Biomedical subjects

B Klosterhalfen

Publications and source records attributed to B Klosterhalfen.

At least 55 records · Page 3Linked to original sources

PubMed2000

Expression of heat shock protein 70 (HSP70) at the interface of polymer-implants in vivo.

Synthetic polymer meshes are widely applied in the modern surgical approach for repairing abdominal wall defects. The implanted material is often observed leading to post-operative complications such as deficient abdominal wall mobility and adhesion formation with the abdominal cavity and/or abdominal organs. However, the functioning of the implant is primarily affected by the wound healing process guided by inflammatory events occurring at the tissue-material interface. This could presumably be influenced by the physicochemical properties of the polymer. With regard to it, the cellular and molecular processes involved in the successful restoration of the abdominal wall function are poorly understood. The present in vivo study, therefore, exemplary investigated in a rat model, the commercially available polymer-meshes Prolene (polypropylene, PP), Mersilene (polyester, PE) and Vicryl (polyglactin 910), as well as new mesh variants consisting either of PP (EB) or a combination of PP and polyglactin 910 (A plus or Vypro). The implanted material was evaluated by light and electron microscopy, immunohistochemistry as well as morphometry over an implantation period of 90 days. The data show that polymers induce heat shock protein (HSP)70, and its expression at the interface correlates inversely with the activity of the inflammatory reaction in vivo. Further, an ascent in HSP70 expression parallels the increasing implantation period and evolving foreign-body granulomas. Accordingly, a major role for HSP70 in modulating the local acceptance of polymers and as an additional marker for in vivo testing of polymers is suggestive.

Journal Article↗

PubMed2000

C1 inhibitor prevents capillary leakage after thermal trauma.

OBJECTIVE: In burned patients, activation of the complement and clotting systems is suggested to play an important role in the development of the capillary leak syndrome and inflammatory tissue destruction. In an animal model of thermal trauma, the possible protective effect of C1 inhibitor (C1Inh), a major control protein of both the complement and clotting systems, was investigated. DESIGN: Prospective, controlled study. SETTING: Animal model. SUBJECTS: Healthy pigs weighing 30 kg. INTERVENTIONS: Pigs were scalded for 25 secs with 75 degrees C hot water to achieve a 30% total body surface deep partial-thickness burn. The treatment group (n = 8) received C1Inh concentrate at an initial dose of 100 units/kg body weight immediately after thermal trauma, followed by three further applications every 12 hrs. Two control groups included animals that were either scalded (n = 8) or not scalded (n = 7) and treated with lactated Ringer's solution. MEASUREMENTS: Before and at various time points after trauma blood samples were analyzed for complement activation (APH50, CH50, SC5b-9, C3). Continuous monitoring of hemodynamic variables was performed and postmortem histologic examination of specimens from lung, heart, liver, kidney, stomach, duodenum, jejunum, ileum, and colon was carried out. Aseptically collected mesenteric lymph nodes were pooled and screened for bacterial translocation. For evaluation of the burn wound, biopsies from defined scalded and not scalded areas were taken daily. As a measure for edema formation, the weight of the animals was recorded every 2 hrs. RESULTS: After C1Inh treatment, which led to a significantly reduced complement activation, the clinical outcome was clearly improved, as indicated by vital signs and as demonstrated by reduced edema formation. Treated animals presented a diminished bacterial translocation. Pathologic alterations were clearly diminished in the burned skin, in shock-related organs, and in the intestines. CONCLUSION: Application of C1Inh appears to be an effective means to prevent capillary leakage and inflammatory tissue destruction after thermal trauma.

Animals↗

PubMed2000

Effects of prolonged partial liquid ventilation, high frequency ventilation and conventional ventilation on gas exchange and lung pathology in newborn surfactant-depleted piglets.

