The role of platelets in tumour growth.

Platelets, as initial responders to vascular injury, play a very important role in the initial stages of the haemostatic process. While the role of platelets in coagulation has been well studied and documented, their role in other physiological and pathological processes is just emerging. Platelets contain many biologically active molecules and, as they adhere to sites of tumour activated or injured endothelium, many of these molecules are released into the local microenvironment leading to platelet-mediated effects on vascular tone, repair and neo-angiogenesis. Platelets are likely play important roles in the tumour microenvironment that may be thought of as "a wound that never heals".

Hyperthermia inhibits platelet hemostatic functions and selectively regulates the release of alpha-granule proteins.

BACKGROUND: Hyperthermia is one of the main disturbances of homeostasis occurring during sepsis or hypermetabolic states such as cancer. Platelets are important mediators of the inflammation that accompanies these processes, but very little is known about the changes in platelet function that occur at different temperatures. OBJECTIVES: To explore the effect of higher temperatures on platelet physiology. METHODS: Platelet responses including adhesion, spreading (fluorescence microscopy), α(IIb)ß(3) activation (flow cytometry), aggregation (turbidimetry), ATP release (luminescence), thromboxane A(2) generation, alpha-granule protein secretion (ELISA) and protein phosphorylation from different signaling pathways (immunoblotting) were studied. RESULTS: Preincubation of platelets at temperatures higher than 37 °C (38.5-42 °C) inhibited thrombin-induced hemostasis, including platelet adhesion, aggregation, ATP release and thromboxane A(2) generation. The expression of P-selectin and CD63, as well as vascular endothelial growth factor (VEGF) release, was completely inhibited by hyperthermia, whereas von Willebrand factor (VWF) and endostatin levels remained substantially increased at high temperatures. This suggested that release of proteins from platelet granules is modulated not only by classical platelet agonists but also by microenvironmental factors. The observed gradation of response involved not only antiangiogenesis regulators, but also other cargo proteins. Some signaling pathways were more stable than others. While ERK1/2 and AKT phosphorylation were resistant to changes in temperature, Src, Syk, p38 phosphorylation and IkappaB degradation were decreased in a temperature-dependent fashion. CONCLUSIONS: Higher temperatures, such as those observed with fever or tissue invasion, inhibit the hemostatic functions of platelets and selectively regulate the release of alpha-granule proteins.

Cell therapy, a new standard in management of chronic critical limb ischemia and foot ulcer.

Fifty percent of diabetics (7% of general population) suffer from peripheral arterial occlusive disease, which may lead to amputation due to critical limb ischemia (CLI). The aim of our study was to prevent major limb amputation (MLA) in this group of patients using a local application of autologous bone marrow stem cells (ABMSC) concentrate. A total of 96 patients with CLI and foot ulcer (FU) were randomized into groups I and II. Patients in group I (n = 42, 36 males, 6 females, 66.2 ± 10.6 years) underwent local treatment with ABMSC while those in group II (n = 54, control, 42 males, 12 females, 64.1 ± 8.6 years) received standard medical care. The frequency of major limb amputation in groups I and II was 21% and 44% within the 120 days of follow up, respectively (p < 0.05). Only in salvaged limbs of group I both toe pressure and toe brachial index increased (from 22.66 ± 5.32 to 25.63 ± 4.75 mmHg and from 0.14 ± 0.03 to 0.17 ± 0.03, respectively, mean ± SEM). The CD34(+) cell counts in bone marrow concentrate (BMC) decreased (correlation, p = 0.024) with age, even though there was no correlation between age and healing. An unexpected finding was made of relative, bone marrow lymphopenia in the initial bone marrow concentrates in patients who failed ABMSC therapy (21% of MLA). This difference was statistically significant (p < 0.040). We conclude ABMSC therapy results in 79% limb salvage in patients suffering from CLI and FU. In the remaining 21% lymphopenia and thrombocytopenia were identified as potential causative factors, suggesting that at least a partial correction with platelet supplementation may be beneficial.

