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Biomedical subjects

Charles James Kirkpatrick

Publications and source records attributed to Charles James Kirkpatrick.

17 recordsLinked to original sources
PubMed2006

Outgrowth endothelial cells isolated and expanded from human peripheral blood progenitor cells as a potential source of autologous cells for endothelialization of silk fibroin biomaterials.

One challenge of particular importance in tissue engineering is to improve vascularization of larger size defects, which would then facilitate a sufficient supply with oxygen and nutrients to the central regions of a larger tissue-engineered construct or in highly vascularized tissues. In this study, we show that outgrowth endothelial cells (OECs) derived from human peripheral blood can serve as a source of human autologous endothelial cells and can be used in combination with fibroin silk fiber meshes for applications in tissue engineering. OEC reveal a highly differentiated endothelial phenotype as well as a high phenotypic stability during their expansion. Furthermore, OEC showed very promising results in the endothelialization of fibroin silk fiber meshes, maintaining their endothelial characteristics and functions. On the fibroin fiber meshes OECs formed differentiated endothelial cell layers covering the single fibers as shown by data from scanning electron microscopy, immunofluorescence, and gene expression analysis. After embedding in a wound-healing matrix, mimicked by fibrin gels, OEC migrated from the fibroin scaffolds into the fibrin and formed a microvessel-like network. Thus, we conclude that OEC could serve as a valuable source of autologous endothelial cells, supporting pro-angiogenic therapies in combination with silk fibroin-based scaffolding materials in the field of tissue engineering and regenerative medicine.

Biocompatible Materials↗
PubMed2006

Retention of a differentiated endothelial phenotype by outgrowth endothelial cells isolated from human peripheral blood and expanded in long-term cultures.

Rapid adequate vascularization by autologous human endothelial cells remains a limiting step in the treatment of ischemic tissues and the generation of new tissues. We have expanded outgrowth endothelial cells (OEC) from human peripheral blood and investigated their phenotypic stability in long-term cultures. Our goal has been to obtain suitable numbers of autologous endothelial cells for pro-angiogenic cell therapies. Mononuclear cells were isolated from human peripheral blood. During culture, cells were characterized for several endothelial and stem cell markers in mono- or in co-culture with mature endothelial cells. In cultures from peripheral blood, we observed cells with a variable ability to assume a differentiated endothelial phenotype. Most of the cells showed markers reported for endothelial progenitor cells or hemangioblasts (CD31, KDR, VE-cadherin, CD34, CD117, CD45) but failed to develop a differentiated phenotype. Caveolin-1 was not detectable in these cells by reverse transcription/polymerase chain reaction (RT-PCR) or immunofluorescence. Another cell type arising from the same cultures expressed a differentiated phenotype and was designated as an OEC. This subset as an OEC was expanded in long-term cultures and analyzed by immunofluorescence, flow-cytometry, and RT-PCR for a stable endothelial phenotype. OEC showed several markers of a differentiated endothelium, such as high levels of caveolin-1 throughout all tested passages, and the ability to form angiogenic sprouts in vitro. Thus, OEC in long-term expansion cultures from blood mononuclear cells are phenotypically highly stable, a feature that is an important prerequisite for using OEC from peripheral blood for autologous endothelial cell therapies.

Antigens, Differentiation↗
PubMed2006

FVIII production by human lung microvascular endothelial cells.

While extrahepatic factor VIII (FVIII) synthesis suffices for hemostasis, the extrahepatic production sites are not well defined. We therefore investigated the ability of the human lungs to produce FVIII. Lungs from heart-beating donors who were declined for transplantation were perfused and ventilated in an isolated reperfusion model for 2 hours. A progressive accumulation of FVIII and von Willebrand factor (VWF) was recorded in the perfusion medium in 3 of 4 experiments. By contrast, factor V, fibrinogen, and immunoglobulin G (IgG) levels remained constant during the perfusion period, indicating that the accumulation of FVIII and VWF was not due to diffusion from the intercellular medium into the vascular system. Purified human lung microvascular endothelial cells produced FVIII during at least 2 passages in vitro. Altogether, these data identify the lung endothelial cells as a FVIII production site in humans.

Endothelial Cells↗
PubMed2006

In vivo release of non-neuronal acetylcholine from the human skin as measured by dermal microdialysis: effect of botulinum toxin.

