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Biomedical subjects

C L Klein

Publications and source records attributed to C L Klein.

At least 19 recordsLinked to original sources
PubMed1998

Current trends in biocompatibility testing.

Biocompatibility remains the central theme for biomaterials applications in medicine. It is generally accepted that this term means not only absence of a cytotoxic effect but also positive effects in the sense of biofunctionality, i.e. promotion of biological processes which further the intended aim of the application of a biomaterial. The national and international standards for testing regimes represent a lowest common denominator for such applications and do not necessarily ensure that optimal function will be achieved. The authors' thesis is that biocompatibility testing has scope for extensive development with respect to biofunctionality. The present paper reviews current trends in the in vitro aspects of biocompatibility testing. As well as a critical appraisal of the recent literature, future trends are also stressed, which the authors regard as essential for a meaningful integration of a modern biological approach into new developments in the material sciences. These include the application of modern techniques of cell and molecular biology, the concepts of tissue remodelling, hybrid organ development and encapsulated cell technology.

Animals↗
PubMed1997

Synthesis, structure, dopamine transporter affinity, and dopamine uptake inhibition of 6-alkyl-3-benzyl-2-[(methoxycarbonyl)methyl]tropane derivatives.

A series of 6-alkyl-3 beta-benzyl-2-[(methoxycarbonyl)methyl]tropane analogues were synthesized and evaluated as cocaine binding site ligands at the dopamine transporter (DAT). The in vitro affinity (Ki) for the DAT of the 6-alkyl-3 beta-benzyl-2-[(methoxycarbonyl) methyl]tropane analogues was determined by inhibition of [3H]WIN 35,428 in rat caudate putamen tissue. The inhibition of dopamine uptake (IC50) was also measured for selected compounds which demonstrated moderate affinity for the dopamine transporter. The unsubstituted enantiopure analogues (-)-19a (Ki = 33 nM) and surprisingly (+)-20a (Ki = 60 nM) were found to be almost equipotent with the high-affinity binding components of cocaine and WIN 35,065-2 and exhibited slightly more potent dopamine uptake inhibition than both cocaine and WIN 35,065-2. In general, substitution at the 6-position of racemic 19a and 20a with alkyl groups was found to result in decreased activity relative to increased chain length of the substituent. The 3 beta-benzyl-2 beta-[(methoxycarbonyl)methyl]-6 beta-methyltropane (21b; Ki = 57 nM) was the only 6-alkyl derivative to exhibit moderately potent activity. The 6 beta-isomer 21b was 4-fold more potent than the 6 alpha-isomer 19b (Ki = 211 nM) and was nearly equipotent with (-)-19a and (+)-20a as well as with cocaine and WIN 35,065-2. The results of this study further demonstrate the steric constraints associated with the C(6)-C(7) methylene bridge of the tropane ring system for molecular recognition of cocaine analogues at the cocaine binding site(s) on the DAT.

Animals↗
PubMed1997

Heavy metal ion induction of adhesion molecules and cytokines in human endothelial cells: the role of NF-kappaB, I kappaB-alpha and AP-1.

