Atrophy and hypertrophy signalling in the diaphragm of patients with COPD.

We investigated whether atrophy and hypertrophy signalling were altered in the diaphragm of chronic obstructive pulmonary disease (COPD) patients. We studied diaphragm fibre dimensions and proportion, expression of markers of the ubiquitin-proteasome pathway, nuclear factor (NF)-kappaB pathways, muscle regulatory factors and myostatin in diaphragm biopsies from 19 patients with severe COPD and 13 patients without COPD. Type I proportion was significantly increased in the diaphragm of COPD patients while type II proportion was decreased. The cross-sectional area of all fibre types was reduced in the COPD patients. In addition, MAFbx mRNA was higher in the diaphragm of COPD patients while Nedd4 mRNA decreased. Cytoplasmatic levels of inhibitor protein IkappaBalpha and IkappaBbeta were decreased in the COPD patients as was NF-kappaB p50 DNA-binding activity. MyoD mRNA and its nuclear protein content were decreased in the diaphragm of COPD patients and myogenin mRNA and protein levels remained unchanged. Myostatin mRNA was decreased but its protein levels in the nuclear and cytoplasmic fraction were significantly increased in the COPD patients. These data show that the ubiquitin-proteasome pathway, the NF-kappaB pathway and myostatin protein were up-regulated in the diaphragm of COPD patients while MyoD expression was reduced. These alterations may contribute to diaphragm remodeling in COPD.

Prevalence of viral genome in endomyocardial biopsies from patients with inflammatory heart muscle disease.

In the report of the 1995 World Health Federation/International Society and Federation of Cardiology (WHF/ISFC) Task Force on the Definition and Classification of Cardiomyopathies, the definition of heart muscle diseases was updated. Idiopathic, autoimmune, and infectious forms of inflammatory cardiomyopathy are now recognized in this definition. Enteroviruses, adenoviruses and cytomegaloviruses are considered as main etiopathological factors in the pathogenesis of inflammatory heart disease. A wide range of different assays have been and are currently being used, either alone or in combination, to assay for the presence of enteroviral RNA and/or DNA of cytomegalo- and adenoviruses in endomyocardial biopsy and explanted heart samples. The prevalence of cardiotropic viruses in endomyocardial biopsies of patients with clinically suspected inflammatory cardiomyopathy varies widely: enteroviral genome was detected in endomyocardial biopsies of 3 to 53% of patients, cytomegaloviral DNA was detected in 3 to 40% of patients with inflammatory heart disease and adenoviruses in 3 to 23% of the patients. This report summarizes the methods that have been used and the results of molecular biological investigation with polymerase chain reaction, which were reported by several groups over the last years. Taking this together it seems to be clear that the improvement of molecular biological techniques and the experience of people working with these methods will lead to more reliable results on prevalence, persistence and the diagnostic value of these investigations. These findings have to be taken into account in future diagnostic and therapeutic studies in the field of cardiomyopathies.

Detection of viral and bacterial protein in endomyocardial biopsies of patients with inflammatory heart muscle disease?

