CD34+ enriched cell products intended for autologous transendocardial CD34+ cell transplantation release significant amounts of angiopoietin-1.

OBJECTIVES: Cell-based therapy has emerged as a promising strategy for the treatment of patients with heart failure. Increasing evidence supports the hypothesis that paracrine mechanisms mediated by soluble factors released by the cells play a predominate role in reparative processes. The aim of our study was to analyze which cytokines are released by CD34+ enriched cell products intended for autologous transendocardial CD34+ cell transplantation in patients with cardiomyopathy. MATERIAL AND METHODS: The peripheral blood CD34+ cells from 12 patients were mobilized with granulocyte colony-stimulating factor, collected via apheresis and enriched by immunoselection. RESULTS: In CD34+ enriched cell population, hematopoietic, but not mesenchymal or endothelial, progenitors were detected. Except for angiopoietin-1, other measured cytokines (FGF1, FGF2, VEGF, PDGF, IL-6, HGH, SDF-1α/CXCL12, NRG1) were not released by CD34+ cells. The average concentration of angiopoietin-1 released by 5×106 CD34+ cells grown in neutral DMEM medium was 213.6±130.0pg/mL (range: 74-448pg/mL). Angiopoietin-1 secretion correlated well with CD34+ cell's capacity for generating colonies derived from hematopoietic progenitors (Pearson's correlation=0.964; P<0.001). CONCLUSION: Our study presents angiopoietin-1 as an interesting candidate and suggests future studies to explore how its release by CD34+ cells might impact the success of autologous CD34+ cell transplantation.

Evaluation of a flow cytometric assay for the confirmation of heparin-induced thrombocytopenia.

INTRODUCTION: Heparin-induced thrombocytopenia (HIT) is an immune-mediated thrombocytopenia associated with heparin therapy. The diagnosis consists of a combination of pretest probability and laboratory testing. The routinely available laboratory antigen binding assays for the detection of specific antibodies have a low HIT-positive predictive value; therefore, to exclude false-positive results, one of the functional assays should be performed. Functional assays evaluate the ability of heparin/PF4 antibodies to activate the platelets. The aim of our study was to validate the flow cytometric functional assay, based on the use of anti-CD61 and anti-CD62 antibodies, as a suitable diagnostic test for HIT. METHODS: Sera from patients with a clinical suspicion of HIT were previously analyzed with screening IgG-specific ELISA, and 41 of those which were positive were selected for the functional assay. RESULTS: Our results were compared to another functional assay - the HIPA (heparin-induced platelet aggregation assay). The diagnostic specificity of the flow cytometric assay was calculated based on HIPA results and was 83%. CONCLUSION: Performing this functional test after the screening assay could significantly improve the specificity of HIT testing as heparin/PF4 antibodies are often not clinically significant.

Effects of a platelet gel on early graft revascularization after anterior cruciate ligament reconstruction: a prospective, randomized, double-blind, clinical trial.

BACKGROUND: Slow graft healing in bone tunnels and a slow graft ligamentization process after anterior cruciate ligament (ACL) reconstruction are some of the reasons for prolonged rehabilitation. AIMS: The purpose of this study was to determine if the use of platelet gel (PG) accelerates early graft revascularization after ACL reconstruction. METHODS: PG was produced from autologous platelet-rich plasma and applied locally. We quantitatively evaluated the revascularization process in the osteoligamentous interface zone in the bone tunnels and in the intra-articular part of the graft by means of contrast-enhanced magnetic resonance imaging (MRI). RESULTS: After 4-6 weeks, the PG-treated group demonstrated a significantly higher level of vascularization in the osteoligamentous interface (0.33 ± 0.09) than the control group (0.16 ± 0.09, p < 0.001). In the intra-articular part of the graft, we found no evidence of revascularization in either group. CONCLUSION: Locally applied PG enhanced early revascularization of the graft in the osteoligamentous interface zone after ACL reconstruction.

Monoclonal platelet antigen capture assays (MAIPA) and reagents: a statement.

This statement concerning the monoclonal-specific immobilization of platelet antigens (MAIPA) has been written on behalf of the International Society of Blood Transfusion--Working Party on Platelet Immunology. The MAIPA technique is considered as the gold standard reference technique in platelet immunology. The assay performed with reagents labelled for 'research only' is acceptable as long as it is regularly evaluated by participation of laboratories in national or international workshops held with reference laboratories.

