Comparative flow cytometry-based immunophenotyping analysis of peripheral blood leukocytes before and after fixation with paraformaldehyde.

Flow cytometry based immunophenotyping provides prime insight into cellular population composition and characteristics, and is widely used in basic and clinical research. Challenges in processing peripheral blood samples in a timely manner necessitate protocol adaptations and utilization of fixatives. Fixation, however, may introduce artifacts to the flow cytometry readout. We performed a comparative flow cytometry immunophenotyping analysis of 13 immune cell populations in the whole blood using a staining protocol with and without fixation step. Freshly procured human peripheral blood samples were stained with a panel of 33 fluorochrome-conjugated antibodies. Samples were processed using a protocol with or without a paraformaldehyde-based fixation step, and matching sample pairs were analyzed by flow cytometry. Our results show that paraformaldehyde-based fixation, in comparison to matched unfixed samples, did not significantly affect population distribution and frequency for: B cells, Plasmablasts, Dendritic cells, NK cells, Granulocytes, Neutrophils, Eosinophils, or Hematopoietic Stem/Progenitor Cells. However, fixation led to significant marker shifts in the subpopulation distribution in CD4, T regulatory, CD8, Monocytes, and Basophils. These results indicate the importance of pre-experimental assessment of fixation-introduced artifacts in the flow cytometry output when considering the feasibility of fresh processing. This is especially important for samples analyzed using comprehensive exploratory immunoprofiling panels.

[MicroRNAs as Biomarkers of Cardiovascular Diseases].

The fact that microRNAs play an important role in the development and pathogenesis of cardiovascular disease is beyond doubt. This article provides a brief overview of recent data that relate to microRNA expression in various cardiovascular diseases. Detecting significant changes in the level of expression of these molecules in various diseases means that microRNAs can be considered to be potential biomarkers of human pathologies including heart failure. Studying the relationship between the mechanisms of cardiovascular disease and the level of expression of a variety of microRNAs, as well as establishing their exact relationships with the genes is an urgent problem and requires further research.

[The fetal proteins in prognosis of development of pneumonia in patients with ischemic stroke].

The searching of laboratory predictors of pneumonia in patients with ischemic stroke is an actual issue. The fetal proteins can be such biomarkers. The study was carried out to determine significance of such fetal proteins as alpha-fetoprotein, cancerous embryonic antigen, CA 19-9, CA 125, CA 15-3, CA 72-4, CYFRA 21-1 for prognosis of development of pneumonia in patients with ischemic stroke. The study included sampling of 216 patients in acute period of ischemic stroke. All patients were measured level of fetal proteins in first day from onset of disease using electrochemiluminescence immunoassay. It is demonstrated that CA 72-4 has the most significance for prognosis of development of pneumonia from all analyzed proteins and complications of ischemic stroke. The probability ratio relatively to other fetal proteins added up to 0.460 (CL 95% 0.267-0.791, p=0.011), to other complications--0.629 (CL 95% 0.433-0.913, p=0.015). The threshold value of CA 72-4 for development of pneumonia added up to 0.82 (CL 95% 0.68-0.96, p=0.011) U/ml. Under lower level of CA 72-4 the risk of development of pneumonia increases. Under higher level of CA 72-4 there is statistical probability of absence of developmnent of pneumonia. The threshold value was lower than reference interval which in the study added up to 0.85-1.42 U/ml. The detection of level of CA 72-4 on first day after onset of stroke in patients can be recommended for establishing of group of high risk of development of pneumonia and implementation of therapeutic activities.

[The alpha-fetoprotein in prognosis of survival of and functional rehabilitation of patients with ischemic stroke].

The study was carried out to determine the prognostic value of alpha-fetoprotein in development of lethal outcome and degree of functional rehabilitation of patients with ischemic stroke. The sampling included 216 patients in acute period of ischemic stroke. At the first day of development of disease they were measured the level of human alpha-fetoprotein. At the second day of disease patients were evaluated the degree of functional rehabilitation and the rate of lethal outcomes was calculated. Previously, the reference interval for alpha-fetoprotein was calculated according the guidelines of the International federation of clinical chemistry and national standard. The reference interval amounted to 0.59-3.78 mE/l. The study results demonstrated that low level of alpha-fetoprotein is related to higher risk of lethal outcome (SE=1.7, p=0.012). The increasing of level of alpha-fetoprotein over mentioned threshold value statistically significant increases probability of survival of patients. The further increasing more than 2.28 mE/l is related to subsequent good functional rehabilitation according the modifies Rankine scale (SE=1.4, p=0.001) and Barthel index (SE=1.49, p<0.001).

