes were analyzed in whole blood and platelet aggregometry was performed with platelet-rich plasma. Upon systemic administration of nanoparticles (1mg/kg) to anesthetized mice, ferric chloride-induced thrombus formation was measured in small mesenteric arteries using in vivo microscopy. In separate experiments, diesel (1mg/kg), titanium dioxide (1mg/kg), or single-walled nanotubes (0.01-1mg/kg) were injected into anesthetized mice and light/dye-induced thrombus formation was investigated in the cremasteric microcirculation. RESULTS: Diesel and titanium dioxide nanoparticles did not activate platelets or exert prothrombotic effects. In contrast, single-walled nanotubes significantly increased platelet P-selectin expression, the number of platelet-granulocyte complexes, and platelet aggregability in vitro, and reduced the occlusion time in mesenteric arteries as well as in cremasteric arterioles. CONCLUSION: Our study shows that single-walled carbon nanotubes, but not diesel or titanium dioxide nanoparticles, induce platelet activation in vitro and exert prothrombotic effects in the microcirculation in vivo.