Analysis of effects of MCB3681, the antibacterially active substance of prodrug MCB3837, on human resident microflora as proof of principle.

The water-soluble prodrug MCB3837 is rapidly converted to MCB3681, active against Gram-positive bacterial species, after intravenous infusion. The aim of this study was to prove the principle that MCB3681 is efficacious in vivo by demonstrating its effect on the resident microflora or colonizers of the human skin, nose, oropharynx and intestine. MCB3837 was infused at a daily dose of 6 mg/kg for 5 days. MCB3681 was active against clostridia, bifidobacteria, lactobacilli, enterococci and Staphylococcus aureus, thus proving the principle that MCB3681 is antibacterially efficacious in vivo without affecting the Gram-negative microflora.

Flow cytometric DNA analysis using cytokeratin labeling for identification of tumor cells in carcinomas of the breast and the female genital tract.

Flow cytometric assessment of DNA-ploidy and S-phase fraction in malignant tumors is compromised by the heterogeneity of cell subpopulations derived from the malignant and surrounding connective tissue, e.g., tumor, stromal and inflammatory cells. To evaluate the effect on quality of DNA cell cycle analysis and determination of DNA ploidy, cytokeratin labeling of epithelial cells was used for tumor cell enrichment in breast, ovarian, cervical and endometrial cancer prior to DNA analysis. In a prospective study, tumor cell subpopulations of 620 malignant tumors were labeled by a FITC-conjugated cytokeratin antibody (CK 5, 6, CK18 and CK 5, 6, 8 and CK 17, respectively) prior to flow cytometric cell cycle analysis. Compared to total cell analysis, detection rate of DNA-aneuploid tumors following cytokeratin labeling was increased from 62% to 76.5% in breast cancer, from 68% to 77% in ovarian cancer, from 60% to 80% in cervical cancer and from 30% to 53% in endometrial cancer. Predominantly in DNA-diploid tumors, a significantly improved detection of S-phase fraction of the tumor cells was shown due to the elimination of contaminating nonproliferating "normal cells". S-phase fraction following tumor cell enrichment was increased by 10% (mean) following cytokeratin staining in ovarian and endometrial cancer, by 30% in breast cancer and even by 70% in cervical cancer compared to total cell analysis. Thus, diagnostic accuracy of DNA-analysis was enhanced by cytokeratin labeling of tumor cells for all tumor entities investigated.

DNA cell-cycle analysis of cervical cancer by flow cytometry using simultaneous cytokeratin labelling for identification of tumour cells.

DNA ploidy and cell-cycle distribution were determined by flow cytometry in fresh tumour tissue of 53 cervical carcinomas. Epithelial cells were labelled by a fluorescein-isothiocyanate-conjugated cytokeratin antibody (CK6, CK18) to study the influence of contaminating stromal and inflammatory cells on results of cell-cycle analysis of tumour cells. Without identification of cytokeratin-positive cells 30/53 (57%) tumours were found to be DNA-aneuploid compared to 43/53 (81%) after gating for cytokeratin. Only 7 of 15 DNA-multiploid tumours could be detected without cytokeratin staining. In addition, cytokeratin-negative cells, which are found in all tumours, can be used as an internal standard for the calculation of ploidy and for quality control (coefficient of variation, linearity) of each individual sample. Cell-cycle analysis revealed significantly higher S-phase and G2M-phase fractions in cytokeratin-gated compared to ungated samples (13.1% versus 10.0% and 8.0% versus 5.4%; P < 0.001). This difference was more pronounced in DNA-diploid than DNA-aneuploid tumours. In conclusion, about 30% of DNA-aneuploid tumours could only be detected after cytokeratin labelling of epithelial cells. Owing to the identification of cytokeratin-positive cells the influence of non-tumoural cell elements on cell-cycle analysis was reduced markedly. Therefore, in cervical cancer, cytokeratin labelling can optimize both the determination of DNA ploidy and cell-cycle analysis.

Flow cytometric DNA analysis of breast cancer by two colour method using cytokeratin labeling for identification of tumour cells.

Flow cytometric assessment of DNA-ploidy and S-phase fraction in breast cancer is compromised by the heterogeneity of cell subpopulations derived from the malignant and surrounding connective tissue, e.g. tumour, stromal and inflammatory cells. To identify tumour cell subpopulations, epithelial cells were labeled by a FITC-conjugated cytokeratin antibody (CK6, CK18) prior to flow cytometric cell cycle analysis in 205 fresh specimens of primary breast cancer. We found 158/205 (77%) DNA-aneuploid tumours compared to 127/205 (62%) without identification of cytokeratin positive cells (P < 0.001). In addition, the number of detected DNA-multiploid tumours rose from 31 (15%) to 51 (25%) after gating for cytokeratin positive cells. In DNA-diploid tumours, S-phase and G2M-phase fractions were significantly higher in cytokeratin positive (tumour) cells compared to total cell populations (4.4% and 5.8% vs. 3.2% and 4.4%; P < 0.001). Cytokeratin negative cells were found in all tumours and can be used as internal standard for calculation of ploidy and for quality control (CV, linearity) of each individual sample. We conclude that at least 20% of DNA-aneuploid tumours would not have been diagnosed without cytokeratin labeling. In addition, influence of non-tumourous cell elements on cell cycle analysis can be markedly reduced. Therefore, both determination of DNA-ploidy and cell cycle analysis can be optimized by cytokeratin labeling.

