Effects of bone marrow or mesenchymal stem cell transplantation on oral mucositis (mouse) induced by fractionated irradiation.

BACKGROUND AND PURPOSE: Oral mucositis is a severe and dose limiting early side effect of radiotherapy for head-and-neck tumors. This study was initiated to determine the effect of bone marrow- and mesenchymal stem cell transplantation on oral mucositis (mouse tongue model) induced by fractionated irradiation. MATERIAL AND METHODS: Daily fractionated irradiation (5 × 3 Gy/week) was given over 1 (days 0-4) or 3 weeks (days 0-4, 7-11, 14-18). Each protocol was terminated (day 7 or 21) by graded test doses (5 dose groups, 10 animals each) in order to generate complete dose-effect curves. The incidence of mucosal ulceration, corresponding to confluent mucositis grade 3 (RTOG/EORTC), was analyzed as the primary, clinically relevant endpoint. Bone marrow or mesenchymal stem cells were transplanted intravenously at various time points within these fractionation protocols. RESULTS: Transplantation of 6 × 10(6), but not of 3 × 10(6) bone marrow stem cells on day - 1, + 4, + 8, + 11 or + 15 significantly increased the ED50 values (dose, at which an ulcer is expected in 50 % of the mice); transplantation on day + 2, in contrast, was ineffective. Mesenchymal stem cell transplantation on day - 1, 2 or + 8 significantly, and on day + 4 marginally increased the ED50 values. CONCLUSION: Transplantation of bone marrow or mesenchymal stem cells has the potential to modulate radiation-induced oral mucositis during fractionated radiotherapy. The effect is dependent on the timing of the transplantation. The mechanisms require further investigation.

[Von Willebrand disease type 1 and pregnancy]. / Von-Willebrand-Syndrom Typ 1 und Schwangerschaft.

The von Willebrand-Jürgens syndrome (VWJS) type 1 is a common hereditary bleeding disorder with a bleeding tendency located especially in the mucous membranes. Women suffering from VWJS type 1 show menorrhagia and prolonged postoperative bleedings. During pregnancy the clinical presentation varies by the increase of the von Willebrand factors. In this article the laboratory findings and the clinical presentation of patients with VWJS during pregnancy was examined. The necessity of interventions during pregnancy and at the time of delivery was under consideration.

Variability of cut-off values for the detection of lupus anticoagulants: results of an international multicenter multiplatform study.

Essentials Between-lab variations of cut-off values in lupus anticoagulant detection are unknown. Cut-off values were calculated in 11 labs each testing plasma from 120 donors with 3 platforms. Major variation was observed even within the same platform. Cut-off values determined in different labs are not interchangeable. SUMMARY: Background Cut-off values for interpretation of lupus anticoagulant (LA) detection are poorly investigated. Aims (i) To assess whether results from healthy donors were normally distributed and (ii) the between-laboratories differences in cut-off values for screening, mixing and LA confirmation when calculated as 99th or 95th centiles, and (iii) to assess their impact on the detection rate for LA. Methods Each of 11 laboratories using one of the three widely used commercial platforms for LA detection was asked to collect plasmas from 120 healthy donors and to perform screening, mixing and LA confirmation with two methods (activated partial thromboplastin time [APTT] and dilute Russell viper venom [dRVV]). A common set of LA-positive or LA-negative freeze-dried plasmas was used to assess the LA detection rate. Results were centralized (Milano) for statistical analysis. Results and conclusions (i) Clotting times or ratios for healthy subjects were not normally distributed in the majority of cases. The take-home message is that cut-off values should be determined preferably by the non-parametric method based on centiles. (ii) There were relatively large inter-laboratory cut-off variations even within the same platform and the variability was marginally attenuated when results were expressed as ratios (test-to-normal pooled plasma). The take-home message is that cut-off values should be determined locally. (iii) There were differences between cut-off values calculated as 99th or 95th centiles that translate into a different LA detection rate (the lower the centile the greater the detection rate). The take-home message is that cut-off values determined as the 95th centile allow a better LA detection rate.

