Biomedical subjects

C J Kirkpatrick

Publications and source records attributed to C J Kirkpatrick.

At least 109 records · Page 6Linked to original sources

PubMed1994

Application of immunogold labelling for light and electron microscopic localization of endothelial leukocyte adhesion molecule 1 (ELAM-1) on cultured human endothelial cells.

This study describes the expression characteristics of E-selectin molecules using immunogold histochemical techniques on cultured human umbilical vein endothelial cells (HUVEC). The expression of E-selectin was induced by tumour necrosis factor-alpha (TNF-alpha, 300 U/ml), phorbol ester (PMA, 10 ng/ml) and bacterial lipopolysaccharide (LPS, 4 micrograms/ml). No expression was demonstrated on control cells. Using the silver-enhanced colloidal gold-labelling technique, at the light microscopical level, HUVEC could be distinctively subdivided into three staining types. The cell labelling index, expressed as the number of 'positively' stained cells as a proportion of all viewed cells was the highest in the LPS group. For transmission electron microscopy (TEM) the preembedding immunocytochemical staining method and embedding in epoxy resin (Agar 100) according to standard procedures was used. In TEM gold particles were localized in close association with the apical plasma membrane, as well as on the surface of microvillus-like projections (the latter by TNF-alpha group). For high resolution scanning electron microscopy (HR-SEM) the secondary (SEI) and the backscattered electron imaging (BEI) modes were used. Gold particles were randomly distributed over the whole cell surface, although they appeared to be denser in the perinuclear zone. The quantitative evaluation on SE and BE viewing (the number of gold particles per cell area in microns 2) demonstrated the highest density of labelling in the LPS-treated group, but there was only a significant difference between LPS and TNF-alpha groups (P < 0.01, t-test). Furthermore, the ultrastructural studies indicated that treatment with substances which up-regulate E-selectin expression was not related to toxic cell damage or significant alterations of cellular ultrastructure.

Cell Adhesion Molecules↗

PubMed1994

Fluid shear induced endothelial cell detachment from glass--influence of adhesion time and shear stress.

In this study, human umbilical vein and human saphenous vein endothelial cells were seeded on glass and exposed to fluid shear in a parallel-plate flow chamber. Cell retention, morphology and migration were studied as a function of shear stress and of adhesion time prior to exposure to shear. Three-hour and 24-h adhesion times gave rise to comparable cell retention values after 2 h of flow for both cell types. Cell retention decreased from 85 to 20% as shear stress increased from 88 to 264 dynes cm-2 (8.8 to 26 Pa). Mean spreading areas decreased after the onset of flow, but subsequently stabilized to plateau values, which were smaller at higher shear stresses. Shape factors increased faster to higher values as cells were exposed to higher shear stresses, without any obvious preference in orientation of the cells with respect to the direction of flow. Migration was unidirectional with flow and linear with time. Migration was faster for cells which had adhered for 24 h than for cells which had adhered for 3 h and was accompanied by the presence of fibrillar structures left behind on the surface upstream of migrating cells. It is concluded that after 3 h adhesion to glass, cells have adhered with an adhesion strength that does not substantially increase during the next 21 h. However, during this time changes in cell-substratum interactions seem to occur judging by the differences in, e.g., migration rates.

Blood Vessel Prosthesis↗

PubMed1994

Treatment of skin defects using suspensions of in vitro cultured keratinocytes.

The in vitro cultivation of keratinocytes and their application in the form of confluent sheets to cover various kinds of skin defects involves a number of problematical steps which could be improved by using single cell suspensions instead. Therefore we developed a method to apply keratinocytes suspended as single cells in a fibrin gel. By testing the feasibility of this method in different experimental animal models we found that it facilitates cultivation as well as application of the cells, moreover, this method allows a much more flexible use of the cells, i.e. it is easier to consider the clinical condition of the patient than by the conventional method.

Animals↗

PubMed1994

Association of different macrophage phenotypes with infiltrating and non-infiltrating areas of tumor-host interface in colorectal carcinoma.

