Biomedical subjects

R Mischke

Publications and source records attributed to R Mischke.

At least 37 records · Page 2Linked to original sources

PubMed2002

Investigations on blood coagulation in the green iguana (Iguana iguana).

The prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time, kaolin clotting time (KCT), dilute Russell's viper venom time (DRVVT) and reptilase time, as well as five different plasma fibrinogen assays [gravimetry, Jacobsson method (extinction at 280 nm), Millar method (heat precipitation), kinetic turbidometry, Clauss method] and resonance thrombography were performed in 26 clinically healthy green iguanas. All assays were carried out in comparison with pooled normal canine plasma. In iguana plasma, the PT [median (x0.50) = 453-831 s, dependent on the reagent], APTT (x0.50 = 170-242 s, dependent on the reagent), thrombin time (x0.50 = 118 - > 1000 s, dependent on thrombin activity), KCT (x0.50 = 274 s), DRVVT (x0.50 = 349 s) and reptilase time (all samples > 1000 s) were widely scattered at the limit of measurability. Only fibrinogen concentrations measured using the Jacobsson method (x0.50 = 4.40 g/l) correlated well (r = 0.91) with gravimetry (x0.50 = 4.22 g/l). The results of this study indicate a limited suitability and a confined diagnostic significance of the selected methods in the green iguana. This may be caused by the species specificity of certain components of the reagents used, as well as a less optimal test system, i.e. relationship of test reagent to clotting factor concentrations in iguana plasma.

Animals↗

PubMed2002

Use of immunocytochemical techniques in canine melanoma.

The staining patterns of the monoclonal antibodies S-100 and Melan-A in canine melanoma were assessed based on cytological specimens of six canine melanomas (four benign, two malignant). In addition, eight regional lymph nodes of the two dogs with malignant melanomas were stained using these markers. For reference, all specimens were also evaluated immunohistochemically using S-100 and Melan-A. To assess the immunocytochemical specificity of both antibodies, various canine tumours and normal tissues were stained. The immunocytochemical staining results of the canine melanomas and the regional lymph nodes showed high conformity with the immunohistochemical reactivity patterns for S-100 and Melan-A. The specificity of Melan-A was higher compared with S-100. Melan-A, in particular, may be helpful for the cytological diagnosis of canine melanoma.

Animals↗

PubMed2002

Reference values for the bone marrow aspirates in adult dogs.

Reference ranges for each cell type and six different ratios (M : E ratio = quotient of number of myeloid cells/erythroid precursors; different maturation ratios) were calculated for bone marrow aspirates of adult dogs. These values were based on 2.5 and 97.5% percentiles of differentials of 1000 cells in bone marrow aspirates of 92 healthy 1-8-year-old dogs. The results of intact male and female dogs were compared. No distinct sex-related differences were found (P > 0.05). A wide physiological range was observed in almost all bone marrow cells resulting in broad reference ranges for the ratios. The clinically relevant M : E ratio varied between 0.45 and 2.87. The accurate cytological examination of bone marrow based on the reference ranges presented in this study requires preparation of high-quality bone marrow films with minimal blood contamination.

Animals↗

PubMed2002

Quantification of thrombopoietic activity in bone marrow aspirates of dogs.

The aims of the study were to investigate possible influences of different numbers of counted fields on slides, and different slides on the counting of platelet precursors, and to establish guidelines for bone marrow examination in dogs. The study was based on bone marrow slides of 131 healthy dogs. The slides were prepared by a squash technique. On each slide the number of thrombopoietic cells at different levels of maturation were counted in all fragment-containing fields. The results of a variance component model revealed no influence of different slides, in contrast to a high variance related to different fields. Between 20 and 25 low power fields (100x magnification) had to be examined to achieve an imprecision of 20%. An imprecision of 10% required the counting of 100 fields. The lowest thrombopoietic activity was seen in the bone marrow of dogs aged one to six years. The total number of platelet percursors per field was significantly higher in adult female (10.4 +/- 2.67) than in male dogs (7.84 +/- 2.04, P = 0.0008) as was the number of megakaryocytes. We recommend assessment of thrombopoiesis on the basis of 20-25 fragment-containing low power fields and that age- and sex-related differences should be considered.