[Fibrinogen concentration in the evaluation of safety and efficiency of thrombolytic therapy in patients with ischemic stroke].

AIM: To identify and investigate threshold fibrinogen concentrations as predictors of hemorrhagic transformation (HT), fatal outcome (FO), and the efficiency of thrombolytic therapy (TLT) in patients with ischemic stroke (IS). SUBJECTS AND METHODS: One hundred and eighty-one patients with IS were examined; all the patients received TLT. Fibrinogen concentrations were determined by the Clauss method on admission, immediately after TLT, and daily during the first 7 days of observation; the efficacy of thrombolysis was evaluated using the NIH stroke scale every day, the Rankin scale, and the Barthel Index on days 14 and 21. RESULTS: The patients with a fibrinogen concentration of below 330 mg/dl showed the lowest frequency of asymptomatic HT (AHT) as hemorrhagic stroke (HS) type 1 in the absence of clinically worsening HT (CWHT), as well as FO and the highest rate of good functional recovery. Those with a fibrinogen concentration of 330-385 mg/dl most commonly displayed AHT as HS types 1 and 2 equally frequently, as well as the highest frequency of a positive effect according to the criteria for good and/or satisfactory functional recoveries. The fibrinogen concentration range of 385-423 mg/dl compared to the above range was characterized by an increased risk for AHT as HS type 2, for CWHT as equally distributed parenchymal hematoma types 1 and 2, by higher death rates and less chance of functional recovery. The elevated fibrinogen concentration above 423 mg/dl was accompanied by high death rates and CWHT as parenchymal hematoma type 2 and the higher frequency of poor outcome in the evaluation of functional recovery. CONCLUSION: The revealed three threshold fibrinogen concentrations of 330, 385, and 423 mg/dl allow one to predict HS, FO, and the efficiency of TLT in patients with IS.

[The balance of markers of regulation vascular tone and fibrinogen in the prognosis of hemorrhagic transformation and fatal outcome in the acute period of ischemic stroke].

The markers of regulation vascular tone, such as rennin, endothelin-1, and C-type natriuretic peptide, are of great value for prognosis of hemorrhagic transformation and fatal outcome of ischemic stroke. A change in the vascular tone in case of hemorrhagic transformation at the affected site precedes activation of the coagulation component of hemostasis as a mechanism preventing blood loss and increasing fibrinogen level. This work was aimed to study the balance of the above markers and fibrinogen in the prognosis of hemorrhagic transformation and fatal outcome in the acute period of ischemic stroke. It included 62 patients receiving no thrombolytic therapy. It was shown that symptomatic hemorrhagic transformation was associated with elevated rennin levels without a marked fall in the level of C-type natriuretic peptide and asymptomatic hemorrhagic transformation with elevated endothelin-1 levels and decreased concentration of natriuretic peptide. Fibrinogen level on day 4 of the observation proved to be a reliable predictor of negative prognosis. Asymptomatic hemorrhagic transformation without fatal outcome was associated with systemic and local vasoconstriction and inhibition of local vasodilation. Symptomatic hemorrhagic transformation with the fatal outcome was accompanied by dysregulation of vascular tone in the form of activation of systemic and local vasoconstriction, insufficient inhibition of local vasodilation and compensatory reaction in the form of activation of hemostatic mechanisms manifest as elevated fibrinogen levels on day 4. The lethal outcome without hemorrhagic transformation was associated with systemic vasoconstriction, activation of local vasodilation and vasoconstriction leading to local "biochemical paralysis" of vascular tone regulation.

Function and viability of human islets encapsulated in alginate sheets: in vitro and in vivo culture.