Autologous bone marrow stem cell transplantation in patients with end-stage chronical critical limb ischemia and diabetic foot.

A total 37 patients suffering from end stage-IV Fontaine (CLI and diabetic foot) with an ulcerated limb in whom all previous therapeutic strategies failed (e.g. surgical revascularization and endovascular repair) were selected and underwent local transplantation of Autologous Bone Marrow Stem Cells (ABMSCs). The efficacy/safety ofthis therapy was assessed by using several endpoints such as (a) prevention of amputation, (b) wound healing and (c) degree of angiogenesis. In order to assess the limb ischemia and hypoxia the several tests and measurements were performed pre- and post transplantation at a variety of time intervals. The measurements include: TP-toe pressure measurements (by Periflux 5000 Laser Doppler and Oxymetry system), SPP-skin perfusion pressure, ABI-ankle brachial index, LDP-Laser Doppler baseline and heat perfusion assessment, TcpO2 without and with O2 provocation inhalation test. In addition, a battery of biochemical and hematological tests of peripheral venous blood samples and bone marrow analysis were performed. Limb salvage was 81% in 30 patients, 7 patients (19%) were amputated for terminal severe ischemia and gangrene progression. In the group of limb salvage patients initial Toe pressure 23.119 (std. error 5.358) increased in 90 days follow-up into 29.888 (std. error 5.99), Toe brachial index increased from 0.1469 (std. 0.0326) to 0.1991(std. 0.401). In LASER doppler and TcpO2, TcpCO2 tissue perfusion examination TcpO2% Increase after O2 provocation inhalation test was elevated from 162.95 (%) to 229.86% which confirmed a very good tissue vasoreactivity after BMSC transplantation.

Single residue (K332A) substitution in Kir6.2 abolishes the stimulatory effect of long-chain acyl-CoA esters: indications for a long-chain acyl-CoA ester binding motif.

AIMS/HYPOTHESIS: The pancreatic beta cell ATP-sensitive potassium (K(ATP)) channel, composed of the pore-forming alpha subunit Kir6.2, a member of the inward rectifier K+channel family, and the regulatory beta subunit sulfonylurea receptor 1 (SUR1), a member of the ATP-binding cassette superfamily, couples the metabolic state of the cell to electrical activity. Several endogenous compounds are known to modulate K(ATP) channel activity, including ATP, ADP, phosphatidylinositol diphosphates and long-chain acyl coenzyme A (LC-CoA) esters. LC-CoA esters have been shown to interact with Kir6.2, but the mechanism and binding site(s) have yet to be identified. MATERIALS AND METHODS: Using multiple sequence alignment of known acyl-CoA ester interacting proteins, we were able to identify four conserved amino acid residues that could potentially serve as an acyl-CoA ester-binding motif. The motif was also recognised in the C-terminal region of Kir6.2 (R311-332) but not in SUR1. RESULTS: Oocytes expressing Kir6.2DeltaC26 K332A repeatedly generated K(+)currents in inside-out membrane patches that were sensitive to ATP, but were only weakly activated by 1 mumol/l palmitoyl-CoA ester. Compared with the control channel (Kir6.2DeltaC26), Kir6.2DeltaC26 K332A displayed unaltered ATP sensitivity but significantly decreased sensitivity to palmitoyl-CoA esters. Coexpression of Kir6.2DeltaC26 K332A and SUR1 revealed slightly increased activation by palmitoyl-CoA ester but significantly decreased activation by the acyl-CoA esters compared with the wild-type K(ATP) channel and Kir6.2DeltaC26+SUR1. Computational modelling, using the crystal structure of KirBac1.1, suggested that K332 is located on the intracellular domain of Kir6.2 and is accessible to intracellular modulators such as LC-CoA esters. CONCLUSIONS/INTERPRETATION: These results verify that LC-CoA esters interact at the pore-forming subunit Kir6.2, and on the basis of these data we propose an acyl-CoA ester binding motif located in the C-terminal region.