At the tumor-host interface (interface) of well differentiated tubulary or tubulopapillary colorectal carcinomas infiltrative, poorly demarcated and non-infiltrative, well bordered areas alternate. The composition of the inflammatory infiltrate within the desmoplastic stroma of the central tumor part and the interface was analyzed, particularly emphasizing differences between infiltrative and non-infiltrative areas of the interface. Of particular interest was the distribution of the following recently identified, functionally different human macrophage phenotypes: the 27E10-positive phenotype, an inflammatory macrophage, the 25F9-positive phenotype, a mature, resident macrophage and the RM3/1-positive phenotype, associated with anti-inflammatory function. It was found that macrophages were the dominating cells in the inflammatory infiltrate of both central tumor part and interface and that the number of B-cells and NK-cells were negligible. The 27E10-positive phenotype revealed a strong association with infiltrative areas at the interface, whereas the resident macrophage together with the RM3/1 was associated with sharply bordered tumor areas dominating within the tumor stroma, particularly in carcinomas with marked desmoplastic stroma response. These findings suggest that different macrophage phenotypes, localized in different regions of the carcinoma, have different effects on tumor cells.

Adenoma↗

PubMed1994

Increased adhesion and activation of polymorphonuclear neutrophil granulocytes to endothelial cells under heavy metal exposure in vitro.

Heavy metals have been implicated in the mechanisms of endothelial damage. Influences of heavy metal ions on diverse cell types have been studied using a variety of in vitro and in vivo methods. Polymorphonuclear neutrophil granulocytes (PMNs) have physiological and pathological functions, including the modulation of adhesion to and destruction of endothelial cells (ECs). PMNs were studied during interaction with human umbilical vein ECs under exposure to zinc, nickel and cobalt using an in vitro model. We studied adhesion processes with the help of a computer-controlled image-analyzing system and examined the activation of PMNs by quantification of leukotriene B4 (LTB4) release. The biphasic effects of the evaluated heavy metals on PMN-EC adhesion, with stimulation at very high and very low molar concentrations, were observed. The release of LTB4 by PMNs increased during exposure to very low metal concentrations. The initiation of these important pathogenetic mechanisms of inflammation at very low metal ion concentrations, which give no morphological changes, must be regarded as potentially significant with respect to the toxic effects of heavy metals.

Cell Adhesion↗

PubMed1994

Comparative studies on vascular endothelium in vitro. I. Cytokine effects on the expression of adhesion molecules by human umbilical vein, saphenous vein and femoral artery endothelial cells.

Endothelial cells (ECs) are very responsive to proinflammatory cytokines. ECs are stimulated by these substances to increase expression of cell surface adhesion molecules, leading to dramatically altered interactions with leukocytes. In these interactions, E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are suggested to play the most important role. Recent evidence has suggested diversity in the responses of ECs from different regions of the vascular system. Human umbilical vein ECs (HUVECs) are the most often used EC culture model, although there are few studies comparing their response with other human EC types from the adult organism. In this study the expression of E-selectin, ICAM-1 and VCAM-1 on cultured human adult ECs from the saphenous vein (HSVECs) and from the femoral artery (HAFECs), as well as HUVECs was studied. Using a cell enzyme immunoassay as well as immunoelectron microscopical methods, we found that both HSVECs and HAFECs respond in a similar way to HUVECs to exogenous stimulation by IL-1 beta, TNF alpha or LPS. IL-1 beta and TNF alpha increased the expression of E-selectin on the cytoplasmic membranes of HUVECs, HSVECs and HAFECs and elicited even similar absolute quantities of this molecule, comparing the different cell types. ICAM-1 and VCAM-1 appeared to be regulated dose dependently by IL-1 beta, independent of the EC type. HUVECs as well as HSVECs and HAFECs gave a reproducible constitutive ICAM-1 expression, whereas E-selectin and VCAM-1 were absent on nonstimulated ECs.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1994

Differential adherence of the human monocyte subsets 27E10 and RM3/1 to cytokine- or glucocorticoid-treated endothelial cells.