Islet encapsulation offers an immune system barrier for islet transplantation, and encapsulation within an alginate sheetlike structure offers the ability to be retrievable after transplanted. This study aims to show that human islets encapsulated into islet sheets remain functional and viable after 8 weeks in culture or when transplanted into the subcutaneous space of rats. Human islets were isolated from cadaveric organs. Dissociation and purification were done using enzymatic digestion and a continuous Ficoll-UWD gradient. Purified human islets were encapsulated in alginate sheets. Human Islet sheets were either kept in culture, at 37°C and 5% CO(2), or transplanted subcutaneously into Lewis rats. After 1, 2, 4, and 8 weeks, the human islet sheets were retrieved from the rats and assessed. The viability of the sheets was measured using fluorescein diacetate (FDA)/propidium iodide (PI), and function was measured through glucose-stimulated insulin release, in which the sheets were incubated for an hour in low-glucose concentration (2.8 mmol/L) and then high (28 mmol/L), then high (28 mmol/L) plus 3-isobutyl-1-methylxanthine (50 µm). Human islet sheets remained both viable, above 70%, and functional, with a stimulation index (insulin secretion in high glucose divided by insulin secretion in low glucose) above 1.5, over 8 weeks of culture or subcutaneous transplantation. Islet transplantation continues to make advances in the treatment of type 1 diabetes. These preliminary results suggest that encapsulated islets sheets can survive and maintain islet viability and function in vivo, within the subcutaneous region.

[Clinical and laboratory assessment of indicators of oxidative status in the cerebrospinal fluid of patients with ischemic stroke].

The objective of the present study was to estimate parameters of oxidative status in the cerebrospinal fluid in the course of ischemic stroke and in the prediction of recovery of neurological functions. Concentration of superoxide dismutase (SOD) as a marker of antioxidant adaptation, the secondary lipid peroxidation products reacting with thiobarbituric acid (PRTBA), cyclic guanosine monophosphate (cGMP) as an indirect product of NO generation, and N-acetylneuraminic acid (NANA) as a marker of destruction of neuronal membranes were studied. One hundred and fifty patients with hemispheric ischemic stroke admitted to a hospital during the first 12 h after stroke were examined. It has been shown that the development of cerebral infarction is accompanied by increased concentrations of oxidative stress markers. Progressive ischemic stroke was characterized by the significantly prolonged increase in PRTBA, cGMP, NANA to the third day from the first symptoms of disease while regressive course was accompanied by the lack of higher production of TBKRP, cGMP, NANA to the third day of disease. The decrease in concentrations of factors of brain damage (PRTBA, cGMP, NANA) in the cerebrospinal fluid, along with the increasing role of processes of antioxidant adaptation, expressed in the growth of SOD concentrations, can be considered as a criterion for the prediction of recovery of disturbed neurologic function to the 21th day of disease.

Ligand binding regions in the receptor for urokinase-type plasminogen activator.

The interaction between urokinase plasminogen activator (uPA) and its cellular receptor (uPAR) is a key event in cell surface-associated plasminogen activation, relevant for cell migration and invasion. In order to define receptor recognition sites for uPA, we have expressed uPAR fragments as fusion products with the minor coat protein on the surface of M13 bacteriophages. Sequence analysis of cDNA fragments encoding uPA-binding peptides indicated the existence of a composite uPA-binding structure including all three uPAR domains. This finding was confirmed by experiments using an overlapping 15-mer peptide array covering the entire uPAR molecule. Four regions within the uPAR sequence were found to directly bind to uPA: two distinct regions containing amino acids 13--20 and amino acids 74--84 of the uPAR domain I, and regions in the putative loop 3 of the domains II and III. All the uPA-binding fragments from the three domains were shown to have an agonistic effect on uPA binding to immobilized uPAR. Furthermore, uPAR-(154--176) increased uPAR-transfected BAF3-cell adhesion on vitronectin in the presence of uPA, whereas uPAR-(247--276) stimulated the cell adhesion both in the absence or presence of uPA. The latter fragment was also able to augment the binding of vitronectin to uPAR in a purified system, thereby mimicking the effect of uPA on this interaction. These results indicate that uPA binding can take place to particular part(s) on several uPAR molecules and that direct uPAR-uPAR contacts may contribute to receptor activation and ligand binding.