Evaluation of the pharmacokinetics of dalteparin in patients with renal insufficiency.

The pharmacokinetics of the low-molecular weight heparin (LMWH), dalteparin, was evaluated after a single intravenous bolus injection of 50 IU anti-Xa/kg in 8 healthy volunteers, 8 patients with moderate/severe renal failure (Cl(crea) 13.1-56.5 ml/min) and 8 hemodialysis patients. Venous blood samples were taken over a 1-day period to determine anti-Xa activity, anti-IIa activity and plasma levels of free tissue factor pathway inhibitor (free TFPI). Plasma anti-Xa and anti-IIa activities were measured using chromogenic assays and free TFPI levels using an ELISA technique. The anti-Xa clearance was significantly decreased (p < 0.05) in both groups with renal insufficiency when compared with healthy volunteers. There was a positive correlation between creatinine clearance and anti-Xa clearance in the healthy volunteers and patients with moderate/severe renal failure. The anti-Ila activity was characterized by 3- to 4-fold lower plasma concentrations and faster elimination compared with the anti-Xa activity. In patients with moderate/severe renal failure the elimination of anti-lla was only slightly decreased, whereas in hemodialysis patients anti-Ila clearance was significantly decreased (p < 0.01). There was no correlation between creatinine clearance and anti-IIa clearance. The baseline mean free TFPI plasma levels in the two groups with renal insufficiency were significantly higher (p < 0.01) than in healthy volunteers. Dalteparin administration induced a transient, 6.0- to 8.1-fold increase in the free TFPI values in the three study groups. Dalteparin induced an increase in C(max) and AUC(0 - infinity) values of free TFPI in the two groups with renal insufficiency that was higher than in healthy volunteers. No bleeding complications occurred during the study. In conclusion, this is the first report showing retarded elimination of dalteparin and enhanced free TFPI plasma levels induced by a LMWH in patients with renal insufficiency.

Impact of the Thr789Ala variant of the von Willebrand factor levels, on ristocetin co-factor and collagen binding capacity and its association with coronary heart disease in patients with diabetes mellitus type 2.

A Thr789Ala variant in the von Willebrand Factor (vWF) gene is associated with increased vWF plasma concentrations and might therefore affect the risk of coronary heart disease (CHD) in the general population. Patients with type 2 diabetes have an increased risk for premature atherosclerosis and are characterized by alterations of the coagulation system. However, it is not known whether the Thr789Ala variant in the vWF gene contributes to the increased CHD risk in patients with type 2 diabetes. We therefore investigated the potential relationship between the Thr789Ala variant in the vWF gene and the occurrence of CHD in 356 patients with type 2 diabetes, either with (DM+/CHD+, n = 204) or without evidence for CHD (DM+/CHD-, n = 152). In addition, two control groups without type 2 diabetes, with (DM-/CHD+, n = 22) or without CHD (DM-/CHD-, n = 100), were investigated. Individuals with the vWF Thr789Ala variant have significantly higher von Willebrand factor plasma concentrations (p < 0.001). In addition, ristocetin co-factor was significantly increased in vWF Thr789Ala variant carriers (p < 0.05). Ristocetin co-factor levels and collagen binding capacity were also increased in individuals affected with either type 2 diabetes, CHD or both (DM+/CHD+, DM+/CHD-, DM-/CHD+) as compared to healthy controls (DM-/CHD-) (p < 0.001). However, we did not find an association between the vWF Thr789Ala variant and the occurrence of CHD in patient with type 2 diabetes (p = 0.34). In conclusion, although the Thr789Ala vWF gene variant is associated with increased plasma concentrations of vWF, ristocetin co factor levels and collagen binding capacity in patients with type 2 diabetes and CHD, a direct effect of this variant on the occurrence of CHD in patients with type 2 diabetes, could not be detected.

Genetic predisposition to bleeding during oral anticoagulant therapy: evidence for common founder mutations (FIXVal-10 and FIXThr-10) and an independent CpG hotspot mutation (FIXThr-10).