1.--Acetylcholine is synthesized in the majority of non-neuronal cells, for example in human skin. In the present experiments, the in vivo release of acetylcholine was measured by dermal microdialysis. 2.--Two microdialysis membranes were inserted intradermally at the medial shank of volunteers. Physiological saline containing 1 muM neostigmine was perfused at a constant rate of 4 microl min(-1) and the effluent was collected in six subsequent 20 min periods. Acetylcholine was measured by high-pressure liquid chromatography (HPLC) combined with bioreactors and electrochemical detection. 3.--Analysis of the effluent by HPLC showed an acetylcholine peak that disappeared, when the analytical column was packed with acetylcholine-specific esterase, confirming the presence of acetylcholine. 4.--In the absence of neostigmine, 71+/-51 pmol acetylcholine (n=4) was found during a 120 min period. The amount increased to 183+/-43 pmol (n=34), when the perfusion medium contained 1 microM neostigmine. 5.--Injection of 100 MU botulinum toxin subcutaneously blocked sweating completely, but the release of acetylcholine was not affected (botulinum toxin treated skin: 116+/-70 pmol acetylcholine/120 min; untreated skin: 50+/-20 pmol; n=4). 6.--Quinine (1 mM), inhibitor of organic cation transporters, and carnitine (0.1 mM), substrate of the Na(+)-dependent carnitine transporter OCTN2, tended to reduce acetylcholine release (by 40%, not significant). 7.--Our experiments demonstrate, for the first time, the in vivo release of non-neuronal acetylcholine in human skin. Organic cation transporters are not predominantly involved in the release of non-neuronal acetylcholine from the human skin.

Acetylcholine↗
PubMed2005

Thyroid transcription factor 1 expression in cystic lesions of the neck: an immunohistochemical investigation of thyroglossal duct cysts, branchial cleft cysts and metastatic papillary thyroid cancer.

Cervical metastases of papillary thyroid cancer (PTC) are of particular diagnostic importance, because they can mimic branchiogenic cysts clinically and histopathologically when undergoing cystic change. Immunohistochemical stains for thyroid transcription factor 1 (TTF-1), which are positive in thyroid cancers, are reported to be valuable in establishing diagnosis. However, TTF-1 may also be expressed in dysontogenetic cysts of the neck. Therefore, immunohistochemical stains for TTF-1 and thyroglobulin (TG) were performed on each of the ten thyroglossal duct cysts, branchial cleft cyst and lymph node metastasis of PTC. Five in ten cases of thyroglossal duct cysts were positive for TTF-1, but all were negative for TG. One of the ten branchial cleft cysts stained for TTF-1, while all cases were negative for TG. All ten cases of lymph node metastases of PTC showed positive for TTF-1, nine of which were positive for TG. Since a positive immunostaining for TTF-1 in cystic lesions of the neck was not only found in metastases of PTC, but also in non-malignant branchiogenic cysts, additional investigations, e.g. an immunostaining for TG, should be added in difficult cases.

Adolescent↗
PubMed2005

The incidence of myelofibrosis in essential thrombocythaemia, polycythaemia vera and chronic idiopathic myelofibrosis: a retrospective evaluation of sequential bone marrow biopsies.

The incidence of myelofibrosis (MF) among the three major Philadelphia chromosome-negative chronic myeloproliferative disorders, i.e. essential thrombocythaemia (ET), polycythaemia vera (PV) and chronic idiopathic myelofibrosis (CIMF), is not well documented since the diagnostic criteria have recently been redefined by the WHO. Therefore we performed a retrospective analysis of follow-up biopsies of 275 patients with ET, PV and CIMF according to the WHO classification of chronic myeloproliferative disorders. In the diagnostic bone marrow biopsies, MF was observed in 57 of the 136 CIMF patients (42%), 4 of the 73 PV patients (5%) and none of the 66 patients with ET. Within a median observation time of 2.9 years, 34 of the 79 patients with CIMF (43%), 13 of the 69 patients with PV (19%) and 1 of the 66 patients with ET (1.5%)--each initially without MF--developed MF regardless of myelosuppressive therapy.

Adolescent↗
PubMed2004

Essential thrombocythemia terminating in pure erythroleukemia.

Transformation into acute leukemia is a rare event in essential thrombocythemia (ET). The blasts are usually of myeloid, rarely of megakaryoblastic differentiation. We present the case of a patient with pure erythroleukemia after a nearly 10-year course of ET, which was treated with hydroxyurea. The patient, a 58-year-old male, presented with an elevated thrombocyte count (926,000/microL) and normal values of hemoglobin and leukocytes. After 10 years of therapy with hydroxyurea, the patient developed acute leukemia of solely erythroid differentiation. Chemotherapy with cytarabine and daunorubicin resulted in incomplete remission. The patient died 2 months after diagnosis of acute erythroleukemia. Transformation of ET into erythroleukemia may demonstrate the pluripotent potential of the neoplastic hemopoietic stem cell, with the ability to cause acute leukemia not only of myeloid or megakaryoblastic but also of erythroid lineage.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2004

Modification of human platelet adhesion on biomaterial surfaces by protein preadsorption under static and flow conditions.