Use of platelet growth factors in treating wounds and soft-tissue injuries.

Tissue repair begins with clot formation and platelet degranulation, which release the growth factors (GFs) necessary for wound repair. Platelet-derived GFs are biologically active substances that enhance tissue repair mechanisms such as chemotaxis, cell proliferation, angiogenesis, extracellular matrix deposition, and remodeling. This review describes the biological background of the topical therapy of wounds and soft-tissue injuries with platelet gel (PG) and PG-derived GFs as well as the success of the clinical studies performed so far. Some other interesting topical applications of PG are also described. Platelet-derivatives represent a promising therapeutic modality, offering opportunities for treatment of wounds, ulcers, soft-tissue injuries, and various other applications in regenerative medicine.

Real-time PCR genotyping of human platelet alloantigens HPA-1, HPA-2, HPA-3 and HPA-5 is superior to the standard PCR-SSP method.

Genotyping of the human platelet alloantigens (HPA) is useful for the diagnosis and therapy of the patients with alloimmune thrombocytopenic syndromes, such as post-transfusion refractoriness to platelets, post-transfusion thrombocytopenic purpura and foetomaternal alloimmune thrombocytopenia. We have developed, optimized and validated a new method for simultaneous genotyping of HPAs - HPA-1, HPA-2, HPA-3 and HPA-5 - by using the real-time polymerase chain reaction (PCR) based on TaqMan technology. Its performances were compared to those of the standard PCR-sequence-specific primers (SSP) method by testing 120 DNA samples. Several discrepancies between the two methods have been observed, especially in the HPA-3 genotyping. Evidently, the PCR-SSP method produced several false positive results due to its technical drawbacks. Based on our comparison, we believe that the new real-time TaqMan PCR assay for the HPA-1, HPA-2, HPA-3 and HPA-5 genotyping is faster, more reliable and reproducible, compared to the standard PCR-SSP.

Are human platelet alloantigens (HPA) minor transplantation antigens in clinical bone marrow transplantation?

The role of human platelet alloantigens (HPA) in clinical bone marrow allotransplantation was investigated. The leading hypothesis was that HPA alloepitopes act as minor histocompatibility antigens and aggravate graft-versus-host disease (GVHD). To exclude the effect of MHC disparity, only HLA identical donor-recipient pairs were entered into the study. The influence of HPA compatibility on overall survival, occurrence of relapses and haematopoietic recovery was also investigated. A total of 223 patients who received a graft from an HLA-identical sibling, genotyped for HPA -1, -2, -3, -4 and -5, were observed over a post-transplant period of 24 months following the protocol recommended by EBMT. The data from patients having received grafts from HPA compatible donors were compared to data from patients having received grafts that were mismatched in HPA allotypes in the GVH direction. Analysis of the incidence of acute and chronic (GVHD), overall survival, relapse incidence, haematopoietic recovery and some other clinical parameters did not reveal any significant difference between the HPA-matched and -mismatched groups of patients, regardless of their age. Our results give no evidence that HPA-1, -2, -3 and -5 alloantigens should be considered minor transplantation antigens in clinical bone marrow transplantation.

Soluble tumor necrosis factor receptor I (sTNFRI) as a prognostic factor in melanoma patients in Slovene population.

Tumor necrosis factor alpha (TNF-alpha) and its receptors (TNFRI and TNFRII) which exist in soluble form as a product of cleavage of the extracellular domain of membrane integrated receptors, still rise debate about their importance. It was reported that TNF-alpha has numerous actions in diseases such as inflammation, autoimmunity, infectious diseases, septic shock and many types of cancer [1, 2]. Several authors have reported the significance of sTNFRI level in serum of cancer patients [3, 4]. This study was performed in collaboration with the Institute of Oncology of Slovenia. At least two different mouse monoclonal antibodies (MAbs) against human sTNFRI have been prepared to obtain a sensitive and reliable sandwich ELISA. It was compared with commercially available R&D and Endogen ELISAs for the determination of sTNFRI. Groups of patients with different stages of melanoma and epithelial ovarian carcinoma were tested and their clinical records were reexamined. Levels of sTNFRI were measured and compared with the normal serum levels of sTNFRI.