The hemopexin-type repeats of human vitronectin are recognized by Streptococcus pyogenes.

The specific binding of vitronectin to Streptococcus pyogenes is believed to play an important role in the infection process by mediating adherence of the bacteria to host cells. The domain of vitronectin involved in the interaction with S. pyogenes is unknown. In the present study, we constructed a vitronectin random epitope phage display library, which was used to pan against intact cells of S. pyogenes. Several phage-displayed vitronectin peptides containing a hydrophobic pentapeptide motif within the hemopexin-type repeats were found to bind to streptococci. These data were supported by competition experiments, in which a representative 23-amino acid synthetic vitronectin peptide comprising part of a hemopexin-type repeat inhibited binding of the bacteria to vitronectin, while a control peptide with identical amino acid composition but a scrambled sequence had no effect. Moreover, cells of S. pyogenes were shown to bind to the synthetic peptide as well as to immobilized hemopexin, whose structural homology to the hemopexin-type repeats in the vitronectin molecule has long been underlined. Soluble vitronectin could inhibit streptococcal binding to immobilized hemopexin. These results provide first evidence for a biological role of hemopexin itself and respective repeats in vitronectin in bacterial binding, suggesting that during an infection process these or other hemopexin-type repeat-containing proteins could be potential targets for bacterial attachment and subsequent colonization.

Identification of novel heparin-binding domains of vitronectin.

Vitronectin is a multifunctional serum protein which provides a unique regulatory link between cell adhesion, humoral defense mechanism and the hemostatic system, and the heparin-binding properties of vitronectin are thought to have participated in various functional aspects. In addition to the carboxy-terminal glycosaminoglycan-binding motif, we report on two novel heparin-binding domains which were identified using phage display technique. One heparin-binding domain is located between amino acids Asp82 and Cys137 at the end of the connector region, while the other is in the second hemopexin-type repeat, between amino acids Lys175 and Asp219 of the vitronectin molecule. Our findings may shed new light to the activities of vitronectin and its binding to cells, which could not be explained solely on the basis of the known heparin-binding domain.

Isolation and characterisation of a vitronectin-binding surface protein from Staphylococcus aureus.

In a previous study we demonstrated that cells of Staphylococcus aureus strain V8 bind 125I-labelled vitronectin in a receptor-ligand type of interaction, and a protein having a molecular mass of 60 kDa was identified as a putative high-affinity staphylococcal vitronectin-binding protein (Liang, O.D. et al. (1993) Biochim. Biophys. Acta 1225, 57-63). In the present communication we report on the isolation and preliminary characterisation of the 60 kDa vitronectin-binding protein. The bacterial cell surface proteins were released by stirring bacteria with 1 M LiCl at 37 degrees C for 2 h and separated on an FPLC Mono-Q column with a gradient of 0-0.5 M NaCl in 20 mM Tris buffer at pH 9.0. Fractions containing vitronectin-binding activity, assayed on microtiter plates with immobilised human vitronectin, were collected and SDS-PAGE analysis showed the content to be a single protein band at the 60 kDa position. In Western blot experiments the protein transblotted onto nitrocellulose membranes could bind soluble vitronectin. Its amino-terminal amino acid sequences showed a striking similarity with those of a 60 kDa heparan sulfate-binding protein from the same staphylococcal strain (Liang, O.D. et al. (1992) Infect. Immun. 60, 899-906), suggesting that they are identical molecules. This was supported by ligand blotting experiments where both vitronectin and heparan sulfate were shown to bind to the same protein band in parallel strips.

Evidence that the heparin-binding consensus sequence of vitronectin is recognized by Staphylococcus aureus.