Biomaterial-induced thrombosis remains one of the main complications of vascular implant devices. Preadsorbed proteins on the biomaterial/blood interface will modify the adhesion and activation of platelets (PTLs) during the initial contact-phase. Our results clearly show that PTL-adherence on biomaterials is influenced not only by protein preadsorption, but also by flow conditions. The covalent coating of TCPS and glass by phosphorylcholine (PC) induces a significant decrease of PTL adhesion but leads to a slight, but nevertheless significant activation of PTL, which was detected by the induction of P-selectin expression using FACS analysis. Methodologically, the visualization of PTL adhesion gave more reliable results for measurement of PTL adhesion than the cell-enzyme immunoassay (EIA) for P-selectin. Human citrated plasma caused an inhibition of PTL. It is probable, that the contained sodium citrate may inhibit PTL adhesion by its calcium ion-binding capacity. The flow experiment as dynamic system is in our view absolutely essential for the evaluation of biomaterials for vascular prosthesis, and is in accordance with the international standards. The results of the experiments also suggest that investigations under static and flow conditions are needed to determine the influence of protein adsorption on mixed blood cell populations, for example, on PTL and PMN mixtures/co-cultures in order to achieve a better simulation of the in vivo situation.

Adsorption↗
PubMed2004

Lung epithelial cell lines in coculture with human pulmonary microvascular endothelial cells: development of an alveolo-capillary barrier in vitro.

We have established a coculture system of human distal lung epithelial cells and human microvascular endothelial cells in order to study the cellular interactions of epithelium and endothelium at the alveolocapillary barrier in both pathogenesis and recovery from acute lung injury. The aim was to determine conditions for the development of functional cellular junctions and the formation of a tight epithelial barrier similar to that observed in vivo. The in vitro coculture system consisted of monolayers of human lung epithelial cell lines (A549 or NCI H441) and primary human pulmonary microvascular endothelial cells (HPMEC) on opposite sides of a permeable filter membrane. A549 failed to show sufficient differentiation with respect to formation of a tight epithelial barrier with intact cell-cell junctions. Stimulated with dexamethasone, the cocultures of NCI H441 and HPMEC established contact-inhibited differentiated monolayers, with NCI H441 showing a continuous, circumferential immunostaining of the tight junctional protein, ZO-1 and the adherens junction protein, E-cadherin. The generation of a polarized epithelial cell monolayer with typical junctional structures was confirmed by transmission electron microscopy. Dexamethasone treatment resulted in average transbilayer electrical resistance (TER) values of 500 Omega cm(2) after 10-12 days of cocultivation and correlated with a reduced flux of the hydrophilic permeability marker, sodium-fluorescein. In addition, basolateral distribution of the proinflammatory cytokine tumour necrosis factor-alpha caused a significant reduction of TER-values after 24 h exposure. This decrease in TER could be re-established to control level by removal of the cytokine within 24 h. Thus, the coculture system of the NCI H441 with HPMEC should be a suitable in vitro model system to examine epithelial and endothelial interactions in the pathogenesis of acute lung injury, infectious lung diseases and toxic lung injury. In addition, it could be used to improve techniques of lung drug delivery that also requires a functional barrier.

Adherens Junctions↗
PubMed2003

Increased acetylcholine levels in skin biopsies of patients with atopic dermatitis.

Recent experimental evidence indicates that non-neuronal acetylcholine is involved in the regulation of basic cell functions. Here we investigated the cholinergic system in the skin of healthy volunteers and patients with atopic dermatitis (AD). The synthesizing enzyme, choline-acetyltransferase (ChAT), was studied by anti-ChAT immunohistochemistry and enzyme assay. Skin biopsies taken from healthy volunteers and from AD patients were separated into the 2 mm superfical (epidermis and upper dermis) and 3 mm underlying portion (deeper dermis and subcutis). ChAT enzyme activity was detected in homogenized skin and subcutaneous fat (about 13 nmol/mg protein/h). ChAT immunoreactivity was expressed in keratinocytes, hair papilla, sebaceous and eccrine sweat glands, endothelial cells and mast cells. In healthy volunteers the superficial and underlying portion of skin biopsies contained 130 +/- 30 and 550 +/- 170 pmol/g acetylcholine (n = 12), respectively. In AD patients (n = 7) acetylcholine was increased 14-fold in the superficial and 3-fold in the underlying biopsy portion. The present study demonstrates the widespread expression of ChAT protein in the vast majority of human skin cells. Tissue levels of acetylcholine are greatly (14-fold) enhanced in the superficial 2 mm skin of AD patients.

