Search PubMed⌕ Search

Biomedical subjects

H Burkhardt

Publications and source records attributed to H Burkhardt.

At least 91 records · Page 5Linked to original sources

Evaluation of granulocyte-releasing products and chemiluminescence during cytapheresis.

During the procedures of centrifugation leukapheresis and plateletpheresis, donors occasionally experience adverse clinical reactions. The possibility of whether the activation of granulocytes and the subsequent release reactions, which may have been triggered by this extracorporeal circuit, were responsible for these adverse effects was evaluated. Six blood samples were obtained at set intervals during cytapheresis. Of these samples, four were taken directly from the donor. The remaining two were drawn from the efferent lines, i.e., those which return blood from the cytapheresis machine to the donor. Reactive oxygen species produced by granulocytes were measured by chemiluminescence (CL) using microamounts of whole blood or isolated granulocytes. Furthermore, secreted granulocyte products such as neutral proteinase elastase, which is present in plasma in a complex with alpha-1-proteinase inhibitor (E-alpha-1-PI), and lysosomal beta-glucuronidase were examined. A complete blood cell count and the values of hemoglobin, hematocrit, lactate dehydrogenase, protein, albumin, and proteinase inhibitors such as alpha-2-macroglobulin and alpha-1-proteinase inhibitor were also determined. Clinical chemical and cytologic values, with the exception of those for E-alpha-1-PI, were 10 to 17 percent lower than values before apheresis. These results can be attributed to inherent plasma volume expansion. Reduced CL was observed on the stimulation of phagocytes in the whole blood assay, as well as with stimulated granulocytes. Unstimulated granulocytes, on the other hand, showed an increased native CL. These data do not indicate a cytapheresis-mediated activation of the oxidative metabolism of granulocytes, and the concomitant discharge of proteolytic enzymes remains, therefore, of no clinical importance.

Blood Cell Count↗

[Immunization of young cattle with gp51 of the bovine leukosis virus and the subsequent experimental infection].

Calves were immunised by means of a native Al(OH)3-absorbed gp51-preparation which had been obtained from a foetal lamb kidney cell line, following bovine leucosis virus (BLV) infection. 9 animals were immunised 3 times, using 300 micrograms gp51. 7 animals underwent test infection, using 2.5 x 10(3) or 2.5 x 10(4) BLV-infected lymphocytes. Serological and virological reactions of all animals, including 3 calves which had received only test infections, were followed up through 40 weeks by means of immunodiffusion test, enzyme-immuno-assay, gp51 antibody radio-immuno-assay, reverse transcriptase test, syncytial test, competitive p24 radio-immuno-assay, and by transmission of whole blood in animal experiments. The results obtained from virological testing showed that 1 animal had been protected by preceding immunisation. 4 in 7 immunised and test-infected animals exhibited transient BLV values, between the 7th and 16th weeks from infection. Typical leucosis infection had been induced to 2 animals. The above findings are discussed and are compared to similar results recorded by other working groups.

Animals↗

Treatment of bovine leukaemia virus-infected sheep with suramin: an animal model for the development of antiretroviral compounds.

Bovine leukaemia virus (BLV) and the human T-cell leukaemia/lymphoma viruses I and II represent a specific group of type-C RNA tumour viruses characterized by the presence between the err gene and the 3'LTR of an "x" region or LOR frame, which codes for a protein that trans-activates the transcription of the viral genome. As BLV can also infect sheep and induces pre B-cell specific tumours in these animals, we were interested in investigating whether suramin, a potent inhibitor of retrovirus-associated reverse transcriptase, may inhibit the in vivo multiplication of BLV in sheep. The sheep were infected with 4 X 10(7) leukocytes from a BLV-infected cow. The animals were maedi-visna virus-negative. Viral p24 antigen and reverse transcriptase appeared at 2 weeks and seroconversion occurred at 4 weeks after infection. Suramin was administered at 20 mg/kg/week from the 10th till the 16th week after infection. During the treatment period the expression of p24 antigen as well as the titre of anti-p24 and anti-gp51 antibodies were followed. Suramin treatment led to a significant, but transient, disappearance of p24 antigen and did not affect the titre of anti-p24 and anti-gp51 antibodies. The BLV-infected sheep may serve as a useful animal model for the investigation of retrovirus inhibitors and the evaluation of different therapeutic regimens.