The recent discovery of five patients with coumarin sensitive FIX-variants due to a missense mutation in the FIX propeptide, either Ala-10Val or Ala-10Thr, has highlighted a novel type of genetic predisposition to bleeding during oral anticoagulant therapy (OAT). In the present study, we report six additional patients with such FIX variants. Haplotype analysis of FIX polymorphisms revealed a founder effect in the five German and Swiss patients with the Val-10 variant. Also, four Thr-10 variants detected in Germany, Switzerland and Great Britain derived from a common founder. Two Thr-10 variants from USA showed an independent de novo origin at a CpG dinucleotide that in general represents a mutation hotspot. These findings implicate the existence of additional subjects with corresponding variants in the populations of various countries. Even though the rare occurrence of these variants does not justify a general aPTT screening during OAT, it is recommended to monitor each bleeding event during OAT in males in order to exclude a genetic predisposition to bleeding by means of the following testing strategy: a) aPTT-testing in each bleeding complication of male patients during OAT, b) if aPTT is disproportionately prolonged, determination of FIX:C, and c) if FIX:C is disproportionately decreased as compared to FII:C, FVII:C and FX:C, sequencing of exon 2 of the FIX gene. This strategy will provide a cost-effective and safe procedure to identify patients that carry the FIX variants. Moreover, such a strategy accumulates data about the prevalence of these FIX mutations in a given population.

The interleukin-1 polymorphism, smoking, and the risk of periodontal disease in the population-based SHIP study.

Several studies have shown a role for interleukin-1 gene cluster polymorphisms in the risk assessment for periodontal diseases. In the Study of Health in Pomerania (SHIP), 3148 subjects were randomly selected from the population and assessed for a broad range of diseases and environmental/behavioral risk factors. From the complete study group in the age 40 to 60 years, N = 1085 subjects were genotyped for the interleukin-1 genotype composite polymorphism in relation to periodontal parameters. The study objective was to elucidate the gene-environment interaction between the risk factors smoking and IL-1 polymorphism. An increased risk of periodontal disease was found for IL-1 genotype-positive smokers: odds ratio adjusted for age, sex, education, and plaque OR = 2.50 (95% C.I. 1.21 to 5.13; p = 0.013). This was not the case with subjects who never smoked: OR = 1.09 (0.73-1.62; p = 0.676). These results support the hypothesis of gene-environmental interaction in periodontitis.

Factor XIII deficiency and postoperative hemorrhage after neurosurgical procedures.

BACKGROUND: Factor XIII is of physiological importance for hemostasis, especially in patients undergoing surgery. It catalyzes the enzymatic cross-linking of fibrin monomers into stable polymers and protects polymers from plasmatic and nonspecific degradation. Postoperative hemorrhage in patients with congenital and acquired Factor XIII deficiencies has been described in various surgical fields. However, there are no data about the incidence and clinical relevance of decreased Factor XIII after neurosurgical procedures. The objective of our study was to investigate the association between Factor XIII deficiency and postoperative hemorrhage after intracranial surgery. METHODS: A total of 1264 patients who underwent intracranial operations were reviewed retrospectively. Standard coagulation parameters were monitored during the perioperative course in all patients. Factor XIII testing was performed postoperatively in 34 patients in whom coagulopathies were suspected despite normal platelets, fibrinogen, prothrombin, and partial thromboplastin time. Data were analyzed to evaluate the association of Factor XIII deficiency and major postoperative hemorrhage. RESULTS: In this series of 1264 patients, a total of 20 patients (1. 6%) suffered from a major postoperative hemorrhage. Of the 34 patients with suspected coagulopathies and postoperative Factor XIII testing, 11 had a major postoperative hemorrhage. Normal levels of Factor XIII, defined as more than 60%, were found in 26 of the 34 patients. Factor XIII deficiency, defined as less than 60%, was found in eight patients. All patients with Factor XIII deficiency (n = 8) had a major postoperative hemorrhage. Of the remaining 26 patients with normal Factor XIII levels only three had a postoperative hemorrhage (p < 0.00001, Fisher's exact test). CONCLUSIONS: Decreased Factor XIII activity may be associated with an increased risk of postoperative hemorrhage after intracranial surgery.

