Laparoscopic versus open splenectomy for nontraumatic diseases.

BACKGROUND: Laparoscopic splenectomy (LS) is the standard procedure for normal size or moderately enlarged spleens; open splenectomy (OS) is preferred in cases of splenomegaly. In this study, indications for and complications of open and laparoscopic splenectomy were analyzed, with the aim to identify patients who will benefit from either technique. METHOD: A consecutive series of 52 patients undergoing elective open or laparoscopic splenectomy between January 2001 and December 2006 was analyzed. Spleen volume was calculated as length x width x depth from the pathologist's measurements. RESULTS: LS was performed in 25 patients with a median age of 41 years (range = 24-65). OS was performed in 27 patients with a median age of 60 years (range = 24-86) (p < 0.001). Conversion to OS was necessary in two patients (8%). Operation time was significantly shorter in LS (p < 0.05). Spleen volume was significantly greater in patients who underwent open (median = 2520 ml, range = 150-16,800 ml) versus laparoscopic (median = 648 ml, range = 150-4860 ml) splenectomy (p = 0.001). In 36% of all laparoscopic procedures, spleen volume exceeded 1000 ml. The underlying disease was mainly immunothrombocytopenia in LS patients and lymphoma and osteomyelofibrosis in OS patients. Five patients died after OS. Significantly more patients were hospitalized longer than 7 days following OS than following LS (p < 0.05). Overall complication rate was higher after OS (LS, 8; OS, 13 patients; p < 0.05). CONCLUSIONS: LS was preferred in younger patients with moderate splenomegaly, while massive splenomegaly mostly led to OS. In view of the absence of technique-related differences, LS can primarily be attempted in all patients.

Routine stenting reduces urologic complications as compared with stenting "on demand" in adult kidney transplantation.

OBJECTIVES: To examine the impact of the chosen surgical technique and of systematic versus "on-demand" placement of a primary stent on the incidence of urologic complications in adult kidney transplantation. METHODS: Data of 497 consecutive patients undergoing kidney transplantation at a single center were retrospectively analyzed with respect to urologic complications. Three different surgical strategies for the ureteroneocystostomy were compared: (1) transvesical anastomosis with stenting "on demand," (2) extravesical anastomosis with stenting "on demand," and (3) extravesical anastomosis with routine stenting. Nine parameters were evaluated by logistic regression for a possible contribution to the development of urologic complications. RESULTS: Routine placement of a stent significantly reduced the number of urologic complications compared with both transvesical or extravesical anastomoses with stenting "on demand" (20.8% in transvesical "on demand," 17.9% in extravesical "on demand," and 5.8% in extravesical "routine"). Logistic regression analysis revealed that routine stenting versus stenting "on demand" (P = 0.001) and living donor transplantation (P = 0.038) are two independent factors associated with a significantly lower incidence of urologic complications. Although routine stenting was not associated with an increased incidence of urinary tract infections, female gender was the only independent factor associated with this complication (P = 0.001). CONCLUSIONS: Routine stenting of the ureteroneocystostomy is superior to stenting "on demand" in adult kidney transplantation, suggesting that the intraoperative decision of whether to stent is insufficient to avoid urologic complications.

Activation of human microvascular endothelial cells with TNF-alpha and hypoxia/reoxygenation enhances NK-cell adhesion, but not NK-Cytotoxicity.

BACKGROUND: Ischemia/reperfusion injury (I/R) and cellular rejection in solid organ transplantation are characterized by adhesion molecule up-regulation on the graft endothelium, a prerequisite for leukocyte recruitment. The contribution of NK cells to I/R and allograft rejection is not well understood. The aim of the present study was to investigate allogeneic interactions between human NK cells and microvascular endothelial cells (MVEC) with special regard to the differential impact of TNF-alpha and hypoxia/reoxygenation in an in vitro model of I/R. METHODS: MVEC were stimulated in vitro for 8 h with TNF-alpha, exposed to hypoxia (1% O2), hypoxia/reoxygenation, and combinations thereof in a hypoxia chamber. Cell surface expression of adhesion molecules on MVEC was analyzed by flow cytometry, and adhesion molecule shedding by ELISA. NK cell adhesion on MVEC was determined under shear stress, and NK cytotoxicity using Cr-release assays. RESULTS: Surface expression of ICAM-1, VCAM-1, and E-/P-selectin on MVEC was up-regulated by TNF-alpha but unaffected by hypoxia/reoxygenation in the absence of TNF-alpha. ICAM-1 expression was further increased by a combination of TNF-alpha and hypoxia/reoxygenation, whereas TNF-alpha-induced E-/P-selectin expression was strongly reversed by hypoxia/reoxygenation. NK cell adhesion increased after exposing MVEC to TNF-alpha and hypoxia/reoxygenation. Susceptibility of MVEC to NK cytotoxicity was enhanced by TNF-alpha and slighty reduced by hypoxia/reoxygenation. CONCLUSIONS: Endothelial activation with TNF-alpha, but not hypoxia/reoxygenation, induced NK cytotoxicity whereas the combination thereof induced the strongest NK cell adhesion. Our findings suggesting a role for NK cells in allograft responses support the development of anti-inflammatory treatment strategies to prevent I/R.