The development of highly sensitive molecular biological methods such as in-situ hybridization and polymerase chain reaction (PCR) made it possible to detect viral/bacterial nucleic acid in human endomyocardial biopsies. However, only a few investigations addressed the problem of latent persistence of viral and bacterial genome and the detection of the corresponding proteins, which could have important consequences for the clinical course of the disease. The purpose of this study was to determine whether protein of various viruses (adenovirus, enterovirus, cytomegalovirus, influenza A and B virus, herpes simplex virus 1 and 2) and bacteria (chlamydia pneumonia) can be detected in endomyocardial biopsies of patients with myocarditis and dilated cardiomyopathy with and without inflammation by use of an immunofluorescence assay and to compare the frequency of its detection with the results of PCR, immunohistology and serology. Thirty-nine patients with myocarditis and dilated cardiomyopathy with and without inflammation were examined by a direct immunofluorescence assay using the endomyocardial biopsy as antigen. Each of the samples was additionally studied by immunohistological methods and PCR for the detection of infiltrating cells and the genome of cardiotropic viruses or bacteria. Fourteen of patients were considered to have myocarditis (group 1), 9 dilated cardiomyopathy with inflammation (group 2), 10 dilated cardiomyopathy (group 3), 6 to have no myocarditis or dilated cardiomyopathy (group 4). Using a direct immunofluorescence assay we could show only that 1 patient without histological myocarditis or dilated cardiomyopathy (group 4) was positive for influenza B and chlamydia pneumonia antigens in the endomyocardial biopsy. In addition we have determined influenza B-specific antibodies, such as IgG (marginal titer) and IgA (high titer) and chlamydia pneumonia-specific antibodies, such as IgG (marginal titer) in serum of this patient. A second patient with dilated cardiomyopathy was found to be positive for protein of chlamydia pneumonia, who was shown to have chlamydia pneumonia-specific antibodies, such as IgG (high titer) in serum. There was no correlation with PCR results, but good correlation with influenza B and chlamydia pneumonia-specific antibodies in sera of these patients. In this investigation we have determined viral/bacterial-specific antibodies using serological methods and proteins of these agents using immunoflourescence. Despite the detection of virus or bacteria-specific antibodies in the sera and detection of viral and/or bacterial protein in the biopsies of some of the patients viral and/or bacterial genome was not found in the biopsy. This may be explained by the focal character of myocarditis and sampling error, because for technical reasons we use different biopsies for immunohistochemical and molecular biological investigations.

[Cell death in inflammatory heart muscle diseases--apoptosis or necrosis?]. / Zelluntergang bei entzündlichen Herzmuskelerkrankungen--Apoptose oder Nekrose?

Cell death can be induced by 2 different mechanisms: necrosis and apoptosis. Necrosis, on the one hand, is usually caused by unphysiological stress factors such as hyperthermia or hypoxia, apoptosis, on the other hand, is part of the normal organ development and controls for example immune responses. Morphologically, necrosis is characterized by swelling of cells and their organelles leading to the disruption of the cell membrane, which in turn causes an inflammatory reaction in the surrounding tissue. Morphological and biochemical criteria (Figure 1, Table 1) of apoptosis are the condensation of chromatin leading to the development of apoptotic bodies or membrane-enclosed vesicles containing oligonucleosomal DNA fragments. Important diagnostic tools of cell death (Table 2), such as the TUNEL test (Figure 2) or gel electrophoresis of extracted DNA (Figure 3) are based on the above mentioned biochemical characteristics, but a reliable differentiation of apoptotic versus necrotic processes is not always possible. Experimental studies in animals and studies in various diseases of the cardiovascular system were able to show that apoptosis in myocytes can be induced, an issue that has long been discussed controversially. Ischemia, reperfusion, and myocardial infarction were also shown to lead to apoptosis in cardiomyocytes, whereas cell destruction was caused mainly by necrosis. Several authors (Table 3) demonstrated apoptotic indices in cardiomyocytes of patients with dilatated cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy and patients with acute infarction from 0.25 to 35% by the use of the TUNEL test. Others were able to demonstrate an elevated expression of Fas-receptor in cells of atheroslerotic plaques in patients with atherosclerosis and high indices of apoptotic cardiomyocytes in patients with chronic heart failure. We investigated endomyocardial biopsies of patients with inflammatory cardiomyopathy, DCM without inflammatory reaction but the presence of adenoviral or cytomegaloviral genome and idiopathic DCM using the TUNEL test. The percentage of apoptotic cardiomyocytes in biopsies of patients with DCMi was 1.03 and in biopsies of patients with adenoviral genome 0.25, whereas in all other groups no apoptosis was found. If apoptosis plays a major role in myocardial diseases such as heart failure, arrhythmia and others, blocking this mechanism will have to be considered as a therapeutical strategy. Therefore, studies on the extent of apoptotic processes in diseased versus healthy cardiac tissue are of great importance.