Partial liquid ventilation (PLV) improves oxygenation in various animal models of respiratory insufficiency. The aim of this study was to compare the effects of conventional ventilation (CV), high frequency oscillatory ventilation (HFOV), and PLV combined with CV or HFOV on gas exchange and histopathology. Thirty anaesthetised newborn piglets (mean weight 1.94 kg, age 1-3 days) were randomized in five groups of six animals: CV, CV + surfactant (S), HFOV+S, PLV/CV, and PLV/HFOV. Thirty min after lung injury had been induced with repeated saline lavage, specific ventilatory treatment was initiated. Three animals of the CV group died within the 24 h study period, whereas none died in any of the other groups. The oxygenation index (OI) and the PaO2/FIO2 ratio improved significantly within 30 min in all groups, but not in the CV group. After 24 h all oxygenation parameters were better in the PLV groups than in CV or CV+S (P < 0.05). No differences in gas exchange were noted between HFOV+S and PLV/CV. The combination of PLV with HFOV led to an increased PaO2/FIO2 ratio when compared with PLV/CV and with HFOV+S (P < 0.05). All PLV treated animals had significantly less lung injury in the upper and lower lobes compared with gas-ventilated animals by histologic semi-quantitative lung injury score (P < 0.01) and in the lower lobes by morphometry (P < 0.001). In conclusion, HFOV+S and PLV either with CV or HFOV are effective techniques to provide adequate gas exchange in S-deficient lungs compared with CV with and without S. However, lung injury was significantly improved in both PLV treated groups compared with HFOV+S and the CV groups.

Animals↗

PubMed2000

Abnormal collagen I to III distribution in the skin of patients with incisional hernia.

The surgical mesh-free repair of incisional hernias has to face recurrence rates of up to 50%. Apart from technical faults this is probably due to collagen metabolic disorders, known to play an important role in the development of inguinal hernia. In particular an altered ratio of collagen types I and III with an increase in collagen type III has been claimed to reduce the mechanical strength of connective tissues. Therefore, we investigated the content of collagen types I and III in the skin of patients with incisional hernia (n = 7) and recurrent incisional hernia (n = 5) in comparison to controls with healthy skin (n = 7) and normal skin scar (n = 7) both by immunohistochemistry and Western blot analysis. Both immunohistochemistry and Western blot analysis revealed a decrease in the ratio of collagen I/III due to a concomitant increase in collagen III. The patients with incisional hernias and with recurrent incisional hernias showed a ratio of 1.0 +/- 0.1 and 0.8 +/- 0.1, respectively, whereas the controls exhibit a ratio of 2.1 +/- 0.2 in healthy skin and of 1.2 +/- 0.2 in normal skin scar, respectively. The decrease was highly significant (p < 0.01) between the patients with either primary or recurrent hernia and the controls or the normal scar, as well as between controls and normal scar, whereas there was not any significant difference between primary and recurrent hernia (p > 0.05). Our data for the first time confirmed that the presence of incisional hernia is accompanied by impaired collagen synthesis in the skin. The decreased tensile strength of collagen type III may play a key role in the development of incisional hernias. Furthermore, it might explain the high recurrence rates of hernia repair by simple closure, as a repetition of the primarily failing technique, and the improvement by the additional use of alloplastic material.

Adult↗

PubMed2000

Effect of artificial valves on intestinal adaptation in the short-bowel syndrome: an integrated study of morphological and functional changes in rats.

Two-third-resections of the proximal or distal small bowel with and without artificial valves were performed in rats. Intestinal adaptation led to a significant increase in bowel diameter, villus height and villus diameter and consequently in absorptive mucosal surface area per unit of serosal area. Additional artificial valve construction did not affect the calculated mucosal surface area after proximal resection, while it significantly decreased the absorptive area by the occurrence of large, plump villi after distal resection. There was no change in small-intestinal absorption of water, glucose and electrolytes per unit mucosa with valve construction. DNA cytometry showed that artificial valves led to mucosal hyperplasia without hypertrophy. These morphological changes coincided with a significant increase in basal and stimulated gastrin release. The body weight was unchanged or even worse in the valve groups than after resection alone, despite a significantly prolonged transit time. Therefore, in our study, artificial valves did not result in functional improvements after small intestinal resections.