We analyzed the influence of heavy-metal ions on human umbilical vein endothelial cells (HUVEC) in comparison to proinflammatory cytokines (TNF-alpha, IL-1beta) and lipopolysaccharide (LPS). Adhesion molecule and cytokine expressions are upregulated by heavy-metal exposure. Expression of E-selectin on the cell surface was strongly induced by 1-mM concentrations of NiCl2 and CoCl2, whereas ZnCl2 and CrCl3 had no influence. Furthermore, it is shown that NiCl2 induces mRNA expression of E-selectin, intercellular adhesion molecule-1, IL-6 and IL-8 in a 1-mM concentration. The transcription factor NF-kappaB is known to be involved in the regulation of adhesion molecule expression in endothelial cells after activation by proinflammatory cytokines. We demonstrated that treatment of HUVEC with Ni2+ and Co2+ ions induces the translocation of NF-kappaB p65 and also p50 into the nucleus. NF-kappaB binding activity is enhanced under the influence of heavy metals as determined by mobility shift analysis. P65 and p50 are components of the NF-kappaB complexes as confirmed by supershift analysis. We could show that activation at the protein level is accompanied by induction of NF-kappaB p65 mRNA expression. HUVEC also express the NF-kappaB inhibitor I kappaB-alpha (MAD-3). In the early phase of activation by Ni2+ and Co2+ ions, disappearance of I kappaB-alpha in the cytoplasm accompanied p65 translocation, followed by its gradual reappearence. Because I kappaB mRNA could be upregulated by NiCl2 as well as by a mixture of cytokines, we suggest that the replenishment of the inhibitor in the cytoplasm is caused by de novo I kappaB gene expression. In addition to the enhanced DNA-binding activity of NF-kappaB, another transcription factor, AP-1, was also augmented in HUVEC stimulated by NiCl2, CoCl2 or by proinflammatory mediators and the phorbol ester PMA. Fos protein is shown to be a component of the activated AP-1 complex, as determined by supershift analysis, suggesting that it consists of Jun/Fos heterodimers.

Blotting, Northern↗
PubMed1997

Physiology and cell biology of the endothelium: a dynamic interface for cell communication.

This manuscript presents a brief overview of the physiology and cell biology of the endothelium, which is the basis for understanding the role of endothelial cells in pathological processes as diverse as atherosclerosis, tumour intravasation and multiple organ failure. Following consideration of general aspects of endothelial function in regulating haemostasis, vascular tone and growth, special emphasis will be placed on endothelial regulation of the inflammatory response, which centres on the microcirculation. A particular role in inflammation is played by cell adhesion molecules (CAM), expressed both on endothelial and blood cells. Cell and molecular biological methods to investigate the expression of CAM in endothelial cells in vitro will be presented, as well as novel data, indicating that cytokine-induced up-regulation of CAM in the endothelium may involve signal transduction pathways other than those culminating in the activation of NF-kappa B. Finally, the phenomenon of angiogenesis will be briefly reviewed as a characteristic of endothelial cell activity of central importance to both physiology and pathology and new experimental data presented from an in vitro model to study the ability of individual endothelial cells to form vessel-like structures. In comparative studies to investigate the roles of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor, the dominant role of VEGF in the formation of capillary networks could be unequivocally demonstrated.

Animals↗
PubMed1996

Synthesis and dopamine transporter affinity of 2-(methoxycarbonyl)-9-methyl-3-phenyl-9-azabicyclo[3.3.1]nonane derivatives.

A series of 9-methyl-3 beta-phenyl-2-substituted-9-azabicyclo[3.3.1]nonane derivatives were synthesized and evaluated as cocaine-binding site ligands at the dopamine transporter (DAT). The conformation of the bicyclic structures and the stereochemistry of the substituents were determined by NMR and X-ray crystallography. The in vitro binding affinity (Ki) of the 9-azabicyclo[3.3.1]nonane derivatives was measured in rat caudate-putamen tissue, and they were found to be 100-fold (Ki = 2-14 microM) less potent than cocaine and other tropane analogs. From these results it is evident that the cocaine-binding site at the DAT is very sensitive to structural modifications of the unsubstituted methylene bridge [C(6)-C(7)] of cocaine and cocaine-like compounds.

Animals↗
PubMed1996

The role of the microcirculation in multiple organ dysfunction syndrome (MODS): a review and perspective.

Major advances in intensive care medicine during the past two decades have altered the spectrum of disease encountered by intensive care physicians, anaesthesiologists, traumatologists and pathologists. One of the most important manifestations of severe trauma or infections is the multiple organ dysfunction syndrome (MODS), a life-threatening condition that often ends in multiple organ failure (MOF) and death. Evidence gathered from clinical and morphological observations in humans, taken together with experimental animal studies and a vast accumulation of in vitro data, clearly indicate that the microcirculation lies at the centre of this complex process, which results in peripheral vascular insufficiency, inadequate oxygen delivery to vital organs, and hence, severe organ dysfunction. The multifunctional nature of the endothelium makes it a prime candidate for study of the pathomechanisms of MODS. This paper reviews the evidence for the hypothesis that the microcirculation, and in particular its endothelial component, has a central role in the pathogenesis of MODS. The evidence is reviewed principally from the standpoints of classical morbid anatomy and cell pathobiology.