Cytomegalovirus-associated heart muscle disease.

Human cytomegalovirus (CMV) can persist in many organs after primary infection. Not only is it suspected to cause morbidity during reactivation in patients under immunosuppression, but it may also induce long-term latency by chronic disease, e.g. in the myocardium. Endomyocardial biopsies of 27 patients with active myocarditis, 35 patients with healing, 41 patients with healed and 25 patients with ongoing myocarditis according to the Dallas Criteria and 52 patients with dilated cardiomyopathy (DCM) and the biopsies of 25 healthy heart donors were studied for persisting CMV-DNA by polymerase chain reaction (PCR) and in-situ hybridization (ISH). CMV-DNA could be assessed in 5-14% of patients in the different stages of myocarditis and in 22% of patients with DCM. Although two biopsies of the control group showed a positive result, studies by in-situ hybridization demonstrated that in patients with heart muscle disease CMV persists in all cell types including myocytes, whereas in the controls it is only found in interstitial cells. CMV antigens could not be detected in the myocardium with our methods. It must be assumed that infection by CMV is more a persistent or latent than an active infection.

Detection of cytomegalovirus genome by in situ hybridization in paraffin embedded endomyocardial biopsy specimens of viral myocarditis.

Cytomegalovirus (CMV) genes were detected by in situ hybridization in 25 Chinese patients with viral myocarditis (VMC). The positive hybridization signals were found in cardiomyocytes (6 cases, 24%), capillary endothelial cells (4 cases, 16%) and interstitial cells (7 cases, 28%). The difference between VMC and control group (16 cases died of brain trauma and 10 cases of congenital heart diseases was statistically significant. There was no definite pathomorphological relationship between the detection of CMV genes and myocardial lesions. The results suggest that CMV infection may be one of the causes of myocarditis and chronic stimulation of the immune system induced by CMV may be a possible pathogenesis of this disease.

Cytomegalovirus associated inflammatory heart muscle disease.

Cytomegaloviruses(CMV) belong to a group of cardiotropic DNA-viruses with well-documented but sporadic cardiac involvement. By in situ hybridization with a biotinylated cDNA probe CMV-DNA was analysed in 2 different series of patients(1982-1988; 1989-1991) in the endomyocardial biopsy specimens of 35 patients with active myocarditis as defined by the Dallas criteria, and of 35 patients with acute perimyocarditis (pericardial effusion and cardiomegaly or segmental wall motion abnormality and/or an endomyocardial biopsy positive for active myocarditis) were analysed. 51% of patients with active myocarditis, 65% positive findings were observed in patients with perimyocarditis when all positive signals in the myocardium were taken into account. Since in interstitial cells and the vascular endothelium HCMV-DNA was also detected in controls we conclude that only HCMV-DNA in the nuclei are specific for HCMV-associated myocarditis. The incidence of positive signals in the myocytes was lower: 14% of all myocarditis patients and 8.5% of all pericarditis patients demonstrated this pattern. The results from in situ hybridization were compared to circulating anti-CMV antibodies from by an ELISA. As possible predisposing immunologic factors or associated alterations of effector functions we found a shift from normal to reduced natural killer cell activity and a marginal increase in B- and activated T-cells in the peripheral blood.

Does CMV infection play a role in myocarditis? New aspects from in-situ hybridization.

Endomyocardial biopsies of 20 patients with active myocarditis, 15 with post-myocarditis and 22 with acute perimyocarditis were assayed for cytomegalovirus (CMV) DNA by in-situ hybridization with a biotinylated DNA-probe. Positive probes were found in 10% of the cases with acute myocarditis and in 7% of those with post-myocarditis; no CMV-positive patients were found in acute peri-myocarditis. The results were compared to anti-CMV antibodies from patients sera detected by ELISA technique.

Determination of the enantiomers of alpha-amino acids and alpha-amino acid amides by high-performance liquid chromatography with a chiral mobile phase.