Adaptation, Physiological↗

PubMed2000

Expression of heat shock proteins 72/73 in human peritoneal mesothelial cells in vivo and in vitro.

Leukocyte accumulation during peritonitis leads to an injurious microenvironment which is involved in the host defense reaction but is also thought to cause peritoneal damage. We tested the hypothesis that mesothelial cells (MC) respond to the injurious microenvironment during peritonitis by an increased expression of heat shock proteins (HSP 72/73), a basic way by which cells are protected against injury. Comparison of resting MC and activated MC during peritonitis in vivo by means of immunohistochemistry revealed an increased expression of HSP 72/73. As assessed by Western immunoblotting, incubation of MC in vitro with tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) caused a time-dependent induction of HSP 72/73 expression, which was maximal 6 h after stimulation. We suggest that the increased HSP 72/73 expression of MC during peritonitis is in part induced by TNF-alpha and IL-1beta and may exert a cell-protective function, lessening MC damage during peritonitis.

Base Sequence↗

PubMed1999

Expression and function of beta(1) and beta(3) integrins of human mesothelial cells in vitro.

Mesothelial cells (MC) and extracellular matrix (ECM) components are thought to play a pivotal regulatory role during the inflammatory-reparative response of serosal membranes. Integrins are known to serve as cellular ECM receptors, but mesothelial integrin expression and its function, particularly its role for attachment to different ECM components, remain to be elucidated. The aim of the present study was to characterize the integrin expression of human omentum majus derived MC (HOMC) in vitro by immunohistochemistry and to investigate their functional significance with regard to HOMC adhesion to fibronectin (fn), vitronectin (vn), collagen IV (coll IV), and laminin (ln). Mesothelial cells in vitro strongly expressed beta(1), beta(3), alpha(2), alpha(3), alpha(5), and alpha(v) chains. A weak reactivity was found for alpha(1) and alpha(6), but no alpha(4) reactivity was detectable. Compared to the control, fn, vn, coll IV, and ln caused a significant 2.6-, 2.2-, 2-, and 1.6-fold increase of HOMC adhesion, respectively. Inhibition studies revealed that HOMC attachment to fn is mediated by alpha(5)beta(1), alpha(v)beta(1), and alpha(v)beta(3), with a synergistic effect of alpha(5)beta(1) and alpha(v)beta(3). Adhesion to vn is mediated by alpha(v)beta(1) and alpha(v)beta(3). Integrins alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) mediate adhesion to coll IV and ln. We suggest that the integrin expression and function of mesothelial cells described here play an important role in the interaction of MC with the ECM, particularly during the acute and chronic inflammatory-reparative response of serosal membranes.

Antigens, CD↗

PubMed1999

[Minimized polypropylene mesh for preperitoneal net plasty (PNP) of incisional hernias].

Repair of incisional hernias requires the extensive implantation of alloplastic materials. The extent of the scar tissue is markedly regulated by the amount and structure of the incorporated material and is responsible for the increased rate of local wound complications. Correspondingly, minimization of the alloplastic implants should be favorable. In a randomized, prospective clinical study, the early results were compared after implantation of either a minimized, low-weight (26.8 g/m2) mesh with a pore size of 5 mm or a common, heavy-weight (90.2 g/m2 polypropylene) mesh with a pore size of 0.8 mm. Indicators for clinical suitability were the rate and volume of seroma, subjective paraesthesia, physical capability, abdominal wall compliance, and the histologically analyzed tissue reaction of samples removed on the occasion of revision operations. As result, the optimized, low-weight mesh showed a remarkable decrease in the rate of seroma, patient complaints, less restriction of abdominal wall mobility, and improved abdominal wall compliance as verified by 3D stereography. These clinical findings corresponded to a pronounced decrease in inflammation and scar reaction, indicating improved incorporation of the alloplastic material. No other major complications except for one recurrence have been found.

