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Biomedical subjects

M Kunc

Publications and source records attributed to M Kunc.

6 recordsLinked to original sources
PubMed2001

Influence of irradiation on neutrophilic granulocyte function.

BACKGROUND: Neoadjuvant radiotherapy is a common treatment modality for patients with Stage II and III rectal carcinoma but, after surgery, often is complicated by local infections. To define a possible influence of radiotherapy on neutrophilic granulocytes in the neighborhood of tumor cells, the authors investigated their function in vitro. METHODS: Density gradient-purified granulocytes from healthy donors were used for all tests. These cells were cocultured with the colon carcinoma cell line HRT-18 and irradiated. Their function was assessed by measuring luminol-enhanced chemiluminescence and migration against the chemoattractant formyl-methionyl-leucine-phenylalanine. RESULTS: Although irradiation decreased, the addition of tumor cells increased reactive-oxygen species release in granulocytes, which was enhanced further by phorbol myristate acid (PMA), even after several hours. All contacts with tumor cells, however, caused immediate radical release that was inversely proportional to the radiation dose. Naïve and irradiated cells were stimulated further by PMA. Migration of granulocytes clearly was inhibited by tumor cells and irradiation, whereas the depth of invasion was enhanced by higher doses of radiation. CONCLUSIONS: The current data show clearly that the influence of radiotherapy on local defense against colorectal carcinoma is limited and cannot explain the increased rate of infectious complications.

Carcinoma↗
PubMed2000

Polymorphonuclear leukocyte functions as predictive markers for infections after organ transplantation.

Infectious complications are still a major cause of morbidity and mortality after organ transplantation, and early therapy would certainly reduce the risk associated with severe infections. We therefore investigated the significance of polymorphonuclear leukocyte (PMN) functional tests as predictive markers for infection in transplant patients under immunosuppressive therapy in a longitudinal study. In 41 patients, blood PMN migration and reactive oxygen species release, the blood levels of PMN elastase, malondialdehyde, neopterin, sICAM-1 and sVCAM-1, and urine neopterine were measured in 3- and 4-day intervals after liver-, kidney-, kidney-pancreas-, and heart and lung transplantation. PMN migration was determined in whole blood and estimated by the amount of PMNs to penetrate into a membrane filter upon FMLP stimulation. Three groups of patients were formed according to their postoperative course. Group I patients (n = 23) had no or only minor local infection, group II patients (n = 11) had infections with distinct systemic involvement, and group III patients (n = 7) developed sepsis. A first elastase-level of over 100 mg/L after surgery, followed by a drop in the amount of blood PMNs ready to migrate, on FMLP stimulation, to below 12 %, turned out to be a marker for impending infection, whereas all other parameters tested were not predictive. In six of seven group III patients, this marker became positive (sensitivity 85.7 %) up to 15 days before clinical manifestation of sepsis. In group I (largely uneventful recovery) only one of 23 patients was positive (specificity 95.6 % ), whereas group II patients were in between (4 of 11 positive). By this method it seems possible to diagnose severe infections in the pre-clinical phase, which may help prevent them if treatment is begun promptly.

Adult↗
PubMed1997

Respiratory defect as an early event in preservation-reoxygenation injury of endothelial cells.