Skeletal muscle partial pressure of oxygen in patients with sepsis.

OBJECTIVE: In order to obtain direct evidence for tissue hypoxia in patients with sepsis oxygen, partial pressure was measured within skeletal muscle. Furthermore, serial intermittent and continuous measurements of skeletal muscle PO2 in patients with sepsis were used to find out whether skeletal muscle oxygenation may change in the course of sepsis and depends on the severity of sepsis. DESIGN: Prospective study. SETTING: Intensive care unit of a university hospital. PATIENTS: Intensive care patients (n = 98) with sepsis (group 1, n = 39; group 4, n = 28), limited infection (group 2, n = 16), and cardiogenic shock (group 3, n = 15). INTERVENTIONS: Pulmonary artery catheterization; standard antibiotic therapy and volume replacement. MEASUREMENTS AND MAIN RESULTS: Skeletal muscle PO2 was determined by polarographic needle electrodes or catheter probes. In patients with sepsis (n = 67), no evidence for skeletal muscle hypoxia was obtained from the PO2 distribution within biceps muscle. Mean skeletal muscle PO2 was increased in patients with sepsis (group 1, 48 torr [6.4 kPa]) compared with patients with limited infection (group 2, 28 torr [3.7 kPa]), p < .001) and with patients with cardiogenic shock (group 3, 22 torr [2.9 kPa], p < .001). Serial measurements of the PO2 distribution during seven consecutive days in another 28 patients (group 4) with sepsis showed that a more severe degree of sepsis was associated with an increase of mean skeletal muscle (p < .001). These results were confirmed by continuous measurements of mean skeletal muscle PO2, using PO2 catheters. CONCLUSIONS: In patients with sepsis, oxygen transport to skeletal muscle was not critically reduced. Serial intermittent and continuous measurements of skeletal muscle PO2 showed that skeletal muscle PO2 increased in relation to the severity of the stage of sepsis. Our findings suggest that oxygen utilization within skeletal muscle decreased with deterioration of sepsis, thereby increasing skeletal muscle PO2.

Repeated administration of a F(ab')2 fragment of an anti-tumor necrosis factor alpha monoclonal antibody in patients with severe sepsis: effects on the cardiovascular system and cytokine levels.

In an uncontrolled clinical trial the effects of repeated administration of the F(ab')2 fragment of a murine monoclonal anti-tumor necrosis factor alpha (TNF alpha)-antibody (MAK 195F) on cytokine levels and the cardiovascular system were studied in 20 patients with severe sepsis. Patients were treated with a total of 11 single dosages of the anti-TNF alpha-antibody intravenously over 5 days using either 1 mg/kg (n = 10) or 3 mg/kg (n = 10). The anti-TNF alpha-antibody was well tolerated in all patients without signs of toxicity and without development of anti-murine antibodies. As assessed by cytokine levels (TNF alpha, Interleukin-6) and hemodynamics there was no evidence that the higher dosage of the anti-TNF alpha-antibody (3 mg/kg per dose) was more effective than the lower dosage (1 mg/kg per dose). Comparison of our data with recent data from phase I or II trials using a complete murine monoclonal anti-TNF alpha-antibody suggest that the F(ab')2 fragments of the murine monoclonal anti-TNF alpha-antibody may be of similar efficacy. Definitive conclusions, however, with respect to improvement of mortality and improvement of the cardiovascular system, await the results of larger ongoing placebo-controlled trials.

Changes in skeletal muscle pO2 after administration of anti-TNF alpha-antibody in patients with severe sepsis: comparison to interleukin-6 serum levels, APACHE II, and Elebute scores.

In 20 patients with severe sepsis, skeletal muscle pO2 was continuously measured in order to assess whether a decrease of skeletal muscle pO2 was accompanied by an improvement of sepsis after repeated administration of F(ab')2 fragments of a murine anti-TNF alpha-antibody. Abnormally high skeletal muscle pO2 decreased from 43.5 +/- 10.9 mmHg (day 0) to 36.4 +/- 10.1 mmHg within 24 h after the first administration of anti-TNF alpha-antibody (day 1, p = .006, n = 20) and remained at 34.6 +/- 7.7 mmHg thereafter (mean day 2-7, p = .004). The decrease of skeletal muscle pO2 within 24 h exceeded 5 mmHg (-7 to -19 mmHg) in 11 patients in contrast to nine patients (-4 to +4 mmHg). Only in the patients showing a decrease of skeletal muscle pO2 did sepsis improve as determined by Elebute score, APACHE II score, and interleukin-6 serum levels. The change of skeletal muscle pO2 within 24 h was associated with a change of interleukin-6 serum levels within 24 h (r = .5, n = 20), with a change of Elebute score (r = .7, n = 20) and of APACHE II score (r = .62). These data suggest that a decrease of skeletal muscle pO2 might be an early indicator of improvement of sepsis after administration of anti-TNF alpha-antibodies.

