[Methotrexate therapy of inflammatory diseases. Reliable effects and recent developments in rheumatology, pneumology, gastroenterology and dermatology].
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Asthma↗
Biomedical subjects
K Dalhoff
Publications and source records attributed to K Dalhoff.
Transport and storage conditions for cultural recovery of Chlamydia pneumoniae.
Chlamydia pneumoniae is characterized by rapidly decreasing viability outside the host cell, and efficient preservation of its infectivity is a prerequisite for subsequent cell culture recovery. Extracellular survival of three C. pneumoniae stock strains and three wild-type strains subjected to simulated conditions of transport was therefore examined in order to establish recommendations for transport and storage of clinical specimens. The presence of fetal calf serum in transport media as well as refrigeration distinctly improved chlamydial retrieval during prolonged transport. Loss of infectivity was kept to a minimum in Eagle's minimal essential medium or sucrose-phosphate-glutamine medium. Storage at 22 degrees C permitted a stock strain recovery of 81% after 12 h. When refrigeration to 4 degrees C was provided, recovery rates of 74% could be achieved after 48 h. Though the strains were from different geographic regions, requirements for good survival were comparable and should therefore apply worldwide. The results indicate that the laboratory strains are not extremely labile. However, comparative examination of the wild-type strains showed less stability: primary isolates were not satisfactorily retrievable beyond 4 h at 22 degrees C or beyond 24 h at 4 degrees C. Further extension of storage times resulted in rapidly decreasing recovery, indicating a requirement to freeze samples at -75 degrees C to preserve viability. Adherence to the shorter storage periods suggested by the data obtained with primary isolates is recommended to ensure successful transport until more extensive testing with clinical materials is available.
Bacteriological Techniques↗
[Pentoxifylline inhibits secretion of O2- and TNF-alpha by alveolar macrophages in patients with sarcoidosis].
Reactive oxygen species (ROS) and cytokines like tumor necrosis factor-alpha (TNF-alpha) play a crucial role as inflammatory mediators in pulmonary sarcoidosis. We examined the antiinflammatory effect of pentoxifylline (POF) on alveolar macrophages (AM) of patients with sarcoidosis in vitro. We could demonstrate that POF (above 4.10(-4) M) inhibited the secretion of superoxide anion and TNF-alpha by AM in a dose-dependent manner via a prostaglandin synthesis-dependent mechanism that was independent of the glucocorticoid receptor. POF is an interesting immunomodulating substance that should be further evaluated in clinical trials.
Adult↗
[Isolation of Chlamydia pneumoniae in atypical pneumonia].
A 33-year-old man, who two months previously had been treated for bronchopneumonia with cefuroxime, fell ill again with cough, dyspnoea and fever up to 39.8 degrees C. Auscultation and lung function tests indicated respiratory tract obstruction. The chest radiograph revealed an infiltrate of the left lower lobe and bronchoscopy showed hypertrophic bronchitis. Empirical antibiotic treatment with clarithromycin (initially 500 mg twice daily, continued on half this dose) lead to rapid improvement of clinical symptoms and the patient could be discharged after 9 days. A follow-up examination 3 months later showed no abnormality. A cell culture procedure using bronchoalveolar lavage fluid obtained during bronchoscopy permitted the continuous demonstration of Chlamydia pneumoniae inclusions in HEp-2 host cells by immunofluorescence microscopy. In parallel, Chlamydia pneumoniae DNA was specifically demonstrated in the lavage fluid by use of the polymerase chain reaction. This is the first reported isolation of a replicative Chlamydia pneumoniae strain in Germany.
Adult↗
Comparison of sample preparation methods for detection of Chlamydia pneumoniae in bronchoalveolar lavage fluid by PCR.