Weak D and partial D in Slovenian population through serology and genotyping.

Weak D red cell phenotype (formerly D(II)) exhibits weaker serological reaction with anti-D antibodies. Weak D occurs in 0.2% to 1% of whites and is caused by qualitatively altered RhD proteins called partial D or normal, only weakly expressed RhD proteins that are called weak D. Partial D genes are hybrid alleles between RHD an RHCE genes. 23 partial RHD alleles are described. Weak D phenotypes with reduced expression are likely to possess the normal RHD gene, but the latest findings indicate that weak D alleles carry at least one point mutation. The aim of the present work was to answer an important question how to approach partial and weak D identification in diagnostic use and if it is possible to distinguish between partial D and weak D using commercially available anti-D reagents for routine use. We also wanted to evaluate D-screen kit for partial D identification. We compared phenotypes identified by serological testing and genotypes identified by RHD Multiplex PCR and D(VII) specific ASPA PCR. Our results showed that it is not possible to distinguish between partial and weak D using commercially available anti-D reagents for routine use. D-screen proved to be useful for D(VI) and D(VII) identification, whereas for partial D(DFR) identification we must look for another set of monoclonal antibodies or simply use genotyping methods. In 44 samples with not interpretable serological results out of 80 we found all RHD specific exons present and we classified the samples as weak D. Fourteen types of weak D with at least one point mutation were recently proposed. Designing of allele specific PCRs for identification of proposed types of weak D is in progress.

Differentiation of autologous ABO, RHD, RHCE, KEL, JK, and FY blood group genotypes by analysis of peripheral blood samples of patients who have recently received multiple transfusions.

BACKGROUND: After multiple transfusions, the serologic typing of autologous blood group phenotypes is difficult, because of mixed RBC populations. The genotyping of ABO, Rh, Kell, Kidd, and Duffy systems could be used to determine autologous blood group antigen status. STUDY DESIGN AND METHODS: Blood samples from patients and donors were analyzed before and after 26 multiple-transfusion events. An average of 6.9 non-WBC-reduced RBC units with an average age of 5.9 days were administered per transfusion event. The average period of blood sampling after transfusions was 5.3 days. All samples were serologically phenotyped for ABO, Rh, Kell, Kidd, and Duffy. Pretransfusion, posttransfusion, and buccal samples from patients were genotyped for the corresponding alleles by a uniform PCR sequence-specific primer protocol that allowed their simultaneous determination within 3 hours. RESULTS: All posttransfusion samples exhibited mixed-cell populations of various blood group systems on serologic testing. Genotyping from peripheral blood produced results identical to the autologous blood group phenotypes, regardless of the amount of blood transfused or of the length of the sampling period after transfusion. CONCLUSION: A fast and reliable PCR-sequence-specific primer DNA genotyping assay for simultaneous determination of autologous ABO, Rh, Kell, Kidd, and Duffy blood groups can be performed on peripheral blood samples, even though the patients have recently received multiple transfusions.

A case of pseudothrombocytopenia after infusion of abciximab in vivo and anticoagulant-independent platelet clumping after rechallenge with abciximab in vitro.

A 45-year old man was treated for unstable angina pectoris with percutaneous transluminal angioplasty and stenting of his left anterior descending coronary artery. The procedure was followed by infusion of abciximab. The patient's automated platelet count in an EDTA-anticoagulated blood sample at admission to the hospital was normal, but dropped to 5 x 10(9)/l three hours after the procedure. The infusion of abciximab was stopped and the patient received platelet transfusions although there were no signs of bleeding. Two days later his platelet count was still low (37 x 10(9)/l) in an EDTA-anticoagulated blood sample, but normal (193 x 10(9)/l) in a heparin-anticoagulated sample. Platelet clumps were present only in the sample anticoagulated with EDTA, and pseudothrombocytopenia was diagnosed. The patient's recovery was uneventful. At follow-up visits four months and one year after discharge from hospital, the patient's blood samples were anticoagulated with three different anticoagulants: EDTA, citrate and heparin. The platelet count was normal in all three samples but after mixing with abciximab in vitro it dropped profoundly due to platelet clumping, regardless of the choice of the anticoagulant. Our report raises two points: (a) one needs to consider the possibility of pseudothrombocytopenia in a patient with a low automated platelet count after infusion of abciximab but without signs of bleeding, and (b) the in vitro results suggest that our patient who had initially responded to abciximab with pseudothrombocytopenia could develop true thrombocytopenia after repeated exposure.