A high-performance liquid chromatographic method for the enantiomeric analysis of a mixture of an alpha-amino acid and the corresponding acid amide is described. Reversed-phase chromatography with copper(II) acetate and N,N-di-n-propyl-L-alanine in the mobile phase are used for the separation. For Val and Val-NH2, several parameters affecting retention and enantioselectivity were investigated. The results indicate that by manipulation of pH, ionic strength, temperature, concentration of CuII, N,N-di-n-propyl-L-alanine and ion-pairing reagent, good control of enantiomeric separation can be achieved. For alpha-amino acid amides a mechanism is proposed which may account for the retention and enantioselectivity. Examples of enantiomeric analysis of mixtures of alpha-amino acids and alpha-amino acid amides with aliphatic, aromatic and polar side-chains are given. The method can be used for the control of the enantiomeric purity of alpha-amino acids and the corresponding acid amides obtained by enantioselective synthesis.

Inhibition of intestinal diamine oxidase by detergents: a problem for drug formulations with water insoluble agents applied by the intravenous route?

One hundred and twenty four water-insoluble drugs were included in a study for their action on diamine oxidase (DAO) after solubilization with 61 detergents. 16 detergents were themselves not water-soluble and were not further investigated. A further 3 detergents affected the extraction procedure and 7 of the remaining 42 detergents themselves inhibited the activity of canine intestinal DAO in vitro. Only 5 detergents fulfilled all prerequisites for our DAO assay, including the solubilization of 76 water-insoluble drugs. Each of these 5 detergents had an individual range of suitability in our test system. 3/76 drugs inhibited DAO in concentrations up to 10(-3) M. This result is in contrast to our study with water-soluble substances, where 16% were DAO inhibitors. Since detergents can block the enzyme which is responsible for histamine catabolism, some of the observed adverse reactions to drugs could arise because of the presence of such detergents in the formulation.

Inhibition of human and canine diamine oxidase by drugs used in an intensive care unit: relevance for clinical side effects?

Three hundred and forty-one drugs, commonly used in intensive care units (ICU), were chosen for an investigation of possible activation or inhibition of the histamine metabolizing enzyme diamine oxidase (DAO). After examination of 164 substances, using both canine and human DAO in an in vitro screening test, 61 agents inhibited DAO activity to various degrees. Of these, 44 inhibited the enzyme from both species, 4 inhibited the canine enzyme only and 13 the human DAO only. No compound tested was able to enhance the enzyme activity. The inhibiting agents included representatives of all major therapeutic groups. A particularly strong inhibition was observed with the neuromuscular blocking drugs d-tubocurarine, pancuronium and alcuronium, however, the other commonly used neuromuscular blocking drug, suxamethonium, was without effect. Similarly with the cephalosporines, cefotiame and cefuroxime caused a marked inhibition of the human DAO activity, whereas another regularly-used substance of this class, cefotaxime, inhibited neither the human nor the canine enzyme in concentrations up to 10(-3) M. The observation that within a given therapeutic group some members inhibit and others do not, could be useful in choosing a therapy concept which minimizes the risk of a more severe 'histamine' reaction in seriously ill patients.

Histamine content, diamine oxidase activity and histamine methyltransferase activity in human tissues: fact or fictions?