[Recurrent coumarin necrosis in type II protein S deficiency]. / Rezidivierende Cumarinnekrose bei Protein S-Mangel vom Typ II.

Coumarin necrosis is a rare but clinical very important complication of therapy with coumarin derivatives. We report a patient with congenital protein S deficiency type II, who developed coumarin necrosis during stabilization of phenprocoumon. Diagnostic problems and therapeutic alternatives are discussed considering the recent literature.

[Activated protein C resistance in patients with central retinal vein occlusion in comparison to patients with a history of deep-vein thrombosis and a healthy control group]. / APC-Resistenz bei Patienten mit Zentralvenenverschluss. Vergleich mit Patienten mit tiefer Becken- un Veinvenenthrombose und gesunder Kontrollgruppe.

BACKGROUND: The recently described "APC resistance" caused by a mutant form of factor V (factor V Leiden) is the most frequent cause of hereditary thrombosis. This study was carried out to investigate the association between activated protein C resistance and central retinal vein occlusion (CRVO). We evaluated the prevalence of APC resistance in patients with CRVO, patients with a history of deep-vein thrombosis, and a healthy control group. PATIENTS AND METHODS: We examined 107 patients with CRVO, 112 patients with deep-vein thrombosis and 70 healthy individuals. The test performed was a modified APC-resistance assay using factor V-deficient plasma. RESULTS: We identified APC resistance in 5.6 % of patients with CRVO and in 5.7 % of the control group. All carriers were heterozygous. In the deep-vein thrombosis group 23.2 % tested positive for APC resistance. Four patients were homozygous and 22 were heterozygous carriers. CONCLUSION: These results indicate that APC resistance has no major role in the pathogenesis of CRVO. Routine testing for the presence of factor V Leiden mutant in CRVO is not necessary.

Clot waveform analysis in patients with haemophilia A.

UNLABELLED: Clot waveform analysis extends the interpretation of aPTT measurement curves. The curve is mathematically processed to obtain information about fibrin formation kinetics including semiquantitative determination of thrombin, prothrombinase and tenase activity. PATIENTS, METHOD: In this study the feasibility of clot waveform analysis for monitoring of haemophilia A was investigated using blood samples from healthy controls as well as haemophilia A patients under various clinical conditions. RESULTS: Thrombin, prothrombinase and tenase activity show a high correlation to factor VIII levels. Tenase activity was found to exhibit a linear relationship to factor VIII levels over a very large concentration range and was able to discriminate patients with severe, moderate and mild haemophilia. CONCLUSION: Clot waveform analysis is an easy, fast and cheap method to access disturbances in clot formation and can be done without any additional measurements beside an aPTT.

[Diagnosis of inherited diseases of platelet function. Interdisciplinary S2K guideline of the Permanent Paediatric Committee of the Society of Thrombosis and Haemostasis Research (GTH e. V.)]. / Diagnose angeborener Störungen der Thrombozytenfunktion. Interdisziplinäre S2K-Leitlinie der Ständigen Kommission Pädiatrie der Gesellschaft für Thrombose- und Hämostaseforschung e. V.

Congenital disorders of platelet function are a heterogeneous group of disorders that are often not detected until bleeding occurs. In clinical settings only a few methods have proven to be useful for identification and classification of inherited platelet disorders. For a rational diagnostic approach, a stepwise algorithm is recommended. Patient history and clinical investigation are mandatory. Von Willebrand disease and other coagulation disorders should always be ruled out prior to specific platelet testing. Platelet count, size, volume (MPV) and morphology may guide further investigations. The PFA-100® CT is suited for screening for severe platelet defects. Platelet aggregometry allows assessment of multiple aspects of platelet function. Flow cytometry enables diagnosis of thrombasthenia Glanzmann, Bernard-Soulier syndrome and storage pool defects. Molecular genetics may confirm a putative diagnosis or pave the way for identifying new defects. We present an unabridged version of the interdisciplinary guideline.