Monocyte-endothelial interactions are of particular importance in the regulation of inflammation. The aim of the present study was to investigate the adhesion of functional different monocyte subsets to human umbilical vein endothelial cells treated with various cytokines or a glucocorticoid. The adherence of monocyte subset 27E10, which is associated with inflammatory processes, increased after endothelial activation with interleukin-1 beta (IL-1 beta), interferon-gamma (IFN gamma), tumor necrosis factor-alpha (TNF alpha), and the glucocorticoid prednylidene (Pred). The adherence at IFN gamma-treated endothelial cells was strong after a coculture duration of 10 min with a slight increase up to 60 min. The peak value after TNF alpha stimulation was reached after 15 min, thereafter quickly decreasing. IL-1 and Pred treatment caused a maximal adherence between 15 and 30 min followed by a slow decrease. TNF alpha and particularly interleukin-6 (IL-6) enhanced the endothelial adhesion of the monocyte subtype RM3/1, which is associated with the downregulation of inflammation. The maximal adherence was found after 15 and 30 min of coculture, respectively. The results show that, through modulation of the adhesive properties of endothelial cells, cytokines and glucocorticoids affect the adherence of monocyte subsets differently. They also suggest that IL-6 plays a role in the downregulation of acute inflammation.

Cell Adhesion↗

PubMed1993

Interaction of human malignant melanoma tumor spheroids with endothelium and reconstituted basement membrane: modulation by RGDS.

Tumor-cell extravasation involves sequential adhesive interactions with vascular endothelium and the subendothelial basement membrane. We have established a 3-dimensional model in vitro to simulate these events and to elucidate targets of the anti-cell-adhesive synthetic peptide RGDS. Tumor spheroids of the melanoma cell line ST-ML-12 served as models of tumor-cell emboli and were transferred onto human umbilical vein endothelial cells. To imitate the vascular anatomy, the latter were grown on reconstituted basement membranes produced by dextran-stimulated bovine corneal endothelial cells. RGDS did not affect the homotypic aggregation of the tumor cells and only minimally inhibited the attachment of the spheroids to the reconstituted vessel. A short-term (20 min) inhibition of adhesion to denuded basement membranes was observed. The attachment was closely associated with damage to the endothelial cells by oxygen-derived free radicals. RGDS retarded endothelial injury for up to 3 hr. The most prominent effect was observed after penetration of the endothelium. RGDS suppressed the emigration of tumor cells from the attached tumor-cell cluster in a dose- and time-dependent fashion. After 12 hr, the inhibitory effect progressively declined. This was not due to loss of activity of the peptide, indicating a resistance mechanism in the melanoma cells. On purified components of the basement membrane, RGDS effectively inhibited the initial spheroid attachment to fibronectin and collagen IV but had no effect on attachment to laminin. By contrast, subsequent migration was significantly suppressed on all substrata. Our model permits the study of dynamic cell-cell and cell-extracellular-matrix interactions and indicates that RGDS might predominantly act on early tumor-cell locomotion after penetration of the endothelium.

Basement Membrane↗

PubMed1993

[Septic shock and multiple organ failure in surgical intensive care. An animal experiment model on the analysis of pulmonary and intestinal dysfunction].

The study deals with an animal model for the problems of surgical intensive care patients. Following repeated applications of E. coli endotoxin WO 111:B4 under standard conditions, specific hemodynamic and biochemical (TNF, TXA2, PGI2, IL-6, PAF) and morphological (endothelium of the lung) alterations were detected. ARDS patterns induced by the sepsis were analyzed by high-frequency measurement of pressure and flow (385 measurements per breathing cycle). The role of the intestine in sepsis was investigated by ion-selective monitoring of surface potassium activity comparing mucosa and serosa. Every injection of endotoxin was followed by a selective increase of the potassium activity revealing relative ischemia induced by the endotoxin. The profile of the potassium levels on the surface correlates both with the cardiac output and with the prostacyclin levels. The continuous narrowing of the difference between mucosa and serosa, potassium during the period of investigation can be regarded as evidence for pathologic change in permeability fostering the septic course.

6-Ketoprostaglandin F1 alpha↗

PubMed1993

Computed tomography and histologic results in the early stages of endotoxin-injured pig lungs as a model for adult respiratory distress syndrome.