Channel density regulation of firing patterns in a cortical neuron model.

Modifying the density and distribution of ion channels in a neuron (by natural up- and downregulation or by pharmacological intervention or by spontaneous mutations) changes its activity pattern. In this investigation we analyzed how the impulse patterns are regulated by the density of voltage-gated channels in a neuron model based on voltage-clamp measurements of hippocampal interneurons. At least three distinct oscillatory patterns, associated with three distinct regions in the Na-K channel density plane, were found. A stability analysis showed that the different regions are characterized by saddle-node, double-orbit, and Hopf-bifurcation threshold dynamics, respectively. Single, strongly graded action potentials occur in an area outside the oscillatory regions, but less graded action potentials occur together with repetitive firing over a considerable range of channel densities. The relationship found here between channel densities and oscillatory behavior may partly explain the difference between the principal spiking patterns previously described for crab axons (class 1 and 2) and cortical neurons (regular firing and fast spiking).

Multicellular gastric cancer spheroids recapitulate growth pattern and differentiation phenotype of human gastric carcinomas.

BACKGROUND & AIMS: Advanced gastric cancer has a poor prognosis and is largely unresponsive to currently available chemotherapeutic drugs. The development of more effective therapies would be aided by better preclinical models. METHODS: An in vitro multicellular gastric cancer spheroid model was established using the liquid overlay technique and compared with the corresponding xenografts in immunodeficient mice. RESULTS: Twelve of 17 (71%) gastric cancer cell lines reflected growth characteristics of their parental gastric carcinomas in three-dimensional culture. Thus, cell lines derived from peritoneal and pleural carcinomatosis grew as single cells (HSC-39, KATO-II, KATO-III) and cell aggregates (SNU-5, SNU-16). Cell lines representing adenosquamous (MKN-1) and tubular differentiation (MKN-28, MKN-74, N87) formed partly compact multicellular spheroids recapitulating the tumor architecture of the respective original tumor. The differentiated phenotype was lost after subcutaneous implantation of the in vitro spheroids in mice. The degree of morphologic differentiation was reflected by the levels of mucin and constitutive E-cadherin expression. Heterogeneous changes of other adhesion molecules (EpCAM, alpha2beta1, CD44s, Le(x), sLe(x)) were observed. In contrast, cell lines derived from poorly differentiated gastric carcinomas (Hs-746T, RF-1, RF-48) formed fully compact spheroids mimicking the poorly differentiated phenotype, were E-cadherin negative, and showed only CD44s up-regulation. CONCLUSIONS: Recapitulating some complexity of their in vivo counterparts, multicellular gastric cancer spheroids may represent a physiologically valid model for studying the biology of this cancer, and testing new therapeutic strategies.

Possible mechanisms of acquired resistance to anti-angiogenic drugs: implications for the use of combination therapy approaches.

The ultimate target of anti-angiogenic drugs is the genetically stable, activated endothelial cell of a newly forming tumor blood vessel, rather than the genetically unstable tumor cell population per se. This led to the notion that acquired resistance to such drugs may not develop as readily, if at all. While there is some evidence that this lack of resistance development may be the case for some direct-acting angiogenesis inhibitors, it is becoming apparent that resistance can develop over time to many types of angiogenesis inhibitors including, possibly, some direct inhibitors, especially when used as monotherapies. Possible mechanisms for such acquired or induced resistance include: (i) redundancy of pro-angiogenic growth factors when the drug used targets a single such growth factor or its cognate endothelial cell-associated receptor tyrosine kinase; (ii) the anti-apoptotic/pro-survival function of growth factors such as VEGF, which, in high local concentrations, can antagonize the pro-apoptotic effects of various angiogenesis inhibitors; (iii) epigenetic, transient upregulation, or induction, of various anti-apoptotic effector molecules in host-endothelial cells; and (iv) heterogeneous vascular dependence of tumor cell populations. It is suggested that long-term disease control with anti-angiogenic drugs can be best achieved by judicious combination therapy. In this regard, the great molecular diversity of anti-angiogenic drug targets, in contrast to chemotherapy, makes this a particularly attractive therapeutic option, especially when approved, commercially available drugs considered to have anti-angiogenic effects are used in such combination treatment strategies.