Acetylcholine↗
PubMed2003

The non-neuronal cholinergic system in humans: expression, function and pathophysiology.

Acetylcholine, a prime example of a neurotransmitter, has been detected in bacteria, algae, protozoa, and primitive plants, indicating an extremely early appearance in the evolutionary process (about 3 billion years). In humans, acetylcholine and/or the synthesizing enzyme, choline acetyltransferase (ChAT), have been found in epithelial cells (airways, alimentary tract, urogenital tract, epidermis), mesothelial (pleura, pericardium), endothelial, muscle and immune cells (mononuclear cells, granulocytes, alveolar macrophages, mast cells). The widespread expression of non-neuronal acetylcholine is accompanied by the ubiquitous presence of cholinesterase and receptors (nicotinic, muscarinic). Thus, the non-neuronal cholinergic system and non-neuronal acetylcholine, acting as a local cellular signaling molecule, has to be discriminated from the neuronal cholinergic system and neuronal acetylcholine, acting as neurotransmitter. In the human placenta anti-ChAT immunoreactivity is found in multiple subcellular compartments like the cell membrane (microvilli, coated pits), endosomes, cytoskeleton, mitochondria and in the cell nucleus. These locations correspond with the results of experiments where possible functions of non-neuronal acetylcholine have been identified (proliferation, differentiation, organization of the cytoskeleton and the cell-cell contact, locomotion, migration, ciliary activity, immune functions). In the human placenta acetylcholine release is mediated by organic cation transporters. Thus, structural and functional differences are evident between the non-neuronal and neuronal cholinergic system. Enhanced levels of acetylcholine are detected in inflammatory diseases. In conclusion, it is time to revise the role of acetylcholine in humans. Its biological and pathobiological roles have to be elucidated in more detail and possibly, new therapeutical targets may become available.

Acetylcholine↗
PubMed2003

Proliferative effect of acetylcholine on rat trachea epithelial cells is mediated by nicotinic receptors and muscarinic receptors of the M1-subtype.

Acetylcholine (ACh), synthesized in mammalian non-neuronal cells such as epithelial cells of the airways, digestive tract and skin, is involved in the regulation of basic cell functions (so-called non-neuronal cholinergic system). In the present experiments rat trachea epithelial cells have been cultured to study the proliferative effect of applied ACh by [3H]thymidine incorporation. ACh (exposure time 24 h) caused a concentration-dependent increase in cell proliferation with a doubling of the [3H]thymidine incorporation at a concentration of 0.1 microM. This effect was partly reduced by 30 microM tubocurarine and completely abolished by the additional application of 1 microM atropine. The stimulatory effect of acetylcholine, remaining in the presence of tubocurarine, was prevented by 1 microM pirenzepine (preferentially acting at M1-receptors), but neither by 1 microM AFDX 116 (preferentially acting at M2-receptors) nor by 1 microM hexahydrosiladifenidol (preferentially acting at M3-receptors). The combination of tubocurarine and pirenzepine halved the basal [3H]thymidine incorporation. In conclusion, ACh produces a proliferative effect in rat trachea epithelial cells, the effect being mediated by both nicotinic receptors and muscarinic receptors of the M1-subtype.

Acetylcholine↗
PubMed2003

Modification of human polymorphonuclear neutrophilic cell (PMN)-adhesion on biomaterial surfaces by protein preadsorption under static and flow conditions.

Biomaterials induce a specific reaction after implantation in the human body. This reaction depends on the chemical and physico-chemical properties of the material as well as on the site and type of implantation. We have used a dynamic model, the parallel-plate flow-chamber, to examine the interactions of different biomaterials with polymorphonuclear neutrophilic cell (PMN) and how these interactions are influenced by protein preadsorption. Our results clearly show that for hydrophobic materials, glass and PE, which induce a prominent adhesion of PMN, the mixture of albumin and fibrinogen induces the best inhibitory effect. On hydrophilic biomaterial surfaces, untreated TCPS and PC-coated TCPS, reveal only a minor influence of adsorbed proteins on PMN adhesion because of a primary low adhesive surface for PMN and proteins as well. Human citrated plasma leads only to a slight inhibition of PMN adhesion. On the hydrophobic materials, glass and PE, bovine serum albumin (BSA) had the best anti-adhesive potential with respect to PMN. The coating using phosphorylcholine is an excellent surface modification to prevent PMN-adhesion and protein adsorption. The results of our experiments suggest that investigations under static and flow conditions are also needed to determine the influence of protein adsorption on other relevant blood cell populations, for example, platelets and monocytes.