Animals↗
PubMed1996

Biomaterial-induced alterations of human neutrophils under fluid shear stress: scanning electron microscopical study in vitro.

Morphological changes of human polymorphonuclear neutrophils (PMN) adhering to hydrophilic (glass) and hydrophobic (FEP-Teflon, polyethylene, polypropylene) surfaces were studied in a parallel-plate flow chamber at the light and scanning electron microscopical levels. The PMN were exposed to a shear stress of 0.19 Pa (1.9 dynes cm-2) or were allowed to adhere without the stress component (static control) during 30 min for all four biomaterials. Observation by light microscopy was performed in situ in the flow chamber at 1, 5, 10, 15, 20, 25 and 30 min. The total number of adherent cells as a function of time and the activation status of the population on the basis of morphological criteria were determined. On the hydrophilic material adhesion of activated PMN was significantly higher (P < 0.05) than on the more hydrophobic surfaces. This effect was most pronounced for the adhesion of neutrophils to glass and polypropylene (PP). Polyethylene (PE) showed only minor adhesion rates. Scanning electron microscopy revealed details of cell shape changes and permitted a more precise classification of populations of neutrophils based on distinctive shapes. As PMN were exposed to shear stress on glass, the majority of cells exhibited surface veils, ridges and ruffles, suggesting a high level of cell migration. In this case, on polymeric surfaces the presence of filopodial networks (FEP-Teflon) and ameoboid cell shapes (PP and PE) was noted. The results suggest that a low shear stress, as well as various chemical and physical properties of biomaterial surfaces, are together responsible for differentiation of PMN populations on solid substrata.

Biocompatible Materials↗
PubMed1996

Local and systemic inflammatory mediator release in patients with acute and chronic posttraumatic osteomyelitis.

The local and systemic release of thromboxane A2, prostaglandin I2, leukotriene B4 (LTB4), tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and interleukin-8 (IL-8) were studied before and after operation in 29 patients with acute and 22 with chronic posttraumatic osteomyelitis. Twenty patients without osteomyelitis, who underwent operations for fractures of the lower extremities, served as controls. Blood and tissue samples from the osteomyelitic and control groups were collected under defined conditions and mediators were determined by radioimmunoassay (thromboxane B2, 6-keto-prostaglandin F1 alpha, LTB4) or by enzyme-linked immunosorbent assay (TNF-alpha, IL-1 beta, and IL-8). In addition, common parameters (leukocyte count, C-reactive protein, temperature) were measured. The best correlation with acute disease activity was given by TNF-alpha, IL-6, IL-8, and LTB4. Plasma levels of these mediators in acute osteomyelitis were significantly increased compared to chronic osteomyelitis and to controls, respectively. Tissue samples from osteomyelitic focus showed significantly increased levels for IL-8, IL-6, TNF-alpha, IL-1 beta, and LTB4 in acute osteomyelitis, whereas the values for TxB2 and 6-keto-prostaglandin F1 alpha were only slightly increased compared to the chronic osteomyelitis group. This study describes the local and systemic liberation of various mediators in acute and chronic posttraumatic osteomyelitis in detail for the first time and provides data for pre- and postoperative monitoring of disease activity and demonstrates new pathogenetic and therapeutic aspects of bone modulation in osteomyelitis.

Acute Disease↗
PubMed1996

PECAM-1 expression in human mesothelial cells: an in vitro study.