[Therapy of inherited diseases of platelet function. Interdisciplinary S2K guideline of the Permanent Paediatric Committee of the Society of Thrombosis and Haemostasis Research (GTH e. V.)]. / Therapie hereditärer Thrombozytopathien. Interdisziplinäre S2K-Leitlinie der Ständigen Kommission Pädiatrie der Gesellschaft für Thrombose- und Hämostaseforschung e. V.

Inherited disorders of platelet function are a heterogeneous group. For optimal prevention and management of bleeding, classification and diagnosis of the underlying defect are highly recommended. An interdisciplinary guideline for a diagnostic approach has been published (AWMF # 086-003 S2K; Hämostaseologie 2014; 34: 201-212). Underlying platelet disorder, platelet count, age and clinical situation modify treatment. Exclusive transfusion of platelet concentrates may be inappropriate as potentially adverse effects can outweigh its benefit. A stepwise and individually adjusted approach for restitution and maintenance of haemostasis is recommended. Administration of antifibrinolytics is generally endorsed, but is of particular use in Quebec disease. Restricted to older children, desmopressin is favourable in storage pool disease and unclassified platelet disorders. Although licensed only for patients with Glanzmann thrombasthenia and alloantibodies, in clinical practice rFVIIa is widely used in inherited platelet disorders with severe bleeding tendency. This guideline aims at presenting the best available advice for the management of patients with inherited platelet function disorders.

[Cardiac surgery in severe haemorrhagical diseases]. / Kardiochirurgische Eingriffe bei schweren Blutungsdiathesen.

Cardiovascular diseases are the most common disorder in the developed countries. Invasive cardiological and cardiosurgical techniques are known therapies. Yet, patients with severe hereditary haemorrhagical diseases (haemophilia, rare deficiencies of coagulation factors) have an increased bleeding risk by the use of anticoagulants. Therefore, the treatment of these patients requires a concomitant therapy. This article shows eight patients with a severe bleeding diathesis and cardiosurgical interventions in the years 2006 to 2011. This case report shall demonstrate that an adequate therapy can be accomplished with the help of a good cooperation between haemostaseologists and colleagues of the cardioinvasive/cardiosurgical disciplines.

Standardisation of thrombin generation test--which reference plasma for TGT? An international multicentre study.

We have previously shown that standardisation and normalization of results improve the intercentre variability of the calibrated automated thrombin generation test (TGT). We suspected that the source of reference plasma (RP) might be a contributing factor to variability and compared 5 commercial RP and a RP provided by the NIBSC, in an international, multicentre study. The detailed composition of the 6 tested plasma samples was determined in the Haemostasis Laboratory in Lyon. The lot to lot consistency, intra-assay, inter-assay variability were calculated for all tested plasmas. The RP and 3 plasma samples (a normal control, a hypocoagulable and a hypercoagulable plasmas) were tested over 6 days, in 5 European centres. Results were normalised against each of the tested RP and intercentre variability of results was compared. All laboratories used the same reagents. Before normalization, the inter-centre variability was 19.8 to 27.3%. After normalization, we observed a significantly improved inter-laboratory variation with all tested RP, despite differences between them. These results clearly demonstrate that the inter-centre variability of TGT can be significantly reduced by using a reference plasma normalization, and that certain RP have a better capacity to reduce this variability than others.

Aspirin resistance in secondary stroke prevention.

BACKGROUND: We investigated the platelet function in stroke patients treated with aspirin [acetylsalicylic acid (ASA)] for secondary stroke prevention during a follow-up period of 1 year. METHODS: In this prospective study 291 patients with first initiated aspirin therapy (300 mg/day) for secondary stroke prevention were included. Platelet aggregation measurements were performed 24 h, 3, 6, and 12 months after starting medication. RESULTS: Twenty-one of 291 patients (7.2%) were identified as primary ASA-non-responders (initial insufficient platelet inhibition) and 4.1% as secondary ASA-non-responders (insufficient platelet inhibition during follow-up). There were no significant differences between ASA-responders and ASA-non-responders concerning age, gender, risk factors, and stroke characteristics. CONCLUSION: Aspirin resistance in stroke patients is not uncommon. The clinical usefulness of routine platelet function tests needs to be proved by further trials.