Chlamydia pneumoniae induces aponecrosis in human aortic smooth muscle cells.

BACKGROUND: The intracellular bacterium Chlamydia pneumoniae is suspected to play a role in formation and progression of atherosclerosis. Many studies investigated cell death initiation versus inhibition by Chlamydia pneumoniae in established cell lines but nothing is known in primary human aortic smooth muscle cells, a cell type among others known to be involved in the formation of the atherosclerotic plaque. Type of cell death was analyzed by various methods in primary aortic smooth muscle cells after infection with Chlamydia pneumoniae to investigate a possible pathogenic link in atherosclerosis. RESULTS: Chlamydiae were found to be localized up to 72 h post infection in aortic smooth muscle cells either as single bacteria or inside of large inclusions. Quantification of host cell death by lactate dehydrogenase release assay revealed strictly dose and time dependent lysis for all tested isolates of Chlamydia pneumoniae. Phosphatidylserine exposure was detected by flow cytometry in Chlamydia pneumoniae infected cells. Ultrastructure of Chlamydia pneumoniae infected human aortic smooth muscle cells showed extensive membrane- and organelle damage, chromatin condensation but no nuclear fragmentation. DNA fragmentation as well as cell membrane permeability was analyzed by TUNEL and NHS-biotin staining and occurred exclusively in cells carrying Chlamydia pneumoniae spots but not in smooth muscle cells with inclusions. These morphological features of cell death were not accompanied by an activation of caspase-3 as revealed by analysis of enzyme activity but involved mitochondrial membrane depolarization as shown by TMRE uptake and release of cytochrome c from mitochondria. CONCLUSION: This study provides evidence that Chlamydia pneumoniae induce a spot like infection in human aortic smooth muscle cells, which results in a chimeric cell death with both apoptotic and necrotic characteristics. This aponecrotic cell death may assist chronic inflammation in atherosclerotic blood vessels.

Dextran sulfate acts as an endothelial cell protectant and inhibits human complement and natural killer cell-mediated cytotoxicity against porcine cells.

BACKGROUND: The innate immune system, including complement and natural killer (NK) cells, plays a critical role in activation and damage of endothelial cells (ECs) during xenograft rejection. The semisynthetic proteoglycan analog dextran sulfate (DXS, molecular weight 5,000) is known to inhibit the complement and coagulation cascades. We hypothesized that DXS may act as an "EC-protectant" preventing complement and NK lysis by functionally replacing heparan sulfate proteoglycans that are shed from the EC surface on activation of the endothelium. METHODS: Binding of DXS to ECs, deposition of human complement, cytotoxicity, and heparan sulfate expression after exposure to normal human serum were analyzed by flow cytometry. The efficacy of DXS to protect ECs from xenogeneic NK cell-mediated cytotoxicity was tested in standard 51Cr-release assays. RESULTS: DXS dose-dependently inhibited all three pathways of complement activation. Binding of DXS to porcine cells increased on treatment with human serum or heparinase I and correlated positively with the inhibition of human complement deposition. This cytoprotective effect of DXS was still present when the challenge with normal human serum was performed up to 48 hr after DXS treatment of the cells. DXS incubation of porcine ECs with and without prior tumor necrosis factor-alpha stimulation reduced xenogeneic cytotoxicity mediated by human NK cells by 47.3% and 25.3%, respectively. CONCLUSIONS: DXS binds to porcine cells and protects them from complement- and NK cell-mediated injury in vitro. It might therefore be used as a novel therapeutic strategy to prevent xenograft rejection and has potential for clinical application as an "EC protectant."

Hypoxia and reoxygenation do not upregulate adhesion molecules and natural killer cell adhesion on human endothelial cells in vitro.

OBJECTIVES: Ischemia/reperfusion injury is characterized by endothelial cell activation leading to increased expression of adhesion molecules such as inter-cellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, endothelial- and platelet-selectin (E- and P-selectin), and to the subsequent recruitment of leukocytes. The aim of the present study was to investigate the respective effects of a proinflammatory cytokine (tumor necrosis factor alpha, TNF-alpha), hypoxia and/or reoxygenation on adhesion molecule expression and natural killer (NK) cell adhesion in an in vitro model of I/R. METHODS: Human aortic endothelial cells (HAEC) were stimulated in vitro for 8h with TNF-alpha (1000 U/ml) and exposed to hypoxia (1% O(2)), reoxygenation (21% O(2)) or different combinations thereof. Cell surface expression of ICAM-1, VCAM-1 and E-/P-selectin on HAEC was analyzed by flow cytometry, and culture supernatants were tested for soluble adhesion molecules by ELISA. Rolling adhesion of NK cells on HAEC was determined using a rotating assay. RESULTS: Untreated HAEC constitutively expressed ICAM-1 on their surface but neither expressed E-/P-selectin, VCAM-1, nor shedded soluble adhesion molecules. Exposure of HAEC to hypoxia or hypoxia and reoxygenation did not upregulate cell surface expression or shedding of adhesion molecules. In contrast, TNF-alpha significantly upregulated cell surface expression of ICAM-1, VCAM-1, and E-/P-selectin and led to the shedding of ICAM-1 and E-selectin. Combined treatment of HAEC with TNF-alpha, hypoxia and reoxygenation reduced E-/P-selectin surface expression and enhanced E-selectin shedding, but did not further influence ICAM-1 and VCAM-1. Soluble VCAM-1 was not detected. NK cell adhesion on HAEC increased 4-fold after TNF-alpha stimulation, but was not affected by hypoxia or hypoxia and reoxygenation. CONCLUSIONS: Both the expression of endothelial adhesion molecules and rolling NK cell adhesion was upregulated by TNF-alpha but not by hypoxia alone or hypoxia followed by reoxygenation supporting the view that anti-inflammatory treatment may reduce ischemia/reperfusion injury.