Mesothelial cells are actively involved in inflammatory processes by expressing a set of cell adhesion molecules (CAMs). Transmigration of leukocytes into inflamed tissues requires a chemotactic stimulus and engagement of platelet-endothelial cell adhesion molecule-1 (PECAM-1). To investigate the kinetics involved in peritonitis, pure cultures of mesothelial cells are necessary. In previous studies, we have found that human mesothelial cells (HOMES) show a weak constitutive expression of PECAM-1, which cannot be further stimulated by cytokines. It is known that all serous cavities and body fluids contain numerous macrophages which strongly express this adhesion molecule. To identify the cells responsible for the expression of PECAM-1, mesothelial cells freshly obtained from omental tissue were isolated using PECAM-1-conjugated magnetic beads by cell sorting. For these studies, the negative as well as the positive fraction of isolated cells were used. As a control, freshly isolated monocytes were studied. Cell cultures were characterized by light and electron microscopy, as well as immunocytochemistry. The negative cell fraction was cultivated and stimulated for different times with tumor necrosis factor-alpha (30 and 300 U/ml), interleukin-1 beta (10 and 100 U/ml) and interferon-gamma (500 U/ml) and PECAM-1 expression was analyzed by a comparative quantitative cell enzyme immunoassay (EIA). The positive cell fraction was treated in the same manner. Both fractions of isolated cells showed strong positivity for cytokeratins 8, 18, 7 and 19, as well as vimentin. CD68, a monocyte marker, was not detected on mesothelial cells. In addition, EIA analysis confirmed the constitutive expression of PECAM-1 obtained from previous studies. This expression on HOMES was not inducible, irrespective of the type and concentration of cytokine studied. These data confirm PECAM-1 expression on mesothelial cells obtained from human omental tissue and suggest a critical role in transmigration of leukocytes during peritoneal inflammation.

Cell Separation↗
PubMed1995

Pentamidine congeners. 3. Crystal structure and molecular modeling studies of trans-1,4-bis(4-amidinophenoxy)-2-butene.

X-ray diffraction was used to confirm the geometry of trans-1,4-bis(4-amidinophenoxy)-2-butene dihydrochloride dihydrate (trans-butenamidine). trans-Butenamidine is a semirigid analogue of pentamidine that has demonstrated good anti-Pneumocystis carinii activity in rats. Molecular modeling studies revealed that unlike pentamidine or propamidine, trans-butenamidine does not discriminate between AT and TA sequences in its binding to the minor groove of DNA. Crystal data: [C18H22N4O2(2+)][Cl(-)]2[H2O]2, triclinic space group, P1, a = 9.443(1) A, b = 11.400(1) A, c = 11.919(1) A, alpha = 62.19(1) degree, beta = 81.10(1) degree, gamma = 72.19(1) degree, V = 1080.3(3) A3, Z = 2, R = 0.054 for 1149 observed reflections with I > 3 sigma (1).

Base Sequence↗
PubMed1995

Synthesis and antimicrobial activity of 6,7-annulated pyrido[2,3-d]pyrimidines.

Four new 6H-indeno[2',1':5,6]pyrido[2,3-d]pryimidines (10-13) were synthesized via cyclocondensation reactions involving chlorovinyl aldehyde 1 or ketoaldehyde 3 and appropriately substituted 6-aminopyrimidines. The regiochemistry of the compounds was established by 1H NMR and 13C NMR spectral data as well as X-ray crystal data. Compounds 10 and 11 and previously reported homologues 14 and 15 were screened for antimicrobial activity. Moderate antimicrobial activity was observed for some of these compounds. Compound 14 was especially active against Staphylococcus aureus. Crystal data for 13 (C14H7N3Cl2) follows: monoclinic space group, P21/n; Unit cell dimensions, a = 7.284(1) A, b = 12.800(1) A, c = 13.108(1) A, beta = 93.98(1) degree, V = 1219.2(2) A3, Z = 4.

Anti-Bacterial Agents↗
PubMed1995

Tissue factor expression during coculture of endothelial cells and monocytes.