Platelet-dependent coagulation assays for factor VIII efficacy measurement after substitution therapy in patients with haemophilia A.

FVIII therapy for haemophilia A is safe and effective, with the problem of individually sufficient efficacy unsettled. Routine one-stage clotting assays and tests employing chromogenic substrates poorly detect individual haemostatic effects of FVIII due to artificial test conditions. In particular, the use of cell-free and diluted plasma samples neglect the crucial role of platelets for thrombin and fibrin formation. To optimize FVIII substitution therapy, we measured in 40 patients with severe to mild haemophilia A before and after FVIII substitution the FVIII activity in cell-free plasma samples using a one-stage clotting assay as well a chromogenic substrate assay and compared the data with those obtained with cell-based coagulation tests, i.e. thrombin generation in platelet-rich plasma (PRP) and thromboelastography (TEG) in samples of citrated whole blood (WB). To determine the maximum ex vivo haemostatic effect we added 1 unit/ml of FVIII to samples of PRP and WB and measured the maximum thrombin generation in the thrombin generation test (TGT) and the maximum clot firmness (MCF) in TEG. After FVIII substitution we observed a nearly linear relation between the individual FVIII activities administered to the patients and the activities measured in the plasma samples. However, data obtained with TGT and TEG revealed a high inter-individual variation and a very poor correlation to the administered FVIII activity. Actually, it could be shown that FVIII substitution yielding in a FVIII plasma activity of about 30% is sufficient to get an ex vivo haemostatic effect of more that 90% as measured by maximum thrombin generation and MCF. FVIII substitution up to a plasma activity of more than 90% did not further enhance the haemostatic effect. Our data clearly demonstrate that the haemostatic effect of FVIII is not only dependent on the activity that is measured in plasma but also depends on the interplay between coagulation and blood cells, in particular with platelets. The use of cell-based coagulation tests such us TGT or TEG may help to optimize FVIII therapy by determining the individual FVIII dosage that produces a maximum haemostatic effect.

Influence of ethanol pretreatment of differing duration on toxic effects of carbon tetrachloride in rats.

Serum activities of alanine-aminotransferase (ALAT, EC 2.6.1.2), aspartate-aminotransferase (ASAT, EC 2.6.1.1), lactate dehydrogenase (LDH, EC 1.1.1.27), and alkaline phosphatase (AP, EC 3.1.3.1) were increased significantly after a dose of 0.16 g/kg/b. w. (ip.) carbon tetrachloride (tetrachloromethane) in rats pretreated with 10% (v/v) ethanol for one and 10 weeks in comparison with water/carbon tetrachloride-treated animals. At the end of 30 and 52 weeks of ethanol consumption these levels were very slightly increased or not detectable. Ethanol treatment alone did not cause an increase in serum enzyme activities or histological liver damage, but caused a diminished intake of fluid and food and in some cases also a reduction of weight gain in the animal body. Significant decrease in body weight after carbon tetrachloride was more evident in rats pretreated with ethanol (1 week greater than 10 greater than or equal to 52 weeks) than in water drinking animals, the lethality caused by carbon tetrachloride was also higher after one and 10 weeks than after 30 to 52 weeks of ethanol pretreatment. The results indicate a decrease of carbon tetrachloride toxicity with increased duration of ethanol pretreatment. This phenomenon could be attributed to reduced sensibility to those alcohol effects which are responsible for increase of carbon tetrachloride toxicity.

Serum enzymes in toxicity of trichloroethylene after subchronic ethanol pretreatment.

In order to establish evidence of serum enzyme activities in toxicological long-term experiments alterations of alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) in the serum of rats were investigated after subchronic ethanol pretreatment and following trichloroethylene exposure. Somewhat lower enzyme activities were found in ethanol treated animals than in those who only got water in nearly all cases. Significant ALAT and ASAT decreases occurred after giving higher ethanol concentrations (5% and 10%, v/v) for 30 weeks. It is possible that this fact among other things could be responsible for the only slight enzyme elevations after trichloroethylene in long-term ethanol pretreated rats.