RATIONALE AND OBJECTIVES: To determine early radiographic changes in diffuse alveolar injury, the authors correlated computed tomography (CT) and histopathology in pigs with recurrent endotoxinemia. METHODS: Five pigs received recurrent endotoxin over a 17-hour period. Three pigs received physiologic saline and served as controls. Hemodynamic and blood-gas data were analyzed. CT was performed immediately before killing the animals. The lungs were cut into 5-mm-thick slices in the same axis as the CT scans and were investigated by light and electron microscopy. RESULTS: Hemodynamic data, blood-gas analysis, and morphologic changes closely simulated the clinical situation of septic shock in the five pigs that had received endotoxin. Results of histologic examination depicted changes similar to those associated with adult respiratory distress syndrome (ARDS). CT clearly demonstrated both interstitial, and to a minor degree, intra-alveolar lesions in the endotoxin-injected group, which correlated well with dilated lymph vessels, thickened interstitium, and areas of dystelectasis on histologic examination. Although there was a rather uniform clinical picture, CT and histologic findings showed different degrees of involvement. CONCLUSIONS: CT clearly depicts changes in endotoxin-injured pig lungs in an early clinical state, which are similar to changes associated with ARDS on histologic examination.

Animals↗

PubMed1993

Macrophages and multicellular tumor spheroids in co-culture: a three-dimensional model to study tumor-host interactions. Evidence for macrophage-mediated tumor cell proliferation and migration.

In a new co-culture model involving multicellular tumor spheroids and different phenotypes of human macrophages, we studied the effects of the latter on migration and proliferation of the human colon carcinoma cell line, HRT-18. The macrophage phenotypes are detectable with monoclonal antibodies and are inducible in culture. 12-O-tetradecanoyl-phorbol-13-acetate-activated macrophages are associated with the phenotype 27E10, which is an acute inflammatory macrophage. The glucocorticoid-induced macrophage phenotype RM3/1 is associated with the down-regulation of inflammation. The phenotype resembling the mature resident macrophage termed 25F9 arises spontaneously in prolonged culture. It could be shown that inflammatory macrophages are localized at invasive areas of the tumor-host interface of colorectal carcinoma, whereas resident and anti-inflammatory macrophages were found in the central tumor region or at well-bordered areas of the tumor-host interface. The results obtained with this co-culture model show that 27E10-associated macrophages stimulate tumor cell migration and inhibit tumor cell proliferation. RM3/1 had only a slight inhibiting effect on proliferation and a slight promoting effect on migration. The 25F9-positive macrophage-stimulated tumor cell proliferation and inhibited migration completely. This investigation indicates that this in vitro system is useful for studying different macrophage effects on tumor cells and that indeed proliferation and migration of tumor cells could be influenced in an opposite manner by different types of macrophages.

Carcinoma↗

PubMed1993

Intracavernosal kinetics of eicosanoids and endothelin during erection. Data from human and animal studies on intrapenile and systemic prostaglandins.

The release of mediator substances of the arachidonic acid cascade is closely related to the functional state of the endothelium. A significant lower prostacyclin/thromboxane A2 ratio in penile plasma of organogenic impotent patients in comparison to patients with psychogenic erectile dysfunction has been described in the literature. We observed the time-related liberation of prostacyclin, thromboxane A2 and the vasoactive peptide endothelin for 16 minutes of a drug-induced erection. We compared kinetics of patients with penile deviation and transsexualism, to patients suffering from severe organogenic impotence. We assessed the usefulness of the prostacyclin-to-thromboxane A2 ratio as a possible indicator of corporal degeneration. An animal model has been created to observe differences between rabbits under 100 days of standard diet alimentation, rabbits under cholesterol enriched diet and rabbits with hereditary hyperlipidemia type II A. Hyperlipidemia is suspected to be one possible factor causing organogenic impotence. Enzyme-immuno-assays were used for the determination of all substances. The systemic prostacyclin-to-thromboxane A2 ratio differed significantly between control rabbits and rabbits with hyperlipidemia. Prostacyclin, thromboxane A2 and endothelin in corpus cavernosum plasma showed a typical profile during spontaneous and drug-induced erection. A significant difference between groups of patients suffering from organogenic or psychogenic impotence could not be found. The value of the determination of the studied substances in differential diagnosis seems to be dubious.