Binding of heparin-binding form of vitronectin to Staphylococcus aureus was inhibited completely by heparin or by the same form of vitronectin. The binding was inhibited only to about 50% by the non-heparin-binding form of vitronectin, indicating an apparent involvement of the heparin-binding properties in the interaction between vitronectin and S. aureus. This was supported by experiments in which a synthetic peptide (Ala347-Arg361, comprising heparin-binding consensus sequences) was found to partly inhibit bacterial adherence to immobilized vitronectin. A bacterial cell surface protein could bind to the quinquedecapeptide, but not to the highly charged peptides consisting entirely of arginine or lysine, immobilized on microtiter plates and the binding could be competitively inhibited by an excess of soluble peptide. Direct binding of radiolabeled peptide to bacterial cells was also demonstrated, which was rapid, saturable, and pH-dependent. Furtherly a bacterial surface protein having molecular mass of 60 kDa was isolated by affinity chromatography on a quinquedecapeptide-HiTrap-NHS column. Our data suggest that the heparin-binding properties of vitronectin play a role in bacterial recognition.

Homogeneous antigen receptor beta-chain genes in cloned CD4- CD8- alpha beta T suppressor cells.

The rearrangements of beta-chain genes of the T cell Ag receptor were examined in 12 CD4- CD8- alpha beta + T cell lines derived from the spleen or thymus of neonatal or adult BALB/c mice. Eleven of the lines were cloned and established from six independent cloning procedures from different mice. Five cloned lines used V beta 9, four cloned lines used V beta 15, and two cloned lines used V beta 7. Nucleotide sequencing of the beta-chain genes showed that clones that used a given V beta were identically rearranged even when they were derived from independent cloning procedures. In the case of V beta 7 and V beta 15 all nucleotides in the V-D-J joining region were in the germ line configuration without N region additions. Rearrangements of the V beta 7, V beta 9, and V beta 15 genes were functional. Each V beta 15 clone also had a homogeneous rearrangement of the V beta 13 gene, which was nonfunctional. The predicted amino acid sequence of the joining regions of the V beta 7, V beta 9, and V beta 15 rearrangements showed homology in four of seven amino acids in the peptide contact region.

Multiple interactions between human vitronectin and Staphylococcus aureus.

Multiple interactions between human vitronectin and Staphylococcus aureus strain V8 were observed. An upward-curved Scatchard plot indicated both high-affinity binding (Kd1 = 7.4 x 10(-10) M) with 260 binding sites per bacterial cell and moderate-affinity binding (Kd2 = 7.4 x 10(-8) M) with 5240 copies per cell. Negative cooperativity of this binding was characterized by its Hill coefficient of less than unity (0.70 +/- 0.08). Up to 60% of the vitronectin-bacteria interaction was unaffected by high ionic strength (i.e., 2.4 M NaCl), and was not inhibited by highly-charged heparin oligosaccharides. Various oligosaccharides (4-20 monosaccharide units) generated by partial deaminative cleavage of heparin were found to affect vitronectin binding to S. aureus. Short-chain-length oligosaccharides increase and long oligosaccharides inhibit vitronectin binding, in accordance with direct association of these saccharides with multimeric vitronectin. A protein having a molecular mass of 60 kDa was identified as a putative high-affinity staphylococcal vitronectin-binding protein. These results indicate that interaction of multimeric vitronectin, mostly present at extracellular matrix sites with multiple recognition sites on the S. aureus surface, may contribute to bacterial colonisation.

Binding of collagen, fibronectin, lactoferrin, laminin, vitronectin and heparan sulphate to Staphylococcus aureus strain V8 at various growth phases and under nutrient stress conditions.

We have examined how Staphylococcus aureus strain V8 cells interact with 125I-labelled extracellular matrix (ECM) and serum proteins (collagen type I and IV), fibronectin, lactoferrin, laminin, vitronectin, and heparan sulphate at various phases of the growth cycle. Maximal binding of these glycoproteins and heparan sulphate to the bacteria occurred after 17 to 20 h in the late stationary phase except for fibronectin-binding, which was maximal after 12 to 14 h. Binding of the glycoproteins and heparan sulphate to S. aureus V8 under nutrient stress conditions exhibited complex patterns based on different starving conditions and various binding ligands. In general, bacteria starved in distilled water and 0.02 M potassium phosphate buffer (pH 7.2) at room temperature showed high susceptibility to all binding ligands within the first 18 h, followed by entering a lower binding period (except for collagen-binding which still remained high). The binding was not correlated to cell surface charge or hydrophobicity of the bacteria. Furthermore, extracellular and cell-associated proteolytic activity of starved cells against ECM and serum proteins was found to be greater than for non-starved cells. Thus, S. aureus could sustain its ability to bind various connective tissue and cell surface components during a long period of time even in the absence of energy-yielding substrates.