Human umbilical cord cells: a new cell source for cardiovascular tissue engineering.

BACKGROUND: Tissue engineering of viable, autologous cardiovascular constructs with the potential to grow, repair, and remodel represents a promising new concept for cardiac surgery, especially for pediatric patients. Currently, vascular myofibroblast cells (VC) represent an established cell source for cardiovascular tissue engineering. Cell isolation requires the invasive harvesting of venous or arterial vessel segments before scaffold seeding, a technique that may not be preferable, particularly in pediatric patients. In this study, we investigated the feasibility of using umbilical cord cells (UCC) as an alternative autologous cell source for cardiovascular tissue engineering. METHODS: Human UCC were isolated from umbilical cord segments and expanded in culture. The cells were sequentially seeded on bioabsorbable copolymer patches (n = 5) and grown in vitro in laminar flow for 14 days. The UCC were characterized by flow cytometry (FACS), histology, immunohistochemistry, and proliferation assays and were compared to saphenous vein-derived VC. Morphologic analysis of the UCC-seeded copolymer patches included histology and both transmission and scanning electron microscopy. Characterization of the extracellular matrix was performed by immunohistochemistry and quantitative extracellular matrix protein assays. The tissue-engineered UCC patches were biomechanically evaluated using uniaxial stress testing and were compared to native tissue. RESULTS: We found that isolated UCC show a fibroblast-like morphology and superior cell growth compared to VC. Phenotype analysis revealed positive signals for alpha-smooth muscle actin (ASMA), desmin, and vimentin. Histology and immunohistochemistry of seeded polymers showed layered tissue formation containing collagen I, III, and glycoaminoglycans. Transmission electron microscopy showed viable myofibroblasts and the deposition of collagen fibrils. A confluent tissue surface was observed during scanning electron microscopy. Glycoaminoglycan content did not reach values of native tissue, whereas cell content was increased. The biomechanical properties of the tissue-engineered constructs approached native tissue values. CONCLUSIONS: Tissue engineering of cardiovascular constructs using UCC is feasible in an in vitro environment. The UCC demonstrated excellent growth properties and tissue formation with mechanical properties approaching native tissue. It appears that UCC represent a promising alternative autologous cell source for cardiovascular tissue engineering, offering the additional benefits of using juvenile cells and avoiding the invasive harvesting of intact vascular structures.

Living, autologous pulmonary artery conduits tissue engineered from human umbilical cord cells.

BACKGROUND: Tissue engineering represents a promising approach to in vitro creation of living, autologous replacements with the potential to grow, repair, and remodel. Particularly in a congenital operation, there is a substantial need for such implantation materials. We previously demonstrated fabrication of completely autologous, functional heart valves on the basis of peripheral vascular cells. Presently the feasibility of creating pulmonary artery conduits from human umbilical cord cells was investigated. METHODS: Human umbilical cord cells were harvested and expanded in culture. Pulmonary conduits fabricated from rapidly bioabsorbable polymers were seeded with human umbilical cord cells and grown in vitro in a pulse duplicator bioreactor. Morphologic characterization of the generated neo-tissues included histology, transmission, and scanning electron microscopy. Characterization of extracellular matrix was comprised of immunohistochemistry. Extracellular matrix protein content and cell proliferation were quantified by biochemical assays. Biomechanical testing was performed using stress-strain and burst-stress tests. RESULTS: Histology of the conduits revealed viable, layered tissue and extracellular matrix formation with glycosaminoglycans and collagens I and III. Cells stained positive for vimentin and alpha-smooth muscle actin. Scanning electron microscopy showed confluent, homogenous tissue surfaces. Transmission electron microscopy demonstrated elements typical of viable myofibroblasts, such as collagen, fibrils, and elastin. Extracellular matrix proteins were significantly lower compared with native tissue; the cell content was increased. The mechanical strength of the pulsed constructs was comparable with native tissue; the static controls were significantly weaker. CONCLUSIONS: In vitro fabrication of tissue-engineered human pulmonary conduits was feasible utilizing human umbilical cord cells and a biomimetic culture environment. Morphologic and mechanical features approximated human pulmonary artery. Human umbilical cord cells demonstrated excellent growth properties representing a new, readily available cell source for tissue engineering without necessitating the sacrifice of intact vascular donor structures.