Aging↗

PubMed2002

[Preclinical studies for the use of the platelet function analyser PFA-100 with the collagen/ADP cartridge in dogs].

In this study, the following three aspects of platelet function analyser were investigated in dogs, using a collagen/ADP cartridge: precision, influence of the cartridge batch and of the sample storage time. Closure time and total volume of blood flow until closure of the capillary were measured. Based on several series of 5 repeated measurements mean coefficients of variation were 5% (3-6%; closure time) or 3% (1-5%; total volume). Neither closure time, nor total volume showed significant differences (p > 0.05) when comparing the results of 6 different batches of the collagen/ADP cartridge. Closure time (p = 0.0211, analysis of variance) and total volume (p = 0.0310) were significantly influenced by storage time, based on the sample material of 6 healthy dogs which was stored for 24 hours. Shortening of the closure time and decrease of the total volume observed in the time interval 1-2 hours after blood collection was followed by a significant prolongation of closure time and increase of the total volume (p < 0.05) starting 8 hours after blood collection. This study shows sufficient reproducibility which is not affected by reagent batch number. The results of the studies on storage indicated nearly identical recommendations for storage time before measurement of canine (0.5-2 hours) and human (0.5-3 hours) sample material.

Adenosine Diphosphate↗

PubMed2001

Possibilities of flow cytometric analysis for immunophenotypic characterization of canine lymphoma.

The purpose of the study presented here is to show the possibilities of flow cytometric analysis for immunophenotypic characterization of canine malignant lymphoma. Aspiration biopsies from lymph nodes of 30 dogs with multicentric lymphoma were stained by four dog-specific antibodies and three human-specific antibodies cross-reacting with canine lymphocytes, and analysed by flow cytometry. Twenty of the 30 lymphoma examined were characterized as B-cell-lymphoma and 10 as T-cell-lymphoma. Flow cytometry is a suitable method for the immunophenotypic analysis of canine lymphoma. Contrary to immunohistochemistry, no surgically produced lymph node biopsy is required and the results are available within a few hours.

Animals↗

PubMed2001

The monitoring of heparin administration by screening tests in experimental dogs.

The objective of this study was to investigate the relationship between different screening tests of haemostasis and amidolytic plasma activities of unfractionated (standard) heparin in dogs. Different doses of intravenous (i.v.) [25, 50 or 100 IU Kg(-1)bodyweight (BW)] and subcutaneous (s.c.) heparin (250, 500 and 750 IU kg(-1)) were given to groups each of five clinically healthy adult beagles. Measurements of heparin activity with a factor Xa-dependent chromogenic substrate, activated partial thromboplastin time (APTT) (two different reagents), thrombin time (TT, two different thrombin activities in the reagent: 3 and 6 IU ml(-1)) and the reaction time of the resonance thrombogram (RTG -r) with two different measuring devices were performed at different times. The relationship between ratio values (actual/baseline values) of the coagulation tests and heparin activity was analysed based on regression analysis and correlation coefficient. The greatest alterations were seen for the TT([3 IU ml(-1)])and the RTG -r which were near or exceeded the upper limit of measuring range, if 25 IU kg(-1)BW heparin were given i.v. at heparin plasma levels of 0.54 +/- 0.13 IU ml(-1). These results show, that only APTT and TT measured with high thrombin activity assay appear suitable for guiding high dose heparin therapy in dogs. Averaged alterations of APTT ratio in canine plasma were less than those observed in people for similar plasma heparin levels, indicating that the guideline extrapolated from people for monitoring high dose heparin therapy using APTT may not be valid for use in dogs. After coagulation times had been converted into ratio values, based on regression analysis and Wilcoxon's test, differences of heparin sensitivity were found not only for TT measured with different thrombin activities but also for different APTT reagents (P < 0.001). The correlation between amidylotic antifactor Xa activity and ratio of coagulation times was only moderate and found to be lower for RTG -r (instrument 1: r(s)= 0.711; instrument 2: r(s)= 0.573) than for the other coagulation tests (r(s)= 0.822 to r(s)= 0.890). This indicates a considerable variability of the ratio values of the screening tests at defined heparin plasma activities. These results show, that blood coagulation tests in general are little or unsuitable for heparin antifactor-Xa activity control.