Amplification inhibitors can lead to false-negative results for PCR. In order to evaluate the reliability of PCR for the detection of Chlamydia pneumoniae, the presence of PCR inhibitors in 75 bronchoalveolar lavage specimens was assessed after treatment by various sample preparation methods. Specimens were collected from patients with acute respiratory infections, including four cases of proven C. pneumoniae infection. Substances inhibitory to the amplification of chlamydial DNA continued to be present in 12% of the samples treated according to the commonly used single-step proteinase K digestion and in 31% of the samples processed by heat treatment. However, the complexing of DNA-contaminating proteins and polysaccharides from digested specimens to cetyltrimethylammonium bromide (CTAB) followed by DNA extraction efficiently removed inhibitors from all experimental samples and provided subsequent identification of all positive clinical samples by PCR. The CTAB method and proteinase K treatment had comparable detection limits of approximately 0.01 inclusion-forming units. CTAB-based DNA purification of respiratory specimens is recommended to increase the diagnostic sensitivity of PCR and confidence in negative results.
Bronchoalveolar Lavage Fluid↗
Oxidative metabolism of pulmonary phagocytes in acute pneumonia.
The oxidative response of activated phagocytes is a primary host defense mechanism in pneumonia, but is in addition capable of causing tissue damage. We evaluated amount and significance of the local oxidant production in immunocompetent and immunocompromised pneumonia patients; the relative contribution of neutrophils and alveolar macrophages to the total oxidant load was differentiated using chemiluminescence (CL) with different amplifiers. Luminol-enhanced CL correlated to neutrophil percentage and myeloperoxidase levels in the bronchoalveolar lavage and was markedly increased in both pneumonia groups. Lucigenin-enhanced CL was produced by both phagocyte types and not significantly increased. In both pneumonia groups elevated levels of serum proteins indicated severe alveolocapillary leakage. In conclusion the oxidative response in acute pneumonia was mainly due to neutrophil recruitment and activation; this defense mechanism was preserved even in severely immunocompromised patients.
Acute Disease↗
Characterization of protein-antiproteinase imbalance in bronchoalveolar lavage from patients with pneumonia.
In order to clarify the mode of inactivation of alpha1-proteinase inhibitor (alpha 1-PI) in pneumonia, 21 immunocompetent patients and 19 immunocompromised patients with acute pneumonia (Groups I and II) were studied. Nine patients successfully treated for pneumonia and 10 healthy volunteers served as controls (Groups III and IV, respectively). The concentrations of alpha 1-PI, elastase and myeloperoxidase (MPO) in bronchoalveolar lavage fluid (BALF) were determined using a luminometric assay. Elastase inhibition capacity was determined using a colorimetric assay. Thus, the functional activity of alpha 1-PI was calculated. Both elastase and MPO were significantly higher in group I, when compared with the other groups. The mean concentration of alpha 1-PI was significantly higher in patients with acute pneumonia (Group I 13 mg.l-1, Group II 4.22 mg.l-1) than in Groups III and IV (2.65 and 0.33 mg.l-1, respectively), whereas, the proportion of active alpha 1-PI was significantly lower in Group I than in the other groups. Only a small proportion was present as a complex with elastase (ca. 5.9%) or in oxidised form (ca. 4.8%), 85% of alpha 1-PI was irreversibly proteolyzed. This resulted in free elastase activity in 7 of the 40 patients (18%) with acute pneumonia. We conclude that functional activity of alpha 1-PI is markedly impaired due to irreversible proteolysis in acute pneumonia, despite high immunological concentrations.
Adult↗
Intercellular adhesion molecule-1 (ICAM-1) is upregulated on alveolar macrophages from AIDS patients.
In acquired immune deficiency syndrome (AIDS) patients, alveolar macrophages (AMs) have an increased ability to serve as accessory cells during the generation of an immune response. In addition to soluble mediators, like cytokines, molecules of the major histocompatibility complex (MHC) class II and adhesion molecules, like intercellular adhesion molecule-1 (ICAM-1), play a major role in the regulation of these cellular interactions. Using an enzyme-linked immunosorbent assay (ELISA) technique and immunocytochemical staining, we investigated ICAM-1 and human leucocyte antigen-DR (HLA-DR) expression on AMs from 20 AIDS (HIV+) patients in context with other parameters of macrophage activation, such as tumour necrosis factor-alpha (TNF-alpha) secretion and the release of superoxide anion, comparing the results to a group of healthy volunteers. In addition, we quantified soluble ICAM-1 in the bronchoalveolar lavage fluid (BALF) using a commercially available kit. We found a nearly twofold increase in ICAM-1 expression (0.81 +/- 0.30 (SD) versus 0.42 +/- 0.12 ELISA units (EU) (mean +/- SD)), whilst the number of HLA-DR+ AMs was slightly decreased in AIDS-patients (80 +/- 5 versus 89 +/- 3%). Furthermore, soluble ICAM-1 in the BALF of these patients was significantly increased (41.9 +/- 26.1 versus 19.1 +/- 5.1 ng.ml-1). ICAM-1 levels on AMs in the patient group correlated strongly with the sodium fluoride triggered release of superoxide anion (O2-) but not with the spontaneous secretion of TNF-alpha by AMs.(ABSTRACT TRUNCATED AT 250 WORDS)
Acquired Immunodeficiency Syndrome↗
A rapid semiautomatical enzyme linked immunoassay identifying intercellular adhesion molecule-1 (ICAM-1) on the alveolar macrophage surface.