Rhd and C/cE/e genotyping in Slovenian population.

The Rhesus (Rh) blood group system is, after ABO, clinically most important. Alloantibodies directed against Rh antigens are the major cause of a haemolytic disease of newborn (HDN) and of transfusion reactions. In search for novel methods for Rh genotyping we started to compare Rh genotypes identified from different tissues and Rh phenotypes. Genotypes determined from blood samples with PCR based RhD, C/c and E/e genotyping methods were compared with serologically identified phenotypes (N=32). With two exceptions the results of phenotyping and genotyping were in concordance. Two Rh serotypes from a Slovenian family that were unexpected according to the Mendelian laws were characterised genotypically. The two family members were suspected to have a chromosomal deletion on RH gene locus.

Genotyping for human platelet-specific antigens HPA-1, -2, -3, -4 and -5 in the Slovenian population reveals a slightly increased frequency of HPA-1b and HPA-2b as compared to other European populations.

Typing of human platelet alloantigens (HPA) is necessary in various clinical situations. The purpose of this study was to type a random sample of the Slovenian population for HPA alleles, in order to obtain genetic population data. A total of 152 unrelated Slovenian blood donors were genotyped for HPA-1, -2, -3, -4 and -5 alleles using a simple method that enables simultaneous and complete determination of HPA genotypes. Ten different polymerase chain reactions employing sequence-specific priming (PCR-SSP), which worked in identical cycling conditions, were used. The allele frequencies were 0.809 for HPA-1a, 0.191 for HPA-1b, 0.891 for HPA-2a, 0.109 for HPA-2b, 0.591 for HPA-3a, 0.407 for HPA-3b, 0.997 for HPA-4a, 0.00 for HPA-4b, 0.934 for HPA-5a and 0.066 for HPA-5b. When compared to results of studies of various other Caucasian populations, our population displayed a slightly but not significantly higher proportion of the HPA-1b and 2b alleles.

Cultivation of mouse-mouse hybridomas producing IgM monoclonal antibodies.

The mouse-mouse hybridomas producing monoclonal antibodies (MAbs) for ABO blood group determination were cultivated for 10 days in 25 cm2 Roux bottles using standard cultivation medium (DMEM) with different foetal-calf serum (FCS) concentrations (2-13%). The highest specific production rates (200-1100 micrograms/10(6) cells/day) for MAbs were measured at the end of the cultivation: this phenomenon could be explained by advanced cell death and liberating the content of the cells into the medium.

Acetaminophen does not decrease hepatic 3'-phosphoadenosine 5'-phosphosulfate in mice.

Capacity-limited sulfation of chemicals is thought to be due to the limited availability of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the cosubstrate for sulfation, which in turn is limited by the availability of its precursor, inorganic sulfate. Because this concept evolved from experimental data obtained from rats, and species differences have also been reported on acetaminophen (AA) sulfation, this study examined the effects of AA on PAPS and sulfate concentrations in mice, another widely used experimental animal. AA lowered serum and liver sulfate concentrations approximately 50% in mice. However, contrary to observations in rats, AA (0-600 mg/kg i.p.) did not decrease hepatic PAPS concentrations in mice. In summary, these studies demonstrate that AA decreases serum and liver sulfate concentrations, but does not decrease hepatic PAPS concentrations in mice. These data indicate that 1) hepatic sulfation of high dosages of AA in mice is not limited by the availability of PAPS, and 2) there are significant species differences in the regulation of AA sulfation between rats and mice.

Sulfation of acetaminophen and acetaminophen-induced alterations in sulfate and 3'-phosphoadenosine 5'-phosphosulfate homeostasis in rats with deficient dietary intake of sulfur.