Animals↗

PubMed2001

Effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on leukocyte count and survival rate of dogs with parvoviral enteritis.

Dogs with clinical signs consistent with parvoviral enteritis and leukopenia (total leukocyte count < 5.0 x 10(9) l(-1)) were included in this randomised double-blind study (treatment group: n = 22; control group: n = 21). The dogs in the treatment group received a subcutaneous daily injection of 10 microg kg(-1) of recombinant human granulocyte colony-stimulating factor (rhG-CSF) for 5 days. Clinical and blood investigations were performed prior to the first injection, daily during the treatment period and on the day after treatment ended, and then once more, 26 days after the first injection. During the study, no significant differences were found between the two groups with respect to survival rate (treatment group: 68 per cent; control group: 71 per cent, P > 0 4, Fisher-Test) and other clinical findings. Similarly the total leukocyte count, neutrophil count and other haematologic and biochemical parameters did not differ significantly between the groups, based on differences from initial values (P > 0 05). Consequently, the use of rhG-CSF in the treatment of dogs with parvoviral enteritis cannot be recommended.

Animals↗

PubMed2001

Amidolytic heparin activity and values for several hemostatic variables after repeated subcutaneous administration of high doses of a low molecular weight heparin in healthy dogs.

OBJECTIVE: To determine effects of SC administration of repeated doses of a low molecular weight heparin (LMWH) in dogs. ANIMALS: 5 healthy dogs. PROCEDURE: Each dog received 6 injections (each injection, 150 U of anti-factor-Xa [anti-FXal/kg of body weight, SC) at 8-hour intervals. Blood samples were collected before and 2 hours after the first, second, third, and sixth injections to measure heparin activity, thrombin time, activated partial thromboplastin time (APTT), antithrombin activity, Hct, and platelet count. RESULTS: Heparin activity varied between 0.36+/-0.10 and 0.77+/-0.08 U of anti-FXa/ml (before and 2 hours after the third injection) and between 0.46+/-0.11 and 0.82+/-0.15 U of anti-FXa/ml (before and 2 hours after the sixth injection). Thrombin time and APTT were influenced only slightly. Platelet count, Hct, and antithrombin activity started to decrease significantly 2 hours after the second LMWH injection. Because of the increased consumption of antithrombin, antithrombin activity continuously decreased from 102.1+/-6.3% before the study to 91.0+/-3.0% at the end of the study. CONCLUSIONS AND CLINICAL RELEVANCE: Heparin plasma activity was only slightly higher than that recommended for LMWH treatment of humans, and none of the dogs had signs of increased bleeding. Thus, administration of heparin in accordance with this dosing regimen can be recommended for use in clinical studies. The screening tests investigated were not suitable for use in monitoring LMWH treatment of dogs. Assays that use chromogenic substrates are necessary to reliably monitor LMWH plasma concentrations in dogs.

Animals↗

PubMed2001

Optimization of coagulometric tests that incorporate human plasma for determination of coagulation factor activities in canine plasma.

OBJECTIVE: To optimize methods used to measure coagulation factor activities in canine plasma, define reference ranges in dogs, and compare activities between canine and human plasma. SAMPLE POPULATION: Human plasma samples (n = 5) and plasma from healthy dogs (140) and dogs with low factor V activity (7), high factor V activity (7), and low factor VIII:C activity (6). PROCEDURE: Coagulometric tests incorporated human plasma deficient in a single coagulation factor (human deficient plasma). Standard curves were generated with pooled plasma from 100 healthy dogs. Effect of sample dilution was evaluated, using plasma from dogs with high or low factor V activity and low factor VIII:C activity. Reference ranges for healthy dogs were established. Activities in human plasma were determined by comparison with standard curves obtained with canine plasma. RESULTS: Activities of factors V and VIII:C in samples diluted < or = 1:20 influenced results of tests for other coagulation factors. Activities of factors V and VIII:C in human plasma were significantly less than in canine plasma. For the other coagulation factors, significant differences in human plasma-to-canine plasma activity ratios were detected among different sample dilutions. CONCLUSIONS AND CLINICAL RELEVANCE: Accurate measurement of coagulation factor activities in canine plasma, using human deficient plasma, requires higher sample dilutions (ie, > 1:20) than typically used for human plasma. Differences in activities between human and canine plasma and nonparallelism of the standard curves emphasize the necessity for use of species-specific standard curves for accurate determination of coagulation factor activity.