Female↗

PubMed1999

Influence of polyglactin-coating on functional and morphological parameters of polypropylene-mesh modifications for abdominal wall repair.

Regarding oversized mechanical properties of most of the currently available materials a new mesh was developed (ETHICON, Norderstedt, Germany) and exactly adopted to the physiology of the human abdominal wall by reducing the amount of polypropylene (weight of <30 g/m2; mesh A). The consecutive increase of pores size as well as the use of multifilaments led to a pronounced increase of flexibility. To improve the handling during operation the initial stiffness of this low-weight large pores mesh was increased by strengthening with different amounts of absorbable polyglactin (combination of glycolide and lactide) in various forms: by coating (mesh B), adding multifilament polyglactin filaments (mesh C, Vypro) or both (mesh D), respectively. To test the consequences of the different supplementary techniques all mesh variants are implanted in a rat model. Over implantation intervals of 3, 7, 14, 21 and 90 days we measured the tensile strength, the resulting stiffness and surveyed the tissue response, particularly in regard to the extent of inflammation and to the induced fibrosis. The results proved a sufficient mechanical stability of the material reduced and pure polypropylene mesh A without restriction of the mobility of the abdominal wall compared with a group that had simple laparotomy and closure. The histological analysis of the interface showed a minor inflammatory reaction and a dense vascularisation. The addition of polyglactin multifilaments (mesh C) reduces the number of macrophages and granulocytes as indicators for acute inflammation, showing generally a scar formation limited merely to the perifilamentary region. The abdominal wall compliance remained unchanged compared with mesh A. The coating of the polypropylene with polyglactin (mesh B and D) appeared to change the tissue reaction remarkably, favouring the formation of a connective tissue capsule around the whole mesh. The mechanical testing revealed an apparent protrusion with an increase of curvature of the artificial abdominal wall at rising intraabdominal pressures. The entire coating of the polypropylene surface with polyglactin induces an all embedding scar plate, filling out the pores and forming a tissue capsule. The complex interaction of tissue and implanted biomaterials with their distinct alterations of the tissue response confirms the necessity of in vivo experiments even after 'minor' modifications. Whereas the addition of polyglactin filaments appears to be favourable, the coating of polypropylene with polyglactin seems to hinder the incorporation of the mesh.

Abdominal Muscles↗

PubMed1999

Experimental treatment of early chronic iliac vein thrombosis with a modified hydrodynamic thrombectomy catheter: preliminary animal experience.

PURPOSE: To evaluate the efficacy and safety of a new hydrodynamic catheter for removal of chronic iliac vein thrombus. METHODS: Unilateral iliac vein thrombosis was induced in seven pigs by combining permanent coil and temporary balloon occlusion. Thrombectomy was performed with a new hydrodynamic catheter (10 F S.E.T.) 3 days after thrombus induction. After thrombectomy, the animals were killed and the iliac veins were examined histologically. RESULTS: Complete thrombectomy (100% thrombus removal) was achieved in three of seven animals, 75% removal in three of seven animals, and only 30% removal in one animal. The average thrombus removal was 75%. Successful re-establishment of flow was achieved in five of seven cases. Histologically, the thrombi were partially organized, meeting the histologic criteria for early chronic venous thrombosis. Minor venous wall damage caused by the thrombectomy procedure without acute hemodynamic consequences was observed in four of seven cases. CONCLUSION: The 10-F S.E.T. catheter was reasonably effective in removing chronic iliac vein thrombus with no hemodynamically significant complications.

Animals↗

PubMed1999

Foreign body reaction to meshes used for the repair of abdominal wall hernias.