Alprostadil↗

PubMed1992

Expression of Cx26, Cx32 and Cx43 gap junction proteins in normal and neoplastic human tissues.

This report concerns the expression of the gap-junction proteins Connexin (Cx)26, 32 and 43 in different malignant and non-malignant human tissues. Affinity-purified polyclonal antibodies against Cx26, 32 and 43 were used for immunohistochemical as well as immunoblot analysis. Cx32, the major gap-junction protein in rat and mouse liver, was detected in human liver and kidney. By contrast, Cx43 was expressed in epithelial and mesenchymal tissues and Cx26 was detected in different epithelia. Whereas all of the benign tumors studied, and some malignant ones, showed stable expression of gap-junction proteins, breast cancer, renal-cell cancer and sarcomas showed a significant decrease in gap-junction proteins as opposed to normal tissue. Cx43, not detected in human normal liver, was found in human hepatocellular carcinoma and Cx26, not detected in human adult skin, was observed in tissue samples of basal-cell carcinoma. In immunoblot analysis, Cx32 antibodies recognized a 27-kDa protein in human liver and hepatocellular carcinoma. A 43-kDa polypeptide was detected in human kidney, renal-cell carcinoma, normal breast, connective tissue of invasive-duct carcinoma of the breast and hepatocellular carcinoma.

Antibodies↗

PubMed1992

Time course of various inflammatory mediators during recurrent endotoxemia.

The time course of thromboxane B2 (TxB2), 6-keto-PGF1 alpha (stable metabolite of prostacyclin), tumor necrosis factor-alpha (TNF alpha), platelet activating factor (PAF), and interleukin-6 (IL-6) formation after three lipopolysaccharide (LPS) infusions was studied in pigs over an 18-hr, period. The Escherichia coli endotoxin W0111:B4 was injected i.v. into 10 of the test group pigs at a dose of 0.5 micrograms/kg over 30 min at 0, 5 and 10 hr of the experiment. Three pigs injected with physiological saline served as controls. At defined time points before and after each LPS administration venous blood was withdrawn (0, 15, 30, 45, 60, 120, 180 min) and plasma levels of TxB2, 6-keto-PGF 1 alpha, PAF, TNF alpha and IL-6 were determined. Pulmonary artery pressure (PAP) and cardiac output (CO) were measured every 15 min. TxB2 and PAF peaked significantly between 30 and 45 min, TNF alpha and 6-keto-PGF 1 alpha between 30 and 60 min, and IL-6 between 120 and 180 min after each LPS injection. The mediators PAF, TNF alpha and TxB2 showed a decreasing three-peak profile whereas 6-keto-PGF1 alpha exhibited an increasing one. IL-6 plasma concentrations increased after each LPS injection. The peak after the third LPS administration, however, was surprisingly low compared to the previous two. The first LPS infusion in our test group led to a significant, sustained rise in mean PAP. After recurrent LPS injections the peak in PAP was not as marked as after the first infusion, indicating the development of a tolerance towards LPS. Initially, CO showed hypodynamic values, whereas the end stage of the experiment was characterized by hyperdynamic CO levels. In conclusion, we believe this porcine model of septic shock to be one of the first large animal models to describe in detail the time-course of various important inflammatory mediators.

6-Ketoprostaglandin F1 alpha↗

PubMed1992

In vitro and in vivo evaluation of a hydrophilized silicone intraocular lens.