Intercellular adhesion molecule-1 (ICAM-1), a member of the immunoglobulin gene superfamily, is a cytokine-inducible adhesion molecule, which plays a central role in leukocyte migration into sites of acute or chronic inflammation. In this article we describe a sandwich immunoenzymometric method which allows rapid, semiquantitative (in "enzyme immunoassay units", EU) identification of ICAM-1 on the surface of alveolar macrophages. We evaluated this method in two groups of patients with pulmonary sarcoidosis (n = 12) or bacterial pneumonia (n = 11) and a group of healthy volunteers (n = 6), comparing the results with those obtained by immunocytochemical staining. ICAM-1 expression on the sarcoid alveolar macrophages surface was significantly elevated, as compared with control alveolar macrophages (0.76 EU +/- 0.27 vs. 0.44 EU +/- 0.12, p < 0.01). ICAM-1 expression on the surface of alveolar macrophages from patients with pneumonia was not elevated (0.48 EU +/- 0.35). Stimulation with tumour necrosis factor-alpha (TNF-alpha) or interferon-gamma (100 kU/l) led to a significant induction of ICAM-1 on the surface of control alveolar macrophages (0.76 EU +/- 0.18, p < 0.005 for TNF-alpha, 0.64 EU +/- 0.10, p < 0.005 for interferon-gamma), whereas alveolar macrophages from both patient groups did not respond to cytokines even at high dosages. ICAM-1 expression on the surface of alveolar macrophages from patients with sarcoidosis correlated with the spontaneous release of TNF-alpha by macrophages (R = 0.77, p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Endobronchial actinomycosis.
Endobronchial actinomycosis was found to be the cause of right-sided atelectasis and haemoptysis in a 57 year old man without predisposing conditions. Fibreoptic bronchoscopy revealed occlusion of the intermediate bronchus by yellow-white masses. The diagnosis was confirmed histologically and by positive Actinomyces culture from bioptic material. Prolonged antibiotic treatment resulted in complete recovery, without need for surgical resection.
Actinomycosis↗
Synthesis rates of glutathione and activated sulphate (PAPS) and response to cysteine and acetaminophen administration in glutathione-depleted rat hepatocytes.
The effects of cysteine and acetaminophen (AA) on the synthesis rates of glutathione (GSH), adenosine 3'-phosphate 5'-phosphosulphate (PAPS, activated sulphate) and the AA metabolites, AA-GSH and AA-sulphate were studied in rat hepatocytes depleted of GSH by diethyl maleate (DEM). The synthesis rates were determined simultaneously by a previously described radioactive tracer method. Preincubation of the hepatocytes with 0.7 mM DEM for 30 min depleted GSH by 59% (P < 0.05) and PAPS by 28% (P < 0.05). Incubation with a toxic AA concentration resulted in GSH synthesis at a rate of 95 nmol/(10(6) cells.min) which increased to 281 nmol/(10(6) cells.min) (P = 0.05) after addition of cysteine. However, increased GSH synthesis was not followed by increased AA-GSH synthesis [4.7 vs 4.8 nmol/(10(6) cells.hr)]. Also, PAPS synthesis increased after cysteine administration [10.2 to 19.1 nmol/(10(6) cells.min)] (P < 0.05) without any change in AA-sulphate synthesis 18.5 vs 18.3 nmol/(10(6) cells.hr)]. Thus, in contrast to hepatocytes with normal GSH concentration, cysteine stimulated both GSH and PAPS synthesis rates in GSH-depleted rat hepatocytes incubated with a toxic AA concentration without stimulation of AA-GSH or AA-sulphate synthesis rates, indicating that the hepatoprotective effect of cysteine on AA toxicity is primarily due to stimulation of a GSH-mediated reduction of the reactive AA metabolite N-acetyl-p-benzoquinoneimine back to AA.