Vitronectin-binding surface proteins of Staphylococcus aureus.

S. aureus strain ISP 546 was selected (of 55 strains tested) to define optimal conditions for expression of vitronectin binding. High binding was expressed when the strain was grown on blood agar and in Todd-Hewitt broth. Binding was optimal in the 6.0 to 7.2 pH range and was unaffected by divalent cations and ionic strength. Binding was partially inhibited by D-mannose, heparin, types I and IV collagen, fibronectin, fibrinogen and vitronectin, but was not affected by other carbohydrates or glycoproteins tested. Cell surface binding components were extracted with the aid of 1 M LiCl (pH 5.0) from strain ISP 546 grown in Todd Hewitt broth. Vitronectin binding proteins were purified by affinity chromatography on heparin-Sepharose. Fractions inhibiting binding of 125I-labelled vitronectin to strain ISP 546 were eluted by 0.01 M NaOH, dialysed, concentrated and subjected to SDS-PAGE. Silver staining revealed one major band (70 kDa) and two minor bands (34 and 36 kDa).

Binding of heparan sulfate to Staphylococcus aureus.

Heparan sulfate binds to proteins present on the surface of Staphylococcus aureus cells. Binding of 125I-heparan sulfate to S. aureus was time dependent, saturable, and influenced by pH and ionic strength, and cell-bound 125I-heparan sulfate was displaced by unlabelled heparan sulfate or heparin. Other glycosaminoglycans of comparable size (chondroitin sulfate and dermatan sulfate), highly glycosylated glycoprotein (hog gastric mucin), and some anionic polysaccharides (dextran sulfate and RNA) inhibited heparan sulfate binding to various extents. Heat treatment (80 degrees C for 10 min) and treatment of the bacteria with pronase E, proteinase K, pepsin, and chymotrypsin considerably reduced their ability to bind 125I-heparan sulfate, but treatment with trypsin and neuraminidase did not affect binding. Scatchard plot analysis indicated the presence of cell surface components with low affinity (Kd = 3 x 10(-5) M) for heparan sulfate. Cell surface components were released by stirring bacteria with 1 M LiCl at 37 degrees C for 2 h. Proteins of this extract that competitively inhibited binding of 125I-heparan sulfate to S. aureus were isolated by affinity chromatography on heparin-Sepharose. Two proteins having molecular masses of approximately 66 and 60 kDa and the ability to bind 125I-heparan sulfate were obtained. The first 9 amino-terminal amino acid residues of the 66-kDa protein are Asp-Trp-Thr-Gly-Trp-Leu-Ala-Ala-Ala, and the first 4 amino-terminal amino acid residues of the 60-kDa protein are Met-Leu-Val-Thr.

Studies of CD4- CD8- alpha beta bone marrow T cells with suppressor activity.

The predominant T cell subset in the bone marrow of specific pathogen-free C57BL/Ka and BALB/c mice expressed the alpha beta+ TCR CD4- CD8- surface phenotype. Purified C57BL/Ka alpha beta+ TCR CD4- CD8- marrow cells obtained by cell sorting suppressed the MLR of C57BL/Ka responder and BALB/c stimulator spleen cells. Although the percentage of typical T cells in the spleen was markedly reduced in adult nude mice or normal neonatal mice as compared to the normal adult, the percentage of alpha beta+ TCR CD4- CD8- cells in the spleen and marrow was not. The percentage of "self-reactive" V beta 5+ T cells in the BALB/c spleen was markedly reduced as compared to that in the C57BL/Ka spleen. However, the percentages in the bone marrow were similar. The results indicate that the predominant subset of marrow T cells in these pathogen-free mice differ with regard to surface marker phenotype, function, dependence on the adult thymus, and deletion of certain self-reactive V beta receptors as compared to typical spleen T cells. The marrow T cells appear to develop directly from marrow precursors without rearranged beta chain genes during a 48 hour in vitro culture.