Animals↗

BLV-p24 expression in BLV infected cattle and detection of BLV-p24 receptors in cattle afflicted with tumorous leukosis in vivo.

Noncultivated and short-term cultivated blood leukocytes of BLV-infected cattle both with and without tumorous leukosis have been investigated for BLV-p24 antigen expression and for their capacity to bind BLV-p24 antigen. In animals with persistent lymphocytosis using cell extracts of 3-6 X 10(8) non-short-term cultivated blood leukocytes, BLV-p24 antigen could be identified by means of competitive RIA. In cattle with tumorous leukosis, the antigen detection in non-short-term cultivated leukocytes may be masked by the presence of antigen-binding receptors. The capacity of p24 antigen binding is likely to be a phenotypical marker for the cells occurring in the tumour phase only.

Animals↗

Separation and chemiluminescence properties of human, canine and rat polymorphonuclear cells.

Human as well as canine and rat polymorphonuclear cells (PMN) were separated from whole blood by centrifugation. Two-step discontinuous Percoll gradients with distinct densities were used. The purity of the preparations was 99.2%, 98.4% and 97.9%, respectively and the corresponding recoveries were 80.1; 66.3% and 69%. The chemiluminescence properties of the isolated PMN and of phagocytes in small quantities of whole blood were compared in luminol-enhanced assays after stimulation with various agents: non-opsonized zymosan (3.5 g/l), phorbol myristate acetate (PMA, 2.8 X 10(-6) M), calcium ionophore A 23187 (10(-5) M) and N-formyl-methionyl-leucyl-phenylalanine (FMLP, 3.5 X 10(-6) M). The isolated cells of the three species responded to all of the various stimuli. Species-related sensitivity followed the order: human greater than canine greater than rat. Responses to the various agents in the human cells was ranked: PMA greater than or equal to A 23187 greater than zymosan greater than FMLP; for the dog: A 23187 greater than PMA greater than zymosan greater than FMLP; and for the rat: zymosan greater than or equal to PMA greater than FMLP greater than or equal to A 23187. Time courses and peak maximum responses were different following stimulation in the absence or presence of autologous plasma. Distinct soluble stimuli resulted in maximum responses below the baseline in the whole blood assays with canine (FMLP) and rat (FMLP, A 23187) phagocytes.

Animals↗

Carbimazole-induced immune haemolytic anaemia: role of drug-red blood cell complexes for immunization.

We report on a patient who developed acute intravascular immune haemolysis while receiving carbimazole. Serological studies revealed a strongly (3+) and a discretely positive (1+) direct antiglobulin test due to C3d and IgG respectively, and a very weak IgG autoantibody in the eluates. Serum from the patient contained specific carbimazole-dependent red blood cell (RBC) antibodies which reacted with all normal human RBC in the presence of free carbimazole as well as with RBC coated with the drug either in vitro or in vivo, although carbimazole itself is not detectable in plasma after oral administration. The results provide direct evidence for the sequence of the drug-RBC-antibody interaction and show that the RBC (and not plasma proteins) function as 'carrier-like' macromolecules in the immune response.

Aged↗

Interaction of polymorphonuclear leukocytes with cartilage in vitro. Catabolic effects of serine proteases and oxygen radicals.

The ability of purified PMN serine proteases as well as oxygen-derived free radicals (ODFR) generated by activated phagocytes to damage cartilage matrix has been thoroughly investigated in vitro. The question in the present study was the extent to which enzymatic and ODFR-mediated mechanisms can contribute to the degradation of bovine cartilage slices by zymosan-stimulated PMN. Tissue destruction as assessed by mechanical parameters of stability as well as by liberation of uronic acids from matrix proteoglycans was not inhibitable by the radical scavengers superoxide dismutase (SOD) and catalase (CAT), while serine protease inhibitors led to a significant reduction of matrix degradation. Thus an enzymatic mechanism may play a major part in PMN-induced cartilage damage. Besides this predominant role of especially serine proteases a direct, non-zymosan-dependent stimulatory effect of cartilage matrix on PMN to release elastase into the incubation medium was detected. Hence an as-yet unknown mechanism of PMN activation is indicated, while unspecific effects by bacterial contamination, complement factors, or endotoxin could be excluded as an explanation for the observed phenomenon.