OBJECTIVE: To investigate the local tissue reactions to meshes that had been removed from humans. DESIGN: Open study SETTING: Surgical department of the technical University, Aachen, Germany. MATERIAL: Samples of 17 non-absorbable meshes (1 polyester, 10 polypropylene, 2 reduced polypropylene, and 4 polytetrafluorethylene, PTFE) and 1 absorbable mesh (polyglactin 910) that had been implanted for repair of abdominal wall defects. INTERVENTIONS: Light and transmission electron microscopy, immunohistochemistry, and histological examination. MAIN OUTCOME MEASURES: Signs of inflammatory response. RESULTS: Light microscopy showed chronic inflammatory tissue reaction, even after years, with pronounced differences among materials. Partial volume of inflammatory cells (%) varied from 32 in polypropylene, to 12 in expanded PTFE, 8 in polyester, and 7 in reduced polypropylene. Formation of connective tissue correlated significantly with the extent of the inflammatory reaction (p<0.01). In meshes implanted for long periods there were still numerous macrophages at the interface between tissue and polypropylene (45%), polyester (45%), expanded PTFE (25%), and reduced polypropylene (22%). There was no difference in time dependent tissue reactions (p = 0.19). CONCLUSION: Inflammation around alloplastic materials used to repair defects in the abdominal wall persists for many years. There was evidence of long term wound complications as a result of persistent foreign body reactions. Further studies are required to evaluate the long term tissue response to these materials.

Abdominal Muscles↗

PubMed1999

Expression of the extracellular matrix proteins collagen I, collagen III and fibronectin and matrix metalloproteinase-1 and -13 in the skin of patients with inguinal hernia.

Although abnormal collagen metabolism has been ascribed an important role in the high recurrence rates after surgical hernia repair, knowledge on tissue sampled in the region affected by inguinal hernias is poor. In the present study, we determined collagen type I and type III in the skin of adult patients with indirect and direct inguinal hernias by both immunohistochemistry and Western blot analysis. In addition, we quantified the immunohistochemical expression of fibronectin and matrix metalloproteinase (MMP)-1 and -13. The results indicated that the ratio of collagen type I/III was significantly decreased in the skin of patients with either indirect (n = 9) or direct hernia (n = 7), with a concomitant increase in collagen type III (p < 0.001 vs. controls, n = 7, without affection of the inguinal region). There was no significant difference between patients with indirect and direct hernia (p > 0.05). MMP-13 was not expressed in any of the skin samples investigated, whereas MMP-1 was found in the epidermis. Fibronectin was predominantly detected at the epidermal-dermal junction. MMP-1, MMP-13 and fibronectin levels were significantly different between patients and controls (p > 0. 05). We conclude that in contrast to the unchanged expression of fibronectin and MMP-1 and MMP-13, the decreased ratios of collagen tpye I/III with the basically increased amount of collagen type III could be of significant importance for the pathophysiology of hernias. The specific ratio collagen I/III probably reflects the altered structural integrity and mechanical stability of the connective tissue in both indirect and direct hernias. Moreover, our findings stress that hernias should be regarded as the manifestation of a systemic disease in the inguinal region with a genetic background, explaining the high recurrence rates after repeated suture repair, as well as the usefulness of surgical meshes in this clinical setting.

Aged↗

PubMed1999

The contact of human macrophages with extracellular matrix proteins selectively induces expression of proinflammatory cytokines.

The interaction of macrophages with proteins of the extracellular matrix (ECM) is important for the regulation of the immune and nonimmune functions displayed by these cells. Little, however, is known about the ability of different ECM proteins to transmit inflammatory signals into macrophages. Here we investigated the effect of the ECM proteins collagen type I, fibrin and fibronectin on the expression of the proinflammatory cytokines interleukin-1beta (IL-1beta) and IL-8 using RT-PCR, Northern and Western blot analysis and ELISA technique. It was found that collagen strongly induced IL-1beta and IL-8 expression in the macrophages. Fibronectin also stimulated cytokine expression, however, the amounts of the specific mRNAs were significantly lower compared to those induced by collagen. On the protein level IL-1beta revealed a close correlation to the mRNA expression. In contrast, fibrin did not elicit any IL-1beta and IL-8 response. These data show that different ECM proteins vary in their ability to induce proinflammatory cytokine expression in human macrophages suggesting that the protein composition of the ECM might be crucial in the initiation of inflammatory processes.