To understand the role of histamine in the aetiology and pathogenesis of human diseases reliable data are urgently needed for the histamine content and for the activities of histamine-forming and -inactivating enzymes in human tissues. In order to make a substantial progress toward this aim a tissue-sampling programme during surgical interventions was carefully conceived and conducted. From March 1982 until January 1983 106 tissue specimens were taken from 56 patients who underwent surgery. Only healthy tissues, not injured or oedematous, and without adherent structures were taken by only one surgeon who was interested in this research and experienced in tissue preparation procedures in biochemistry. The times of 'warm' ischaemia during the operative procedures were visually estimated, the times between resection of the organs or specimens and deep-freezing of the tissues were precisely recorded. Compared to previous work in the literature and especially to our own work using the same assays for determination higher histamine contents were found in this study in most of the tissues, in particular in the gastrointestinal tract. Also the diamine oxidase activities were considerably higher in many organs, e.g. 3-4 times higher in the gastrointestinal tract when compared with those in publications of our group who used always the same analytical test. However, the histamine methyltransferase activities in this study were not at variance to those determined in previous investigations. Many of them were reported in this communication for the first time. Since the methods for histamine determination and those for measuring enzymic activities were not different in this study and in previous communications of our group we are convinced that the optimized tissue-sampling and -preparation techniques were responsible for the higher values in this communication. But the problem of the 'warm' ischaemia period could not be solved by sample-taking procedures of this type during operations. There are good reasons to prefer biopsy specimens for the analysis of histamine storage and metabolism in human tissues in health and disease, but - unfortunately - they are not always available.

Gastric histamine methyltransferase: different methylation rates for enantiomers of side-chain methylated histamine analogues using a highly purified enzyme preparation.

The inhibitor/activator and substrate properties of enantiomers of two methylated histamines (MH) were investigated using a histamine methyltransferase preparation which was purified 1207-fold from pig fundic mucosa by ultracentrifugation, ion-exchange chromatography on DEAE-cellulose and preparative electrofocusing. In 1-100 microM concentrations, S-alpha-MH and R-alpha-MH were acceptor substrates as good as histamine itself. When substrate concentrations were increased to 1 mM these substances were methylated to an even greater extent than histamine, since they did not exert substrate inhibition on HMT. Introduction of a further methyl-group into the N alpha-position reduced acceptor substrate properties drastically. A difference in methylation was then seen since R-alpha,N alpha-DMH was a better substrate than S-alpha,N alpha-DMH. Whereas alpha-MH's could not activate HMT the alpha,N alpha-DMH's did. The poorer the substrate affinity of the investigated substances was, the better they were able to activate HMT.

Assay and identification of histamine in human gastric aspirate by a fluorometric--fluoroenzymatic technique. Its application in patients with chronic duodenal ulcer.

Histamine assays can be unreliable in individual subjects or samples even though the particular method is in general working very well. Therefore the specificity and accuracy of histamine determination in the gastric aspirate of individual duodenal ulcer patients was thoroughly examined and shown to be satisfactory. Pitfalls of the fluorometric assay were investigated. A native (non-histamine) fluorescence in gastric aspirate which occurs before the addition of OPT was not removed by the original Shore procedure. In the combined assay (Dowex 50 + butanol extraction) this fluorescence no longer interferes with the assay. For the identification of histamine in a single gastric aspirate of an individual duodenal ulcer patient, the reversed blank (3 M HCl added to the reaction mixture before OPT instead after OPT), excitation and fluorescence spectra, the heating test with spectra recorded and the HMT test were found to be reliable. The formaldehyde test and the heating test without recording the spectra were useless since they gave false negative results. Since the HMT test was regarded as a reference method it was thoroughly investigated both by theoretical considerations (enzyme kinetics) and by a series of measurements in a single patient as well as in a group of nine subjects. Samples from the period of peak acid output in response to pentagastrin showed an average histamine concentration of about 8 ng/ml and a histamine output of 1.5 microgram/30 min.

[Histamine level and histamine metabolism of the human liver in biliary tract diseases]. / Histamingehalt und Histaminstoffwechsel der menschlichen Leber bei Erkrankungen der Gallenwege.

In a control group (n = 12) the histamine content of the liver was 1.42 microgram/g tissue and the activity of the histamine-degradating enzyme, HMT, 421.5 pmol/(min . mg protein). In patients with affections of the biliary tract (n = 12) the histamine content was significantly raised to 3.09 microgram/g tissue and the HMT activity slightly diminished [349 pmol/(min . mg protein)]. The lowest values of HMT activity were found in the livers of those patients with histologically proven damage of liver cells.