Journal Article↗
PubMed2003

Effects of sex hormones, forskolin, and nicotine on choline acetyltransferase activity in human isolated placenta.

The activity of choline acetyltransferase (ChAT) was investigated in the human placenta before and after long-term incubation (24 h) to test the effects of sex hormones, nicotine and forskolin. ChAT activity differed considerably between the amnion (0.03 micromol/mg protein/h) and the villus (0.56). After long-term incubation, ChAT activity persisted in the latter but declined in the amnion. Neither sex hormones (beta-estradiol, testosterone, progesterone; 10 or 100 nM each) nor follicle stimulating hormone and luteinizing hormone (FSH/LH; 8.4 U/ml each) modified ChAT activity. Also nicotine (1 nM-100 microM) did not affect ChAT activity. Forskolin, an activitor of adenylyl cyclase, reduced ChAT activity in the villus but not in amnion. The present model offers the possibility to investigate ChAT regulation in intact tissue under long-term incubation. The risks of maternal smoking during pregnancy cannot be attributed to an effect of nicotine on placental ChAT activity. Differences in the regulation of ChAT appear to exist between neuronal and nonneuronal cells.

Amnion↗
PubMed2002

GM-CSF expression by human lung microvascular endothelial cells: in vitro and in vivo findings.

Recently, many findings indicate that granulocyte-macrophage colony-stimulating factor (GM-CSF) plays an important role in the pathogenesis of acute and chronic lung diseases. In the present paper, the production of this cytokine in human pulmonary microvascular endothelial cells (HPMEC) is investigated. In an in vitro study, quiescent HPMEC did not express GM-CSF, either at the transcriptional or at the protein level. After activation for 4 h with tumor necrosis factor (TNF)-alpha (30/300 U/ml), lipopolysaccharide (LPS; 0.1/1 microg/ml), or interleukin (IL)-1 beta (100 U/ml), a significant release of GM-CSF was measured by enzyme-linked immunosorbent assay, with a time-dependent increase over 72 h. IL-8 (4, 16, or 64 ng/ml) or IL-1 beta at a concentration of 10 U/ml did not induce the release of GM-CSF. Human umbilical vein endothelial cells (HUVEC) and the angiosarcoma cell line HAEND served as reference cell lines. GM-CSF release in HPMEC was significantly (P < 0.025-0.05) less inducible by IL-1 beta than in HUVEC. A constitutive expression of GM-CSF by HAEND was observed. Additionally, GM-CSF expression in vivo by the lung microvasculature was confirmed by immunohistochemistry in lung tissue. To our knowledge, this is the first report of the ability of human pulmonary endothelial cells to synthesize and release GM-CSF. These results support the hypothesis that the lung microvasculature via the production of GM-CSF is a potential contributor to the cytokine network in lung diseases. This could be of particular importance in the pathogenesis of the acute respiratory distress syndrome in which endothelial dysfunction plays a central pathogenetic role.

Cells, Cultured↗
PubMedDate not supplied

Differential endothelial CAM-expression after stimulation with supernatants of LPS- and cytokine-stimulated HT-29 and ST-ML-12 tumor cells growing as monolayer cultures and multicellular spheroids.

The mechanisms of tumor metastasis in vitro have been principally investigated using monolayer culture systems. In vivo, however, multicellular clusters of tumor cells rather than individual cells appear to penetrate the microvasculature. Thus, the multicellular tumor spheroid model represents a more suitable tool to study tumor cell-endothelial interactions. Our interest has centered on the role of inflammation on tumor intra- and extravasation. The expression of three endothelial cell adhesion molecules (CAM), ICAM-1, VCAM-1 and E-selectin, on HUVEC after incubation with supernatants of previously LPS-, TNF-alpha- or IL-1 beta-stimulated HT-29 (colon adenocarcinoma) and ST-ML-12 (melanoma) tumor cells growing as monolayers and spheroids, was compared using cell enzyme immunoassay. The results indicated that important differences in the expression of CAM between monolayers and spheroids exist depending on the tumor cell line. The possible significance of LPS for colon carcinomas was underlined by the spheroid-model results. With respect to the influence of intestinal bacteria on the behaviour of colon carcinomas, the spheroid model could be a useful in vitro system for a more realistic simulation of in vivo conditions.

Cell Adhesion Molecules↗