Sulfation of drugs depends on the availability of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), which requires inorganic sulfate for its synthesis. Therefore, decreased alimentary intake of inorganic sulfate or its precursor, cysteine, may compromise sulfation of xenobiotics. To test this hypothesis, separate groups of rats were maintained for 5 days on synthetic diets, which lacked sulfate, or cysteine, or both sulfate and cysteine. These dietary restrictions did not cause growth retardation or depletion of glutathione in liver. Under anesthesia, the animals were injected with acetaminophen (0.5 mmol/kg, i.v.) and elimination of acetaminophen from blood and excretion of acetaminophen metabolites in urine and bile was simultaneously quantified. Deficient intake of inorganic sulfate or cysteine alone did not significantly change elimination and biotransformation of acetaminophen. Combined nutritional deficiency of sulfate and cysteine, however, resulted in a 40% reduction in the excretion of acetaminophen-sulfate, quantitatively the most significant metabolite. Concomitantly, these animals eliminated acetaminophen from blood at a slower rate and converted more acetaminophen to its toxic intermediate, as indicated by increased excretion of acetaminophen-thioether conjugates. Serum and tissue sulfate concentrations were decreased to significantly lower levels in rats on sulfate and cysteine deficient diets, than in rats with a sufficient sulfur supply. Thus, reduced sulfation is apparently caused by diminished availability of inorganic sulfate for PAPS synthesis, even though hepatic and renal PAPS levels were not depleted more by acetaminophen in rats with deficient dietary supply of sulfate and cysteine than in rats with adequate sulfur intake.(ABSTRACT TRUNCATED AT 250 WORDS)

Homeostasis of sulfate and 3'-phosphoadenosine 5'-phosphosulfate in rats after acetaminophen administration.

Acetaminophen (AA) is a drug whose biotransformation by sulfation is easily saturated. We have previously demonstrated in rats that its dose-dependent kinetics appear to be due to depletion of 3'-phosphoadenosine 5'-phosphosulfate (PAPS). In order to determine if the depletion of PAPS might be due to a lack of inorganic sulfate, we characterized the effect of AA not only on the homeostasis of PAPS but also on its precursor, sulfate. The maximum excretion of AA-sulfate was observed after 75 mg/kg of AA, i.p., and higher dosages did not increase its excretion. AA dosages between 150 to 600 mg/kg, i.p., 2 hr after dosing depleted 60 to 80% of hepatic PAPS. Hepatic PAPS levels returned to control values 16 to 20 hr after dosing with 600 mg/kg of AA. AA decreased serum sulfate to a similar degree (80%) and duration (16 hr) as did hepatic PAPS. AA also lowered sulfate concentrations in liver, but to a somewhat lesser extent (65%) than in serum. Hepatic sulfate levels returned to control values at 16 to 24 hr after dosing with AA. Even though AA did not alter renal PAPS concentrations, it did produce a 65% decrease in the renal sulfate levels. In summary, these studies demonstrate that AA markedly depletes PAPS concentrations in liver, but not in kidney, and drastically decreases serum and tissue sulfate concentrations. Our findings indicate that the capacity-limited sulfation of AA is due to the limited availability of hepatic PAPS, which in turn is limited by the availability of sulfate in liver.

Homeostasis of sulfate and 3'-phosphoadenosine 5'-phosphosulfate in rats with deficient dietary intake of sulfur.

This study was designed to determine the role of dietary organic and inorganic sulfur on 3'-phosphoadenosine 5'-phosphosulfate (PAPS) homeostasis. Organic sulfur was altered by adding various amounts of methionine (0.15, 0.3, 0.6, or 1.2%) to a sulfhydryl-deficient diet. Inorganic sulfur was altered by providing rats with no sulfate or sulfate in their diets (0.12%) and distilled or tap water. Rats received these diets for 5 days. The two lowest methionine-containing diets produced a 60% reduction in liver glutathione concentrations, and the addition of sulfate to the diets did not restore hepatic glutathione levels. Urinary sulfate excretion was reduced by 95% in rats fed the three low-methionine diets. Addition of sulfate to these diets increased the urinary excretion of sulfate, but did not return sulfate levels to control values. The three low-methionine-containing diets decreased serum and liver sulfate concentrations about 50% and addition of sulfate to these diets largely restored them to control levels. Hepatic PAPS concentration was decreased (10%) only in the group receiving the lowest methionine content in their diet, and addition of sulfate had no effect on hepatic PAPS. In summary, dietary alterations of sulfur lowered the glutathione concentration in the liver as well as decreased sulfate levels in serum, liver, and urine, but had minimal effect on hepatic PAPS concentrations. Therefore, it appears that hepatic steady-state PAPS levels are not highly dependent on the sulfur content of the diet.