Animals↗

[Differential diagnosis of jaundice and fever of unknown origin: ruptured aortic aneurysm with obstruction of the bile ducts].

A 65-year-old man with fever of unknown origin developed progressive extrahepatic cholestasis. Radiological examination documented a suprarenal abdominal aortic aneurysm. Fatal intraabdominal bleeding occurred, leading to death. Post-mortem examination revealed an earlier retroperitoneal rupture of the aortic aneurysm and massive perianeurysmal inflammation. The blood masses, reaching the porta hepatis, caused progressive obstruction of the extrahepatic bile ducts. Surgical treatment, delayed because of cholestatic and febrile complications, could have prevented the outcome.

Aged↗

Purification and characterization of a serine proteinase inhibitor from human articular cartilage.

An inhibitor of serine proteinases from human articular cartilage was purified to homogeneity by sequential ultrafiltration and ion exchange chromatography on CM-Sephadex C-50. The apparent molecular weight of the cationic glycoprotein (pI greater than 10) was determined to be 16.5 X 10(3) by SDS gel electrophoresis. The inhibitor blocked the activity of leukocyte elastase, cathepsin G and trypsin but not leukocyte collagenase. In kinetic studies for the interactions with leukocyte elastase a firm enzyme-inhibitor binding was obtained. Amino acid analyses did not reveal homologies with other serine proteinase inhibitors already purified from human tissues.

Amino Acids↗

Interference of cartilage surface with interaction of granulocyte elastase with alpha 1-proteinase inhibitor. An in vitro model of enzyme inhibition in the joint space.

Synovial fluids of patients suffering from rheumatoid arthritis contain elevated levels of granulocyte (PMN) elastase in complex with alpha 1-proteinase inhibitor (alpha 1-PI), whereas free-elastase activity is usually not detectable. This absence of free enzymatic activity in joint effusions has cast some doubt on the pathophysiological relevance of PMN elastase in inflammatory joint destruction. Our in vitro experiments using bovine nasal cartilage demonstrate that incubation with elastase and alpha 1-PI in equimolar concentrations to or even in excess of the serum proteinase inhibitor resulted in significant tissue destruction as assessed by histological staining for proteoglycans, release of uronic acid from the matrix and loss of mechanical stability. Though in the supernatants containing alpha 1-PI, free-elastase activity was not detectable, immunofluorescent staining for elastase evidenced penetration of the enzyme into the matrix. Simultaneous measurements of the incubation media employing a sandwich enzyme-linked immunoadsorption assay (ELISA) revealed PMN elastase in complex with alpha 1-PI but without correlation to the parameters of tissue degradation. In comparison with the results obtained using the chromogenic substrate Suc-Ala-Ala-Ala-pNA (SAPA) for titration of alpha 1-PI against elastase, the employment of cartilage matrix showed that a fourfold increase in inhibitor concentration was necessary to achieve 100% enzyme inhibition. Hence, cartilage surface obviously interferes with the interaction between alpha 1-PI and elastase. Measurements of elastase-inhibitor concentrations or free enzymatic activity in synovial fluid seem to have limited value in predicting cartilage destruction.

Animals↗

Mechanisms of non-opsonized zymosan-induced and luminol-enhanced chemiluminescence in whole blood and isolated phagocytes.