Acetaminophen↗
[Generalized nocardiosis with meningoencephalitis in a nonimmunosuppressed female patient].
Four weeks after an attack of pneumonia of unknown aetiology a 40-year-old woman was hospitalized because of a nonpurulent, predominantly basal meningoencephalitis and infratentorial abscesses. She had dysarthria, mild right-sided motor hemiparesis and central paresis affecting the 7th cranial nerve. An area of fluctuating resistance, about 3 cm in diameter, was noticed over the left thigh. Serology indicated inflammatory disease, but there was no immunodeficiency. The CSF showed lymphocytic pleocytosis with mild protein increase but no evidence of infective agent. As tubercular meningitis was suspected she was treated with rifampicin (300 mg i.v. twice daily), isoniazid (300 mg i.v. once daily), streptomycin (800 mg i.m. once daily), cefotaxime (2.0 g i.v. three times daily), fluconazole (200 mg i.v. once daily) and dexamethasone (16-8-8 mg i.v.). She suddenly died two days after admission, probably as the result of central regulatory failure. Generalized nocardiosis involving lung, subcutaneous tissue and brain was revealed at autopsy. Although nocardiosis occurs predominantly in patients under immunosuppression, this infection should be considered in the differential diagnosis of treatment-resistant pneumonia and meningoencephalitis without obvious predisposition.
Adult↗
Simultaneous measurements of glutathione and activated sulphate (PAPS) synthesis rates and the effects of selective inhibition of glutathione conjugation or sulphation of acetaminophen.
The aim of the present study was to examine the effects of the hepatotoxic drug acetaminophen (AA) on the synthesis rates of glutathione (GSH), activated sulphate (PAPS; adenosine 3'-phosphate 5'-phosphosulphate) and the AA metabolites AA-GSH and AA-sulphate after selective inhibition of GSH biosynthesis or sulphation in isolated rat hepatocytes. Selective inhibition of the two interdependent metabolic pathways was accomplished by buthionine sulphoximine (BSO) and 2,6-dichloro-4-nitrophenol (DCNP). The synthesis rates of GSH and PAPS were determined simultaneously by a previously described method based on trapping of radioactivity (35S) in the pre-labelled GSH and PAPS pools. Pre-incubation with 10 mM BSO for 30 min depleted GSH by 38% (P < 0.05) and PAPS by 27% (P < 0.05). The depletion resulted in increased PAPS synthesis at low, non-toxic [5-19 nmol/(10(6) cells.min)] (P < 0.05) and at high, toxic [7-30 nmol/10(6) cells.min)] (P < 0.05) AA concentrations. In both cases sulphur is diverted from GSH biosynthesis to sulphoxidation and PAPS synthesis, thereby maintaining the PAPS pool and preserving the sulphation capacity. This corresponds to the finding that AA sulphation was unaffected by BSO irrespective of AA concentration [6 vs 5 and 20 vs 17 nmol/(10(6) cells.hr), respectively]. Even though the GSH synthesis was halved after BSO pre-incubation, the GSH conjugating capacity of AA was well preserved. Incubation with 200 microM DCNP and 5 mM AA diminished PAPS synthesis from 24 to 10 nmol/(10(6) cells.min) (P < 0.02) and reduced AA-sulphate synthesis by 67% compared to experiments without DCNP incubation [4.8 vs 14.7 nmol/(10(6) cells.hr)] (P < 0.05). GSH and AA-GSH synthesis rates did not change compared to control experiments in which sulphation was not inhibited [1165 vs 1487 nmol/(10(6) cells.min), respectively] and [1.7 vs 1.7 nmol/(10(6) cells.hr), respectively]. This indicates that increased sulphur availability due to decreased PAPS synthesis is unable to raise the cysteine pool and stimulate the gamma-glutamyl cycle and GSH synthesis.