The role of monocytes as initiators of coagulation through the expression of tissue factor has been well documented in vitro, and the relationship of monocyte tissue factor to the thrombotic complications of atherosclerosis has been suggested. Tissue factor antigen has been identified in the plasma membranes of monocytes adherent to the vascular endothelium overlying atherosclerotic plaques and the presence of tissue factor in adherent mononuclear cells correlates with the polymerization of fibrin at these same sites. To further understand the relationship of cellular adhesion to tissue factor expression, human monocytes were cocultured for periods ranging from 30 min to 24 hr with endothelial cells isolated from human umbilical veins (HUVEC). Tissue factor antigen, as assayed by both ELISA and immunogold electron microscopy, was minimal on either monocytes or HUVEC maintained in homogeneous cultures or on the cells when cocultured for 1 hr or less. This was true whether the HUVEC were in a native state or if they had been stimulated with interleukin-1 (IL-1 beta) or lipopolysaccharide (LPS) prior to monocyte adhesion. Typically, less than 13% of the cells in short-term cocultures were positively labeled through anti-tissue factor immunogold microscopy. The level of tissue factor, however, was increased 3-fold above baseline when monocytes were cocultured with unstimulated HUVEC for 4 hr, and it was more than double this if the HUVEC had been exposed to IL-1 beta or LPS (7-fold increase). By 24 hr, the expression of tissue factor antigen was nearly 50-fold higher in cocultures involving stimulated HUVEC, and at later times greater than 70% of the cells were labeled with immunogold. Through the use of quantitative immunogold electron microscopy, the increase in tissue factor was most pronounced on monocytes which had three times greater increase in tissue factor than HUVEC in the same cultures. These studies document the stimulation of tissue factor expression by monocytes upon coculture with endothelial cells, and the data document an enhancement of this coculture effect upon HUVEC stimulation with cytokines. These observations have relevance to atherosclerotic disease by suggesting that interaction of monocytes with dysfunctional endothelial cells overlying atherosclerotic plaques would be sufficient to induce tissue factor and by so doing predispose to localized thrombotic events.

Cell Adhesion↗
PubMed1995

Comparative studies on vascular endothelium in vitro. 2. Hypoxia: its influences on endothelial cell proliferation and expression of cell adhesion molecules.

Recent studies have presented evidence that the processes of hypoxaemia and reperfusion are involved in several pathogenetic mechanisms of atherosclerotic lesions. The ability of hypoxaemia to activate circulating white blood cells (WBCs) and enhance WBC-endothelial cell (EC) interactions is suspected to be a major factor in deleterious processes in the blood vessel wall. Various groups have suggested that cell adhesion molecules (CAMs), such as ICAM-1, VCAM-1 and E-selectin and their leukocyte ligands are involved in intercellular activities of the relevant cell types. We studied the effects of different oxygen tensions, simulating normoxic conditions, hypoxia and hyperoxia in vitro with the help of an umbilical vein EC model in order to determine the effects of oxygenation on CAM presentation on vascular ECs with and without further cytokine and endotoxin (lipopolysaccharides; LPS) stimulation. Semiquantitative analysis of ICAM-1, E-selectin and VCAM-1 was performed using cell enzyme immunoassay techniques. The presentation of ICAM-1, E-selectin and VCAM-1 remained on the whole unaffected by both hypoxia and hyperoxic conditioning after both 7 and 24 h. Stimulation of ICAM-1 by cytokines and LPS was only marginally influenced by the oxygen tension. Cytokine induction of E-selectin was not affected after 7 h and was even reduced under hypoxia, compared to the control culture after 24 h, while stimulation was increased by hyperoxia. VCAM-1 was reduced in both the hypoxic and hyperoxic culture, while being maximally stimulated by cytokines and LPS after 7 h. In general, an effect of hypoxia was not found without any further stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Adhesion Molecules↗
PubMed1995

Comparative studies on vascular endothelium in vitro. 3. Effects of cytokines on the expression of E-selectin, ICAM-1 and VCAM-1 by cultured human endothelial cells obtained from different passages.