'Accidental' anti-angiogenic drugs. anti-oncogene directed signal transduction inhibitors and conventional chemotherapeutic agents as examples.

A number of drugs currently being tested in clinical trials as possible angiogenesis inhibitors were not originally developed with the intention of suppressing tumour angiogenesis. Thalidomide and interferon alpha are obvious examples of such drugs. This list of 'accidental' angiogenesis inhibitors may include established agents such as conventional cytotoxic chemotherapeutic drugs as well as the new generation of anticancer drugs known as anti-oncoprotein signal transduction inhibitors. With respect to the former, the potential of such drugs to inhibit angiogenesis could be the result of their ability to cause collateral damaging effects on cycling endothelial cells found in newly formed blood vessels, or inhibiting other vital endothelial cell functions necessary for angiogenesis. The antitumour vascular side-effects of chemotherapy may be optimised by administering such drugs continuously on a more frequent (e.g. weekly or even daily) basis at levels well below the maximum tolerated dose (MTD), especially when this is done in combination with newly developed anti-angiogenic drugs such as vascular endothelial cell growth factor (VEGF) receptor blocking antibodies. This strategy may minimise or delay the problems of host toxicity and acquired drug resistance. The possibility of anti-angiogenic effects mediated by signal transduction inhibitors such as ras farnesyltransferase inhibitors (ras FTI's), or drugs which block receptor tyrosine kinases (e.g. ErbB2/neu) such as Herceptin, may be the consequence of such oncogenes inducing or upregulating various pro-angiogenic molecules such as VEGF (vascular endothelial cell growth factor) in tumour cells. Hence, treatment of tumour cells with such drugs can lead to downregulation of tumour cell-associated VEGF expression and this can contribute to an anti-angiogenic effect of the drug in vivo. In addition, some of these drugs may also affect certain 'activated' endothelial cell functions directly so as to block angiogenesis. An awareness of the potential of such conventional or experimental anticancer drugs to affect tumour growth through blockade or suppression of angiogenesis has implications for how anticancer drugs may be used clinically, either alone, or in combination with other drugs to optimally treat cancer.

Continuous low-dose therapy with vinblastine and VEGF receptor-2 antibody induces sustained tumor regression without overt toxicity.

Various conventional chemotherapeutic drugs can block angiogenesis or even kill activated, dividing endothelial cells. Such effects may contribute to the antitumor efficacy of chemotherapy in vivo and may delay or prevent the acquisition of drug-resistance by cancer cells. We have implemented a treatment regimen that augments the potential antivascular effects of chemotherapy, that is devoid of obvious toxic side effects, and that obstructs the development of drug resistance by tumor cells. Xenografts of 2 independent neuroblastoma cell lines were subjected to either continuous treatment with low doses of vinblastine, a monoclonal neutralizing antibody (DC101) targeting the flk-1/KDR (type 2) receptor for VEGF, or both agents together. The rationale for this combination was that any antivascular effects of the low-dose chemotherapy would be selectively enhanced in cells of newly formed vessels when survival signals mediated by VEGF are blocked. Both DC101 and low-dose vinblastine treatment individually resulted in significant but transient xenograft regression, diminished tumor vascularity, and direct inhibition of angiogenesis. Remarkably, the combination therapy resulted in full and sustained regressions of large established tumors, without an ensuing increase in host toxicity or any signs of acquired drug resistance during the course of treatment, which lasted for >6 months. This article may have been published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.

Oncogenes and angiogenesis: signaling three-dimensional tumor growth.