A luminol-dependent non-opsonized zymosan-induced chemiluminescence method for phagocytes in small quantities of whole blood (40 microliters; final dilution: 1:14) is described. It was characterized with reference to cellular and humoral components, and also applied to isolated neutrophils, eosinophils and monocytes. Normal values for whole blood chemiluminescence and for neutrophils, eosinophils and monocytes are presented. From the chemiluminescence characteristic of distinct phagocytes and their frequency distribution pattern in whole blood, it is concluded that whole blood chemiluminescence has its source predominantly in neutrophils. The question as to the origin of chemiluminescence in phagocytes of whole blood and isolated neutrophils is investigated. The results support the importance of the myeloperoxidase-H2O2-halide system, but also go beyond this. The release of arachidonic acid by phospholipase A2 and of diacylglycerol and inositol trisphosphate by phospholipase C, the metabolism of arachidonic acid by the cyclooxygenase and lipoxygenase pathway, the activation of membrane NADPH oxidase by diacylglycerol and the calcium mobilisation by inositol trisphosphate are necessary for the chemiluminescence reaction. Inhibition of either mechanism suppresses the chemiluminescence response. The interaction of non-opsonized zymosan with plasma opsonins, phagocyte Fc- and complement receptors, respectively, for the initiation of chemiluminescence, was investigated. Non-opsonized zymosan initiates a chemiluminescence response in blood phagocytes in the absence of opsonin from the interaction of the zymosan polysaccharide component glucan with the complement receptor type 3. In the presence of plasma this receptor type also mediates the major chemiluminescence response brought about by the zymosan-coated cleavage products of complement fraction three, iC3b and to a minor degree C3b, while immunoglobulin G-coated zymosan interaction with the Fc-receptor is in this case of minor importance.

Eosinophils↗

[Dose-dependent serum concentration of ISDN and its metabolites following administration of ISDN-retard. Studies under steady-state conditions].

In a randomized cross-over trial of four groups of patients (total 49 patients) the serum concentrations of isosorbide dinitrate (ISDN), isosorbide-2-mononitrate (IS-2-MN) and isosorbide-5-mononitrate (IS-5-MN) were measured under steady-state conditions after administration of 20, 40, 60 and 80 mg, respectively, of two ISDN preparations, the two being bioequivalent. At the studied dosage there was a linear relationship between dose and serum concentration of ISDN and its two metabolites in the proportion of 1:6:70. Because the serum concentration of IS-5-MN is so high under steady-state conditions, there is the potential problem of tolerance developing. The drug should therefore be administered at a lower dose twice daily.

Aged↗

Oxygen radicals as effectors of cartilage destruction. Direct degradative effect on matrix components and indirect action via activation of latent collagenase from polymorphonuclear leukocytes.

Degradation of intact cartilaginous tissue (bovine nasal cartilage) by oxygen-derived free radicals (ODFR) generated enzymatically by xanthine oxidase and hypoxanthine was studied. The degree of tissue destruction was determined by measuring the indentation under a defined compression force as well as by the loss of uronic acid- and hydroxyproline-containing matrix components. Cartilage slices altered by prior elastase treatment were more susceptible to oxygen radical attack than were intact tissue specimens. Degradation of cartilage matrix by ODFR was strongly inhibited by superoxide dismutase or catalase. Coincubation of latent collagenase from polymorphonuclear leukocytes with the ODFR-generating system led to activation of collagenolytic activity, resulting in marked degradation of the bovine cartilage slices. In further studies, activated polymorphonuclear leukocyte-collagenase was shown to degrade intact human articular cartilage to a degree of mechanical insufficiency. Thus, our assay system serves as an in vitro model of tissue damage, which may be relevant to pathophysiologic states such as rheumatoid arthritis.

Animals↗

Activation of latent collagenase from polymorphonuclear leukocytes by oxygen radicals.

Polymorphonuclear leukocytes (PMN) accumulating at inflammatory sites have the potential to degrade collagen by releasing the metalloproteinase collagenase (EC 3.4.24.7), which is stored within the specific granules of these cells in a latent, inactive, form. In order to elucidate the activation mechanism the latent enzyme (molecular weight 91,000) was purified from human PMN and incubated with the oxygen radical-generating system of xanthine oxidase (EC 1.1.3.22) and hypoxanthine. This coincubation resulted in the activation of the latent enzyme as assessed by the collagenolytic attack on human and bovine cartilaginous tissue. Two parameters for collagenolysis were used: loss of hydroxyproline-containing fragments, and mechanical measurements reflecting the stability of tissue specimens. Superoxide dismutase (EC 1.15.1.1) as well as catalase (EC 1.11.1.6) were capable of inhibiting the activation of latent PMN collagenase by the oxygen radical-generating system. The results indicate the hydroxyl radical to be the final oxidant responsible for the activation of latent PMN collagenase. Thus a new activation mechanism of latent collagenase is presented in this paper and discussed together with the potential relevance in pathophysiologic states of acute and chronic inflammation.

Animals↗