Acetaminophen↗
Inhibition of acetaminophen oxidation by cimetidine and the effects on glutathione and activated sulphate synthesis rates.
The aim of the present study was to examine the effects of the hepatotoxic drug, acetaminophen, on the synthesis rates of glutathione, activated sulphate (PAPS, adenosine 3'-phosphate 5'-phosphosulphate) and the acetaminophen metabolites, acetaminophen-glutathione and acetaminophen-sulphate after inhibition of cytochrome P-450 drug oxidation by cimetidine in isolated rat hepatocytes. The synthesis rates of glutathione and PAPS were determined simultaneously by an established method based on trapping of radioactivity (35S) in the prelabelled glutathione and PAPS pools. Preincubation of the hepatocytes with 60 micrograms/ml cimetidine for 30 min. did not affect PAPS (1.71 versus 1.78 nmol/10(6) cells) nor glutathione concentration (16.0 versus 16.4 nmol/10(6) cells). The subsequent incubation with 5 mM acetaminophen resulted in decreased PAPS synthesis in the cimetidine treated cells [0.79 x 10(3) versus 0.92 x 10(3) nmol/(10(6) cells.hr)] (P < 0.05). There was no difference in PAPS concentration or acetaminophen-sulphate synthesis [1.73 versus 1.79 nmol/10(6) cells and 13.0 versus 12.9 nmol/(10(6) cells.hr), respectively]. Decreased PAPS synthesis may be related to decreased ATP supply or may be the result of a feed-back regulation due to diversion of sulphur from glutathione synthesis to sulfoxidation. The glutathione synthesis was not significantly affected by cimetidine treatment [57 x 10(3) versus 27 x 10(3) nmol/(10(6) cells.hr)]. As expected acetaminophen-glutathione synthesis decreased by 38% [1.66 versus 2.68 nmol/(10(6) cells.hr)] (P < 0.01). Also the glutathione concentration was lower in cimetidine treated cells [15.2 versus 15.9 nmol/10(6) cells] (P < 0.05). We have previously shown that glutathione synthesis was reduced if substrate availability decreased (acetaminophen concentration lowered).(ABSTRACT TRUNCATED AT 250 WORDS)
Acetaminophen↗
Intercellular adhesion molecule 1 (ICAM-1) in the pathogenesis of mononuclear cell alveolitis in pulmonary sarcoidosis.
BACKGROUND: Alveolitis in pulmonary sarcoidosis is characterised by an accumulation of highly activated macrophages and CD4+ lymphocytes in the alveolar compartment. The role of intercellular adhesion molecule 1 (ICAM-1) expression on alveolar cells has been studied in this context. METHODS: Using a sandwich ELISA technique, ICAM-1 expression on alveolar macrophages from 17 consecutive untreated patients with pulmonary sarcoidosis and six healthy normal volunteers was quantified. In addition, parameters of macrophage activation (tumour necrosis factor alpha (TNF alpha) and superoxide anion release) were evaluated. RESULTS: Significantly elevated expression could be demonstrated on alveolar macrophages from patients with pulmonary sarcoidosis compared with healthy controls (mean (SD) 0.74 (0.24) ELISA units (EU) v 0.46 (0.12) EU). On subdividing the patients into those with active and those with inactive disease, only the former showed increased ICAM-1 levels on alveolar macrophages (0.82 (0.27) EU) compared with control alveolar macrophages. No differences were detected in serum levels of soluble ICAM-1 between patients and controls. ICAM-1 expression on alveolar macrophages from patients with sarcoidosis correlated with the spontaneous release of TNF alpha but not with the release of the superoxide anion by the activated macrophages. There was no correlation with the percentage of lymphocytes or the absolute number of CD4+ cells in bronchoalveolar lavage fluid. CONCLUSIONS: Increased ICAM-1 surface expression on alveolar macrophages reflects disease activity in the pulmonary compartment. Considering the significance of adhesion molecules during antigen presentation and lymphocyte activation, ICAM-1 expression on alveolar macrophages may have an important role in the immune process of pulmonary sarcoidosis.
Adult↗
Drug metabolism and genetic polymorphism in subjects with previous halothane hepatitis.