Three-dimensional tumor growth is dependent on the perpetual recruitment of host blood vessels to the tumor site. This recruitment process (mainly via angiogenesis) is thought to be triggered, at least in part, by the very same set of genetic alterations (activated oncogenes, inactivated/lost tumor suppressor genes) as those responsible for other aspects of malignant transformation (e.g., aberrant mitogenesis, resistance to apoptosis). Potent oncogenes are able to deregulate expression of both angiogenesis stimulators and inhibitors in cancer cells. For example, mutant ras expression is associated with increased production of vascular endothelial growth factor (VEGF) and downregulation of thrombospondin-1 (TSP-1). Upregulation of VEGF and angiogenesis can also be induced by constitutive activation of other oncogenic proteins (e.g., EGFR, Raf, MEK, PI3K) acting at various levels on the Ras signaling pathway. The mode and the magnitude of such proangiogenic influences can be significantly modified by cell type (fibroblastic or epithelial origin), epigenetic factors (hypoxia, changes in cell density), and/or presence of additional genetic lesions (e.g., preceding loss of p16 or p53 tumor suppressor genes). Activated oncogenes (e.g., ras, src, HER-2) induce co-expression of angiogenic properties concomitantly with several highly selectable traits (increased mitogenesis, resistance to apoptosis), a circumstance that may accelerate selection of the angiogenic phenotype at the cell population level. On the other hand oncogene-induced reduction in growth requirements may also endow tumor cells with a diminished (albeit not abrogated) dependence on (close) proximity to blood vessels, i.e., with reduced vascular dependence. Thus, oncogenes can impact several interconnected aspects of cellular growth, survival, and angiogenesis. Experimental evidence suggests that, in principle, many of these properties (including angiogenesis) can be simultaneously suppressed (and tumor stasis or regression induced) by effective use of the specific oncogene antagonists and signal transduction inhibitors.

Impact of oncogenes and tumor suppressor genes on deregulation of hemostasis and angiogenesis in cancer.

We postulate that oncogenes and tumor suppressor genes may influence tumor angiogenesis not only directly (e.g. by upregulating vascular endothelial growth factor) but also through their impact on expression (and/or function) of tissue factor and other elements of the hemostatic system.

An oral ivermectin regimen that eradicates pinworms (Syphacia spp.) in laboratory rats and mice.

Pinworm infection, a common problem in laboratory rodent colonies, is difficult to control because anthelmintics like ivermectin eliminate adult worms but have no effect on ova, which can survive ex vivo for prolonged periods. On the premise that repeated treatments with ivermectin would keep rodents parasite-free until all ova matured into ivermectin-susceptible worms in vivo or died in vivo or ex vivo, 80 rats and 25 mice heavily infected with pinworms (Syphacia obvelata and S. muris) were randomized to receive two to five courses of ivermectin 3 days apart or no treatment. During each treatment, ivermectin was given for 4 days in the drinking water; based on water consumption, the mean ivermectin dose was 2.9 and 4.0 mg/kg of body weight per day in rats and mice respectively. Ova production was monitored by weekly cellophane tape tests; 29 to 32 weeks after treatment ended, all rodents were euthanized, and their evacuated large intestinal contents were examined for adult pinworms and ova. Despite intermittently negative cellophane tape test results in untreated rodents (10 rats and 5 mice), all were infected with parasites at the end of the follow-up period. These findings underscore the limitations of the tape test for diagnosis of pinworm infection. After two courses of ivermectin, 1 of 10 rats and four of five mice were infected, whereas after three courses only 1 of 40 rats and one of five mice had parasites. In contrast, none of the 20 rats or 10 mice given either four or five courses of ivermectin had parasites at 30 to 32 weeks of follow-up evaluation. This simple and well-tolerated ivermectin regimen may help to treat and control pinworm infection in laboratory rodent colonies.

Dietary sodium restriction and pressor responsiveness to tyramine in spontaneously hypertensive rats.