Animals↗

PubMed2001

Investigation of the fibrinolytic system during nonpregnant and pregnant oestrous cycles of bitches.

The fibrinolytic system was studied during the oestrous cycle of nonpregnant (n = 12) and pregnant (n = 12) bitches. Blood samples were taken during late pro-oestrus, at ovulation, at days 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 90 and 120 of metoestrus, and during anoestrus. The concentrations of fibrinogen and fibrin(ogen) degradation products, the euglobulin lysis time, the activities of plasminogen and alpha 2-plasmin inhibitor and the haematocrit were determined. Concentrations of oestradiol and progesterone were measured in additional blood samples in late pro-oestrus, at ovulation, at days 30, 60, 90 and 120 of metoestrus, and in anoestrus, for retrospective control of oestrous cycle stages. In the pregnant bitches, significantly higher values (P < 0.05) were found for the concentrations of fibrinogen and fibrin(ogen) degradation products from day 15 to day 60, for the euglobulin lysis time from day 20 to day 50, for the plasminogen activity from day 15 to day 40 and for the alpha 2-plasmin inhibitor activity from day 20 to day 30 of metoestrus compared with values for nonpregnant bitches. The significant increase in fibrinogen concentrations observed in the pregnant bitches is thought to be a local reaction of the coagulation system due to alterations of the uterine epithelium and endothelium induced by placentation. The slight increase in the concentration of fibrin(ogen) degradation product combined with the slight increase in the activities of plasminogen and alpha 2-plasmin inhibitor are indicative of a local rather than systemic hyperfibrinolysis during canine pregnancy. The prolonged euglobulin lysis time, which is indicative of decreased fibrinolytic activity in pregnancy, is thought to be a laboratory artefact caused by increased fibrinogen concentrations.

Anestrus↗

PubMed2000

Intracellular action of the cytokine MIF to modulate AP-1 activity and the cell cycle through Jab1.

Cytokines are multifunctional mediators that classically modulate immune activity by receptor-mediated pathways. Macrophage migration inhibitory factor (MIF) is a cytokine that has a critical role in several inflammatory conditions but that also has endocrine and enzymatic functions. The molecular targets of MIF action have so far remained unclear. Here we show that MIF specifically interacts with an intracellular protein, Jab1, which is a coactivator of AP-1 transcription that also promotes degradation of the cyclin-dependent kinase inhibitor p27Kip1 (ref. 10). MIF colocalizes with Jab1 in the cytosol, and both endogenous and exogenously added MIF following endocytosis bind Jab1. MIF inhibits Jab1- and stimulus-enhanced AP-1 activity, but does not interfere with the induction of the transcription factor NFkappaB. Jab1 activates c-Jun amino-terminal kinase (JNK) activity and enhances endogenous phospho-c-Jun levels, and MIF inhibits these effects. MIF also antagonizes Jab1-dependent cell-cycle regulation by increasing p27Kip1 expression through stabilization of p27Kip1 protein. Consequently, Jab1-mediated rescue of fibroblasts from growth arrest is blocked by MIF. Amino acids 50-65 and Cys 60 of MIF are important for Jab1 binding and modulation. We conclude that MIF may act broadly to negatively regulate Jab1-controlled pathways and that the MIF-Jab1 interaction may provide a molecular basis for key activities of MIF.

COP9 Signalosome Complex↗

PubMed2000

Influence of factor VIII:C and factor IX activity in plasmas of haemophilic dogs on the activated partial thromboplastin time measured with two commercial reagents.

The present study is based on 145 plasma samples with a reduced activity of factor VIII:C (range: 0.009-0.62 IU mL-1) and 28 samples with a reduced factor IX activity (range: 0.035-0.55 IU mL-1). The samples were collected from dogs with haemophilia A (n=22) or haemophilia B (n=3), some of these during substitution therapy. For all samples the activated partial thromboplastin time (APTT) was measured with two commercial reagents containing kaolin as a contact activator. In each case, the deficiency of factor VIII:C or IX was reflected in abnormal results of the APTT. This was true for both reagents. A significant correlation (P < 0.001) was found between factor VIII:C activity and APTT (reagent 1, Pathromtin(R); Spearman's rank correlation coefficient, rS=-0.731, reagent 2, PTT-Reagenz; rS=-0.875) as well as between factor IX activity and APTT (reagent 1, rS=-0.819; reagent 2, rS=-0.955]. In each case, the relationship between coagulation factor activity and APTT could be proven most precisely by geometric regression. The results of this study illustrate the applicability of commercial APTT test kits as a sensitive screening test of factor VIII:C and IX deficiencies in canine plasma.