The surface of a silicone-disc intraocular lens (IOL) was hydrophilized by plasma etching (oxygen plasma) and compared to an untreated but otherwise identical IOL. Various methods of surface analysis were used to characterize the modification (e.g., X-ray photoelectron spectroscopy, contact angle estimation). A cytotoxic effect of the modified surface was excluded by cell culture experiments evaluating cell spreading, cell morphology, DNA and protein synthesis. In vivo experiments on rabbits indicated that the postoperative foreign-body reaction was not significantly affected by the hydrophilization of the IOL surface. Throughout the entire follow-up (12 weeks) we found less induced posterior synechias in the eyes with hydrophilized lenses than in those with untreated lenses (P = .009). While the IOL dislocations out of the capsular bag and the posterior capsular opacification rate did not differ significantly between the two groups of eyes, we did see special patterns of posterior capsular opacification on the posterior capsules of eyes with the hydrophilized IOL.

Animals↗

PubMed1992

[EBV in malignant lymphomas].

PCR and in-situ-Hybridization were used to detect EBV-DNA within Hodgkin- and Non-Hodgkin-Lymphomas. By the use of primers for the Bam H1 W fragment we could show EBV-DNA to be associated with 45% Hodgkin- and 25% T-Lymphomas, whereas we could not find EBV-DNA within B-Lymphomas. In about one third of the PCR-positive cases we could localize EBV-DNA mostly in the tumor cell nuclei by in-situ-hybridization.

Base Sequence↗

PubMed1992

[Electrocardiographic diagnosis of acute graft rejection after heart transplantation].

In order to evaluate the diagnostic value of standard-ECG (ST-ECG) and precordial mapping-ECG with 63 unipolar leads (PM-ECG) for detection of acute cardiac allograft rejection, 15 patients (12 male, 3 female; age range 24-64 years) were studied. ST-ECG and PM-ECG were recorded along with 94 endomyocardial biopsies. Twenty-four acute rejections were detected histologically. Using the ST-ECG, a reduction of the QRS-amplitude-sum (lead I, II, III, V1 and V6) greater than or equal to 5% in comparison with the ECG obtained 1 week before was found to be the best diagnostic criterion (sensitivity 63%, specificity 74%, positive predictive value 48%, negative predictive value 85%). By analysing the PM-ECG a drop of the QRS-amplitude greater than or equal to 12% in greater than or equal to 14/63 precordial leads was determined to be the most reliable parameter (sensitivity 79%, specificity 71%, positive predictive value 49%, negative predictive value 91%). In contrast to the high sensitivity of PM-ECG, ST-ECG was less suitable for detection of acute rejection. However, taking into account the high negative predictive value of PM-ECG, acute rejection could be excluded with high probability, if the QRS-amplitudes of the PM-ECG remained stable. This may lead to a lower frequency of routinely performed endomyocardial biopsies.

Adult↗

PubMed1992

Interaction of human malignant melanoma (ST-ML-12) tumor spheroids with endothelial cell monolayers. Damage to endothelium by oxygen-derived free radicals.

Clinical and experimental observations suggest that tumor-induced endothelial cell injury may be one of several initial events in the establishment of tumor metastases. To test this hypothesis, the authors have analyzed the interaction of malignant melanoma (ST-ML-12) multicenter tumor spheroids with endothelial cell monolayers in a three-dimensional coculture system. After 1.5 hours of interaction, the authors observed a toxic effect on endothelial cells in the perispheroid region. The latter was demonstrated by testing membrane integrity with the fluorescent probes acridine orange/ethidium bromide and resulted in sensitivity to shear stress of the damaged cells. The endothelium then underwent a regenerative cycle to replace the denuded halo. Addition of the oxygen radical-scavenging enzyme superoxide dismutase to the culture medium prevented this endothelial cell damage in a dose-dependent manner for up to 12 hours. By contrast, catalase, deferoxamine mesylate, allopurinol, and the proteinase inhibitors soybean trypsin inhibitor and aprotinin were not protective under the same conditions. The endothelial damage was dependent on the attachment of the spheroids. Medium conditioned by ST-ML-12-spheroids proved to be ineffective. A similar, but less prominent, deleterious effect was seen when human peritoneal mesothelial cells were used in place of the human umbilical vein endothelial cells. Spheroids of the uroepithelial cell line HU-609 were used as control. No toxicity was observed in these cocultures. Melanin biosynthesis is associated with the production of oxygen-derived free radicals. The results suggest a possible implication of these free radicals in metastasis formation of malignant melanoma.

Cell Communication↗