Blotting, Northern↗

PubMed1999

[Meshes within the abdominal wall].

The marked reduction in recurrence rates following reinforcement of the abdominal wall by meshes in incisional hernia has promoted their increasingly widespread use. The primary suture in technique failed more than half of the cases; therefore, the closure method needs to be changed and improved, particularly with regard to a possibly underlying defect in collagen metabolism. After more than 100 years of mesh development they are mainly placed in a sublay or onlay position, ePTFE, polyester and polypropylene are preferred. In any case the mesh has to overlap the defect sufficiently because of wound contraction. On the basis of our experience and reports in the literature, the advantages and disadvantages of various mesh techniques and mesh materials are discussed. However, because long-term studies are missing, the relevance of the cumulative risk for long-term complications such as mesh migration and fistula formation, the extent of patient complaints or the potential risk of a persistent foreign-body reaction cannot yet be ascertained. Nevertheless, because there are no surgical alternatives, meshes represent an improvement in hernia surgery that cannot be overestimated.

Follow-Up Studies↗

PubMed1999

Synthesis of type I and III collagen, expression of fibronectin and matrix metalloproteinases-1 and -13 in hernial sac of patients with inguinal hernia.

Since many years the importance of a weakness of the soft tissue for the development of hernias is discussed controversially. The tensile strength of the tissue is supposed to depend largely on the varying proportion of type I collagen with its high tensile strength and the immature type III collagen. Their relation is regulated by several collagenases, mainly matrix metalloproteinases-1 and -13 (MMP-1 and MMP-13), whereas fibronectin plays a key role for the adherence of cells within the extracellular matrix. The aim of this study was to investigate whether an alteration in type I and type III collagen synthesis, amounts of MMP-1 and MMP-13 and the expression of fibronectin were associated with the development of inguinal hernia. We analysed the hernial sac of patients with indirect (n = 9) and direct (n = 7) inguinal hernias and peritoneum in controls (n = 7) by immunohistochemistry and Western blot analysis. The results showed that the ratio of relative amount of I/III collagen was markedly decreased in patients with either indirect or direct hernias as compared with controls (p < 0.001) with a concomitant increase in type III collagen synthesis. MMP-13 was expressed neither in the hernial sac nor in the peritoneum of the controls, but the positive reactions of MMP-1 were found in the surface of the subserosa of the hernial sac in patients with indirect or direct hernias without any difference compared to controls. Furthermore, the relative amount of fibronectin in patients with either indirect or direct hernias is not significantly different from controls (p > 0.05). In regard to the known alterations of the collagen metabolism in fascia and skin of hernia patients the changed collagen I/III ratio with its increase of type III collagen in hernial sacs support the presence of a systemic disturbance of collagen metabolism. The absence of changes of the expression of collagenases (MMP-1, MMP-13) and the constant levels of fibronectin underline the central role of collagen synthesis for the development of indirect or direct hernias.

Aged↗

PubMed1998

Functional and morphological evaluation of different polypropylene-mesh modifications for abdominal wall repair.