Animals↗

PubMed2000

Sensitivity of the reaction time of the resonance thrombogram for factor VIII:C and factor IX deficiencies in the blood of dogs with haemophilia A or B.

The sensitivity of the reaction time of the resonance thrombogram (RTG-r) was examined for two instruments based on 105 canine samples with a reduced factor VIII:C activity and 26 samples with a reduced factor IX activity. These samples were taken from dogs suffering from haemophilia A (n=18) or B (n=3) before and at different times after the substitution therapy with fresh frozen plasma. The RTG-r sensitivity was low; with reference to the total of all examined samples, it was between 0.39 and 0.65 for the factor VIII:C activity and between 0.46 and 0.62 for the factor IX activity, depending on the instrument and the testing procedure used. Low sensitivity was shown especially by the fact that even some of the samples with a factor VIII:C activity < 5% showed false negative results. Moreover, the correlation found between RTG-r and factor VIII:C activity (Spearman's rank correlation coefficient, r(S)=-0.556 to -0.700) as well as between factor IX activity and RTG-r (r(S)=-0.380 to -0.612) was only moderate. The low sensitivity of the RTG towards a reduced activity of the haemophilia factors VIII:C and IX contradicts the exclusive use of the RTG for monitoring haemophilic dogs as well as a global screening test for the haemostasis of dogs.

Animals↗

PubMed2000

Dissection of the enzymatic and immunologic functions of macrophage migration inhibitory factor. Full immunologic activity of N-terminally truncated mutants.

Macrophage migration inhibitory factor (MIF) is a cytokine with broad regulatory functions in innate immunity. MIF belongs to the few cytokines displaying catalytic activities, i.e. MIF has a Pro2-dependent tautomerase and a Cys-Ala-Leu-Cys (CALC) cysteine-based thiol-protein oxidoreductase activity. Previous studies have addressed the roles of the catalytic site residues and the C-terminus. The two activities have not been directly compared. Here we report on the N-terminal mutational analysis and minimization of MIF and on a dissection of the two catalytic activities by comparing mutants P2AMIF, Delta4MIF, Delta5MIF, Delta6MIF, Delta7MIF, Delta8MIF, and Delta10MIF with the cysteine mutants of MIF. As N-terminal deletion was predicted to interfere with protein structure due to disruption of the central beta sheet, it was surprising that deletion of up to six N-terminal residues resulted in normally expressed proteins with wild-type conformation. Strikingly, such mutants exhibited full MIF-specific immunologic activity. While mutation of Pro2 eliminated tautomerase activity, the CALC cysteine residues had no influence on this activity. However, mutant C81SMIF, which otherwise has full biologic activity, only had 32% tautomerase activity. Deletion of four N-terminal residues did not interfere with insulin reduction by MIF. By contrast, reduction of 2-hydroxyethyldisulfide (HED) was markedly affected by N-terminal manipulation, with P2AMIF and Delta2MIF exhibiting 40% activity, and Delta4MIF completely failing to reduce HED. This study constitutes the first comparison of the two catalytic activities of MIF and should assist in understanding the molecular links between the catalytic and immunologic activities of this cytokine and in providing guidelines for N-terminal protein minimization.

Amino Acid Sequence↗

PubMed2000

The correlation between plasma anti-factor Xa activity and haemostatic tests in healthy dogs, following the administration of a low molecular weight heparin.