Characterization of preservation injury in endothelial cells has been primarily accomplished by measurement of cell viability. To analyze early events and cellular mechanisms of preservation-reoxygenation injury, we developed high-resolution respirometry for the study of mitochondrial function in endothelial cells, to provide a quantitative marker for sublethal stress. Cultured human umbilical vein endothelial cells were stored for 4 and 8 hr at 4 degrees C under an atmosphere of 95% N2 and 5% CO2 in University of Wisconsin (UW) and histidine-tryptophan-ketoglutarate (HTK) solutions. Respiration of suspended cells, measured after reoxygenation in growth medium at 37 degrees C, was significantly reduced in all treatments in comparison to controls not subjected to cold preservation. In contrast, trypan blue staining was unchanged after 4 hr of preservation and was significant only after 8 hr. After 8 hr of cold storage in UW and HTK solutions, respiration was 64+/-5% and 49+/-6%, respectively, of controls (46.5+/-3.3 pmol O2 x s(-1 x 10(-6) cells), indicating significantly better protection by UW solution than HTK solution. A titration regimen with substrate (succinate), uncoupler (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), and inhibitors of complexes I and III (rotenone and antimycin A) resulted in identical respiratory response patterns in all treatments. The plasma membrane remained impermeable to succinate. Inner mitochondrial membrane function was preserved as indicated by a constant relative increase of respiration after uncoupling. These results demonstrate that loss of catalytic capacity for respiration constitutes an early event in preservation-reoxygenation injury, whereas membrane damage is not a primary defect. Respirometric evaluation of sublethal cell injury and localization of cell damage may provide selective guidelines for further optimization of strategies in organ preservation.

Adenosine Triphosphate↗
PubMed1996

Oxygen dependence of respiration in coupled and uncoupled endothelial cells.

We studied the oxygen dependence of respiration in cultured human umbilical vein endothelial cells by use of high-resolution respirometry. The rate of oxygen consumption varied from 30 to 50 pmol O2.s-1.(10(6) cells)-1 over a sixfold range of cell densities. Respiration was stimulated up to 3.5-fold by uncoupling with carbonyl cyanide p-trifluoromethoxyphenylhydrazone or 2,4-dinitrophenol, and the PO2 at half-maximal respiration (P50) increased from 0.05 to 0.12 kPa (0.3 to 0.9 Torr) with respiratory rate. P50 decreased to a minimum of 0.02 kPa when uncoupled cells were inhibited to control levels. Differences in cell size explained a variation of approximately 0.015 kPa in P50 at similar respiratory rates per cell. Oxygen diffusion to mitochondria contributed maximally 30% to the regulation of P50 in coupled cells, as deduced from the shallow slope of the flux dependence of P50 in uncoupled-inhibited cells compared with the slope in coupled cells. Therefore 70% of the flux dependence of P50 in coupled cells was caused by changes in metabolic state, which correlated with respiratory rate.

Cell Hypoxia↗
PubMedDate not supplied

Optimization of phagocyte chemiluminescence measurements using microplates and vials.

In order to cope with large amounts of samples for chemiluminescence (CL), vials were replaced with microplates. Although various types of plates have been commercially available for quite some time and the free-plate mode is advocated by the producer of the counter, little is known about their impact on the outcome of CL measurements. We tested two different 24-well microplates and six different 96-well microplates in two different luminometers, and results were compared with those achieved with vials. Before these comparative tests, we attempted to optimize measurement conditions. CL sensitivity was highest with luminol concentrations of 0.8-3.3 micromol/L, PMA concentrations of 0.06-80 micromol/L, a pH value of 10 and a temperature of 20 degrees C. An indirect correlation was found between fluid volume and yield in counts: the lower the volume, the higher the counts. With regard to sensitivity and cross-talk, the 96-well Isoplatetrade mark was superior to all other plates tested. While all white plates tested gave acceptable results, usage of the black 96-well plates resulted in an extremely low sensitivity. Plates designed for cell culturing gave even lower counts and a cross-talk of up to 31%. All attempts to reduce cross-talk and improve sensitivity, such as aluminium foil or grids, irrespective of the position of the photomultiplier, did not give results comparable to the original 96-well isoplate. Our results suggest that, with the exception of black 96-well microplates and cell culture plates, all other plates tested have a sufficient sensitivity when compared to vials and acceptable cross-talk, the 96-well Isoplatetrade mark being the best. Both types of luminometers used gave reproducible results, Wallac having a somewhat higher sensitivity, Canberra Packard somewhat less cross-talk.

Humans↗