Modern surgical hernia repair depends increasingly on synthetic meshes for the reconstruction of the abdominal wall. Despite the undisputed advantages of the polypropylene (PP) meshes currently available (Marlex, Prolene), reports of complications after implantation are increasing. Although, serious complications such as perforation and fistula formation are rare, minor and local complaints such as seromas, misfeelings and a decreased abdominal wall mobility are observed in about one-half of the patients. In regard to the exaggerated strength of the currently available mesh modifications a reduction of the material should improve the integration of the meshes into the artificial abdominal wall. In the present study, the commercially available basic mesh Prolene has been compared to two newly constructed PP-mesh modifications with reduced amounts of PP. The modifications have gradually been adopted to the physiological requirements of abdominal wall stability and mobility by reducing the amount of PP to 64% (E-BLUE) and 24% (variant A) of the Prolene mesh (developed by ETHICON, Norderstedt, Germany). All PP-mesh variants have been implanted in a rat model and studied by 3D-photogrammetry, tensiometry, light- and electron microscopy, as well as morphometry over implantation intervals of 3, 7, 14, 21 and 90 days. The data show that current constructions of PP-meshes are oversized and definitely restrict abdominal wall mobility in the present model. Sufficient stability of the artificial abdominal wall is even guaranteed by PP-mesh modifications with a reduction of PP-quantity to about 25% of the Prolene mesh. The degree of fibrosis directly correlated with abdominal wall restriction, whereas the formation of connective tissue in the interface PP-fibre/host-issue depends on the amount and activity of the inflammatory reaction. The quantity and quality of inflammation, again, directly relies to the amount of PP and to the surface area in contact with the recipient tissues. Altogether, the present study suggests that a modification of the PP-meshes could be helpful to prevent major and minor complications of surgical PP-meshes.

Abdominal Muscles↗

PubMed1998

Septic shock.

1. Septic shock is still a major complication of patients in intensive care units (ICUs) and, therefore, a main goal of ICU-related research. 2. After the unsatisfying results of multicenter clinical trials in blocking proinflammatory mediators such as tumor necrosis factor and interleukin-1 or endotoxin itself, research has focused on other important members of the cytokine pathways--in particular, anti-inflammatory cytokines such as interleukin 10. Moreover, there is an increasing knowledge of intracellular activation and deactivation pathways, inaugurating the theoretical advantage to block, attenuate or manipulate the inflammatory response at an early stage of disease or in a more specific fashion. 3. This review points out new developments in our understanding of the pathophysiology of septic shock and gives an example of a more recent approach in septic shock research--the heat shock response.

Clinical Trials as Topic↗

PubMed1998

Induction of apoptosis in the centre of multicellular tumour spheroids of colorectal adenocarcinomas--involvement of CD95 pathway and differentiation.

Multicellular tumour spheroids (MCTS) are three-dimensional cell culture systems which are widely used in cancer research. They are characterized by an outer zone of proliferating cells, an inner region of differentiating quiescent cells and an area of so-called necrotic cell death in their centre. The exact cause of this cell death, a controversy for many years, was the aim of the present study. Our data show that cell death in the centre of MCTS of three colorectal adenocarcinoma cell lines (HRT-18, HT-29 and CX-2) was induced by apoptosis. Apoptotic cells were initially distributed at random but accumulated very quickly in the quiescent and central area at day 4-5, suggesting a time- rather than size-dependent synchronization of apoptosis parallel to the formation of the proliferation gradient in MCTS. To study mechanisms inducing apoptosis, the Fas-pathway was investigated. A cell-cell contact-dependent expression of CD95 was found in all MCTS. FasL was not detected in monolayer cultures, but was expressed in spheroids of HRT-18 and CX-2. We found that TNF alpha and TGF beta 1 activated the CD95 pathway in all three cell lines. Since both TNF-alpha and TGF-beta are known to be inducible by hypoxia in a variety of cell types, we suggest that these hypoxia-induced factors sensitize the CD95 pathway in the quiescent area of MCTS. Furthermore, a loss of the heat shock proteins 27, 32, 60, 73 and 90 was observed in the quiescent area of spheroids. This suggests that tumour cell differentiation in the inner region of MCTS may be an additional factor inducing apoptosis.

Journal Article↗