The aim of the study was to examine how activated partial thromboplastin time (APTT, two different reagents), thrombin time (TT, thrombin activity in the reagent: 3 or 6 IU ml(-1)) and reaction time of the resonance thrombogram (RTG-r) in healthy dogs are influenced by low molecular weight heparin (LMWH). Three different LMWH doses were given subcutaneously or intravenously to groups, each of five healthy dogs. Mean plasma anti-FXa activities of 0.43, 0.88 and 1.86 anti-FXa IU ml(-1)were measured 2 min after intravenous injection of 25, 50 or 100 anti-FXa IU kg(-1). At this time, a dose-dependent increase of the coagulation times, above the baseline values (P < 0.05), was observed for all haemostatic tests. The significant prolongation of coagulation time lasted 10 minutes to 3 hours, and it was dependent on the test employed and LMWH dose. After subcutaneous LMWH injection of 50, 100 and 200 anti-FXa IU kg(-1), significant changes of the coagulation time above initial values were limited to the period around the time when maximum anti-FXa activities (0.23, 0.43 or 0.90 anti-FXa IU ml(-1)) were observed. For the tests which were less affected by the LMWH (APTT, TT([6 IU ml)(-1)(])), only small increases (< 4 seconds) were observed even after the highest subcutaneous LMWH dose. The correlation between plasma heparin activity and the relative alteration compared to the initial value (ratio), of the different coagulation tests was only moderate and considerably lower for RTG-r (r(s)= 0.526) than for the TT (r(s)= 0.711([6 IU ml(-1)]), r(s)= 0.780([3 IU ml(-1)])) and APTT (r(s)= 0.667([reagent 1]), r(s)= 0.727([reagent 2])). The low degree of prolongation, which was found particularly for the group tests APTT and TT([6 IU ml)(-1)]), reflects the low anti-thrombin activity of LMWH. The results indicate that measurement of anti-FXa activity with chromogenic substrates is the method of choice to control LMWH therapy in dogs, as is the case in humans.

Animals↗

PubMed2000

Activated partial thromboplastin time as a screening test of minor or moderate coagulation factor deficiencies for canine plasma: sensitivity of different commercial reagents.

To determine the sensitivity for detection of coagulation factor deficiencies by commercial reagents for canine plasma, 5 commercial activated partial thromboplastin time (APTT) reagents with different types of contact activator and phospholipid of various origin were examined. Thirty canine plasma samples with minor or moderate deficiencies of coagualition factors that influence the APTT were examined. Significant differences were found for the sensitivity of various reagents, but no correlation was found with the type of contact activator. Following the test instructions provided by the manufacturers, the number of APTT results that were prolonged beyond the reference range varied between 20 and 30 (sensitivity = 0.67-1.00); the number of corresponding results using a standardized test protocol varied between 19 and 28 (sensitivity: 0.63-0.93). The most sensitive reagent contained kaolin as a contact activator and a human placental thromboplastin. The results of this study indicate that the APTT test optimized for human plasma is also a sensitive screening test of the intrinsic system of canine plasma, provided that a suitable reagent is used.

Animals↗

PubMed2000

[Effect of a high single subcutaneous dose of unfractionated heparin on platelet function in dogs].

The influence of heparin on different tests of platelet function was investigated in 5 healthy dogs receiving subcutaneously 1000 I.E./kg BW of a commercial unfractionated Sodium heparin preparation. Blood samples were collected before and 4 hours after heparin injection. Besides plasma activity of heparin platelet count, capillary bleeding time with two different methods, platelet aggregation according to Breddin and to Born with the inductors ADP, collagen, and thrombin as well as in vitro bleeding time were measured. The activity of heparin four hours after the subcutaneous heparin application was 1.20 +/- 0.11 I.E./ml. Compared to the starting values no significant influence of this heparin level could be demonstrated on platelet count, platelet aggregation according to Born with the inductors ADP and collagen, platelet aggregation according to the Breddin method as well as in vitro bleeding time (p > 0.05). Only one of the two methods used for measuring the capillary bleeding time showed a slight prolongation compared to the starting value (mean = 77 sec) to 88 sec (p < 0.05). The most significant influence was seen on the platelet aggregation induced by 1 I.U./ml thrombin, whereby the aggregation maximum decreased from 92.0% (mean) to 12.2% (p < 0.001). Whereas the latter result has to be interpreted primarily as a consequence of the anti-thrombin effect of heparin, the results of the other tests in summary illustrate the clinical unimportant influence of heparin on platelet function in dogs.

Adenosine Diphosphate↗