To test the hypothesis that halothane hepatitis is caused by a combination of altered drug metabolism and an immunoallergic disposition, the metabolism of antipyrine, metronidazole, sparteine, phenytoin, and racemic R- and S-mephenytoin was investigated in seven subjects with previous halothane hepatitis. The HLA tissue types and the complement C3 phenotypes were also determined. The metabolism of antipyrine and metronidazole was within normal range in all subjects, and they were all fast or extensive metabolizers of sparteine, mephenytoin, and phenytoin. HLA tissue types were unremarkable. Five of the seven subjects had complement C3 phenotypes F or FS. In the general population phenotype S is the most common, but the difference in complement C3 phenotypes is not statistically significant (p = 0.07). We conclude, although in a limited number of patients, that subjects with previous halothane hepatitis do not appear to be different from controls with regard to drug metabolism and HLA tissue type. The possibility of a higher frequency of complement C3 phenotype F and FS needs further investigation.
Adult↗
Effects of cysteine and acetaminophen on the syntheses of glutathione and adenosine 3'-phosphate 5'-phosphosulfate in isolated rat hepatocytes.
The aim of the present study was to introduce and validate a radioactive tracer method in which adenosine 3'-phosphate 5'-phosphosulfate (PAPS) and glutathione (GSH) are measured simultaneously in isolated hepatocytes. PAPS and GSH are co-substrates in sulphation and GSH conjugation, and both are dependent on sulphur deriving from cysteine. The effect of cysteine on the syntheses was investigated at non-toxic and toxic concentrations of the hepatotoxic drug acetaminophen (AA). Administration of AA trapped radioactivity (35S) in the pre-labelled PAPS and GSH pools by formation of the metabolites, AA-sulphate and AA-GSH. Turnover rates were determined from the decline of AA-sulphate and AA-GSH specific activity. Syntheses of PAPS and GSH were calculated by multiplying the rates with the concentrations of the respective co-substrates. Increasing AA concentration from non-toxic to toxic levels resulted in increased median PAPS and GSH syntheses (8 to 11 and 311 to 2218 nmol/10(6) cells/min, respectively) (P less than 0.05). Addition of cysteine did not alter median PAPS synthesis (5 to 3 nmol/10(6) cells/min) but decreased median GSH synthesis (666 to 261 nmol/10(6) cells/min) (P less than 0.05) in experiments with non-toxic AA concentrations. In experiments with toxic AA concentrations opposite effects of cysteine were seen, i.e. median PAPS synthesis was reduced (3 to 2 nmol/10(6) cells/min) (P less than 0.05) while median GSH synthesis was unchanged (23 to 16 nmol/10(6) cells/min). The present method provides a tool in which two important detoxification pathways can be measured simultaneously and the data suggest that the two pathways are regulated by substrate availability.
Acetaminophen↗
[Oxygen radical formation in pulmonary sarcoidosis. The signs of early macrophage activation].
Oxygen radical formation of alveolar macrophages (by luminogenic substrate-intensified chemiluminescence) and the concentrations of phagocyte products myeloperoxidase, elastase and lactoferrin, as well as alpha-proteinase inhibitor and albumin were measured in bronchoalveolar lavage fluid of 28 patients with pulmonary sarcoidosis. There were 15 men and 13 women (mean age 41 [18-62] years), 10 of them with sarcoidosis I, 10 with clinically active sarcoidosis II and 8 with inactive sarcoidosis II. Six healthy persons served as controls. The purpose of the study was to demonstrate the extent of correlation between macrophage activity and stage of sarcoidosis. Patients in stage I had significantly higher luminescence (346 +/- 253 relative light units [RLU] per second) than the controls (117 +/- 29 RLU/s; P less than 0.02). But the difference between controls and patients in clinically active stage II (294 +/- 75 RLU/s) was not significant and the luminescence in patients with clinically inactive stage II was within normal range (119 +/- 33 RLU/s). The concentrations of proteins measured in the lavage fluid was increased, independent of the site of formation. These data indicate that in pulmonary sarcoidosis there exists, early and independent of stage, a marked activation of alveolar macrophages with increased production of oxygen radicals and a resulting increase in permeability of the alveolo-capillary membrane.
Adolescent↗