Preservation of regional myocardial function and myocardial oxygen tension during acute ischemia in pigs: comparison of selective synchronized suction and retroinfusion of coronary veins to synchronized coronary venous retroperfusion.

OBJECTIVES: The efficacy of selective synchronized suction and retroinfusion of coronary veins was compared with synchronized coronary venous retroperfusion in preventing ischemic reduction of regional myocardial function and myocardial oxygen tension. BACKGROUND: Because incomplete protection by synchronized coronary venous retroperfusion during ischemia might result from nonselective retroinfusion and only passive drainage of the veins, a suction device was added to a retroinfusion system. METHODS: Regional myocardial function (ultrasonic crystals) and myocardial oxygen tension (polarographic electrodes) were studied in 30 pigs during 10-min occlusion of the left anterior descending coronary artery (ischemia), followed by reperfusion. During ischemia, group A (n = 10) was supported by selective synchronized suction and retroinfusion; group B (n = 10) was supported by synchronized coronary venous retroperfusion, and group C (n = 10) was not supported by retroinfusion. RESULTS: In group A, subendocardial segment shortening decreased from 21 +/- 4% (mean +/- SD) before ischemia to 11 +/- 5% during ischemia. In contrast, systolic dyskinesia was observed in group B (-2 +/- 4%, p < 0.001) and group C (-2 +/- 5%, p < 0.001). During ischemia, the decrease in intramyocardial oxygen tension was less pronounced in group A (41 +/- 15 vs. 27 +/- 12 mm Hg) than in group B (40 +/- 10 vs. 19 +/- 10 mm Hg, p = 0.1) or group C (33 +/- 11 vs. 12 +/- 8 mm Hg, p = 0.002). During ischemia, myocardial surface oxygen tension was preserved > 0 mm Hg only in group A. CONCLUSIONS: Preservation of regional myocardial function and myocardial oxygen tension was substantially higher by selective synchronized suction and retroinfusion of coronary veins than by synchronized coronary venous retroperfusion in pigs.

Desensitization of rat cardiomyocyte adenylyl cyclase stimulation by plasma of noradrenaline-treated patients with septic shock.

The purpose of this study was to test the possibility that the mechanisms of catecholamine-induced desensitization of cardiac beta-adrenoceptor stimulation are modified in septic shock. Exposure of neonatal rat cardiomyocytes for 48 hr to plasma of noradrenaline-treated patients with septic shock led to a down-regulation of beta-adrenoceptors by 35%, an increase in the level of inhibitory G protein alpha-subunits by 60%, and a decrease in isoproterenol-stimulated adenylyl cyclase activity by 50% in membranes prepared from the rat cardiomyocytes. Similar alterations were observed following pretreatment of the cells with plasma of adrenaline-treated patients with cardiogenic shock. In contrast, exposure of the cardiomyocytes to plasma of intensive care patients without shock, and to plasma of dopamine-treated patients with septic shock did not induce alterations of the cardiomyocyte adenylyl cyclase system. The dosage of the catecholamines had to be increased within the first two days of treatment in the noradrenaline-treated patients, but not in the dopamine-treated patients with septic shock. Thus, this observed tolerance to noradrenaline in the treatment of septic shock may, in part, be due to a desensitization of cardiac beta-adrenoceptor stimulation induced by the beta-adrenoceptor stimulatory effect of noradrenaline.

[Continuous measurement of peripheral oxygen availability in skeletal muscle of patients with infection]. / Kontinuierliche Messungen der peripheren Sauerstoffverfügbarkeit im Skelettmuskel bei Patienten mit Sepsis.

In patients with sepsis, a pathologic oxygen uptake supply dependence due to an oxygen extraction defect was suggested to result in tissue hypoxia--a hypothesis which is discussed controversially. In order to determine more directly whether the oxygen transport to tissue was reduced in patients with sepsis, the distribution of skeletal muscle pO2 was measured intermittently by polarographic needle electrodes in 28 patients with sepsis for 7 days. Comparison of intermittent with continuous measurements by pO2 catheters showed a close linear relation (r = 0.88; p < 0.001) and acceptable agreement. Therefore, continuous measurement of skeletal muscle pO2 by pO2 catheters can be used as an estimate of mean skeletal muscle pO2. Neither by intermittent nor by continuous measurements, skeletal muscle hypoxia was detected in patients with sepsis. In contrast, the skeletal muscle pO2 was statistically significantly higher on days with septic state (44.1 +/- 10.3 mm Hg) than on days with intermediate (30.1 +/- 8 mm Hg) and nonseptic (26.8 +/- 5.1 mm Hg) states. From our data we infer that the hypothesis of tissue hypoxia in sepsis must be questioned at least for the skeletal muscle. The increase of skeletal muscle pO2 in sepsis, however, suggested that oxygen utilization within skeletal muscle decreased the more severe the stage of sepsis was. A decreased oxygen utilization within tissue, which may result from a 'downregulation' of oxygen-dependent metabolic pathways, might account for a decreased oxygen extraction of peripheral tissue in sepsis.