Endothelial cells (EC) are very responsive to proinflammatory cytokines, e.g. interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha), as well as to bacterial lipopolysaccharide. EC are stimulated by these substances to secrete chemotactic factors and to increase expression of cell adhesion molecules (CAM), leading to dramatically altered interactions with leukocytes, e.g. granulocytes and monocytes. In these interactions E-selectin, ICAM-1 and VCAM-1 are known to play an important role, as they are presented by the EC and interact with corresponding ligands on the white blood cell membranes. These adhesion molecules have been studied worldwide in a variety of in vitro experiments using cultured EC. Different passages and mixtures of passages have been used in these experiments, often without any regard to the comparability of the results. In this study the expression of E-selectin, ICAM-1 and VCAM-1 on cultured human umbilical vein EC (HUVEC) obtained from different passages (passages 1-6) was studied after 4, 8 and 24 h of exposure to IL-1 beta and TNF alpha. In previous studies, we have shown that IL-1 beta and TNF alpha increase the expression of E-selectin and ICAM-1 on the cytoplasmatic membranes of HUVEC and human adult EC from the saphenous vein and femoral artery in a similar fashion. Using a comparative quantitative cell enzyme immunoassay, we found that the expression of the adhesion molecules was significantly reduced with increasing passages. There was also a decreased persistence of CAM comparing different periods of stimulation between 6 and 24 h in the different passages. These data indicate that the number of passages plays an important role in the expression of adhesion molecules on EC. The results are relevant for the meaningful planning of comparative in vitro studies on EC presentation of CAM.

Cells, Cultured↗
PubMed1995

Effects of cytokines on the expression of cell adhesion molecules by cultured human omental mesothelial cells.

Cultured mesothelial cells (HOMES) are very responsive to the proinflammatory cytokines, interleukin (IL)-1 and tumor necrosis factor-alpha (TNF-alpha). E-selectin, ICAM-1 and VCAM-1 are known to play an important role, because they are presented by diverse cell types, for example endothelial cells (ECs), and interact with co-responding ligands on white blood cell membranes. In this study, the expression of ICAM-1, VCAM-1, E-selectin as well as PECAM-1 on cultured HOMES was studied over 5, 24, 48 and 72 h exposure to IL-1 beta, interferon-gamma and TNF-alpha. In previous studies we have shown that IL-1 beta and TNF-alpha increase the expression of ICAM-1, E-selectin and VCAM-1 on the cytoplasmatic membranes of HUVECs, HSVECs and HAFECs (ECs from human umbilical vein, saphenous vein and femoral artery, respectively). Using a comparative quantitative cell enzyme immunoassay, we found that expression of the adhesion molecules ICAM-1 and VCAM-1 was significantly increased on HOMES in a dose- and time-dependent manner, compared to nonstimulated cells. Thus, ICAM-1 increased dramatically after 5 h incubation with TNF-alpha. Values of about 450% of the control level were measured. VCAM-1 was similarly stimulated after 24 h incubation with the same cytokine, although its level of expression was significantly lower than that of ICAM-1. In contrast to findings in the literature, VCAM-1 was not found to be expressed constitutively. E-selectin was neither constitutively expressed nor markedly inducible on HOMES. Only weak expression was found after 24 h incubation with high-dose IL-1 beta. PECAM-1 was expressed constitutively, as became evident in antibody dilution studies. These data indicate that HOMES respond to inflammatory stimuli, in some ways in a similar fashion to vascular endothelial cells, but also show a specific pattern of antigen presentation. The results are important for a better understanding of inflammatory processes in serous cavities. The data are also relevant for the improvement of antithrombogenous surfaces of the lumina of vascular prostheses by cell seeding.

Cell Adhesion Molecules↗
PubMed1994

(-)-Norcocaine.

The title compound, [2R,3S-(2 beta,3 beta)]-methyl 3-(benzoyloxy)-8-azabicyclo[3.2.1]octane-2-carboxylate, C16H19NO4, is a metabolite of the tropane alkaloid cocaine. The molecule crystallized as the free base with the piperidine ring in a chair conformation. The tropane ring system and its methoxycarbonyl and benzoyl groups are rigid; only rotational flexibility is allowed in the conformation of the substituents.

Cocaine↗