OBJECTIVE: To determine in vivo whether in young spontaneously hypertensive rats (SHR) dietary sodium restriction decreases adrenergic transmitter release from the sympathetic nerve terminal. DESIGN: Dietary sodium restriction was initiated in young and mature SHR and Wistar-Kyoto (WKY) rats, and subsequently changes in pressor responsiveness to norepinephrine and to the indirectly acting sympathomimetic tyramine were determined in relation to their effects upon plasma catecholamines. RESULTS: In young SHR sodium restriction for 3-6 weeks prevented the development of hypertension, whereas in mature SHR sodium restriction did not affect blood pressure. Sodium restriction caused modest decreases in pressor responsiveness to the exogenous alpha-agonist, not different in young and mature SHR compared with WKY rats. In contrast, sodium restriction markedly inhibited pressor-responses to tyramine in young SHR and WKY rats, but not at all in mature rats. Tyramine increased plasma norepinephrine 5-10-fold. However, sodium restriction did not affect this response. The pressor response to tyramine was related to increases in total peripheral resistance, with minimal changes in cardiac output, and could be blocked by alpha 1-receptor blockade in rats on either control or low-sodium diets. CONCLUSIONS: These results show that sodium restriction causes only a small decrease in the pressor response to norepinephrine, but a more marked inhibition of the pressor response to tyramine in young SHR and WKY rats without affecting the plasma norepinephrine response to tyramine. These results suggest that dietary sodium can indeed affect presynaptic functions in vivo, but that plasma norepinephrine responses to tyramine may not reflect changes in arterial norepinephrine release, or that sodium restriction affects a co-transmitter rather than norepinephrine release per se.

Dietary sodium restriction and blood pressure response to sympathetic blockade in young versus adolescent spontaneously hypertensive rats.

The effects of dietary sodium restriction on the maintenance of blood pressure (BP) by sympathetic tone were evaluated in young versus more mature spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Sympathetic activity was assessed by BP responses to alpha 1-receptor blockade (prazosin), central inhibition of sympathetic outflow (clonidine), and by ganglionic blockade (hexamethonium). On regular sodium intake, SHR showed elevated BP and increased BP responses to sympathetic blockade at both 10 and 16 weeks of age. Sodium restriction to 9 or 17 mumols Na+/g food prevented or blunted development of hypertension in SHR when started at 4 weeks of age but did not affect BP when started at 10 weeks of age. Sodium restriction initiated in young SHR also prevented development of increased BP responses to sympathetic blockade. However, sodium restriction in more mature SHR did not decrease the increased BP responses to sympathetic blockade. We conclude that prevention of development of sympathetic hyperactivity in young SHR represents a major mechanism in the antihypertensive effect of sodium restriction in young SHR.

Dietary sodium restriction and blood pressure response to alpha-receptor blockade in young spontaneously hypertensive rats.

Dietary sodium restriction (9 mumol/g food) was started in 4-week-old spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY), and was continued for 3 or 6 weeks. Low sodium intake for 3 weeks prevented the development of hypertension in SHR. More prolonged restriction caused hypotensive blood pressure levels in both SHR and WKY. At 7 weeks of age, SHR and WKY on a control diet showed similar blood pressure decreases after prazosin; at 10 weeks the response was significantly larger in SHR. Sodium restriction from 4 to 7 weeks of age did not inhibit the prazosin effect, whereas after more prolonged restriction the response was significantly inhibited in SHR compared with control SHR as well as with sodium-restricted WKY. These results indicate that sodium restriction may affect blood pressure in several ways, and that inhibition of the alpha 1-receptor mediated pressor effect is specific to SHR.

Mitral valve replacement in dogs. Surgical technique and postoperative management.

Although techniques for the perioperative management of patients undergoing mitral valve replacement have been well established in humans, the use of these techniques has not been widely accepted in veterinary practice. The purpose of this study is to demonstrate that low morbidity and mortality could be achieved in the dog undergoing mitral valve replacement. Nine mongrel dogs (25-45 kg body weight) were subject to left thoracotomy and mitral valve replacement using cardiopulmonary bypass (CPB). The average time on CPB was 1 hour with an aortic cross-clamping time of 40 minutes using chemically induced cardiac arrest. CPB was performed under conditions of moderate systemic hypothermia (28-30 degrees C) and hemodilution (hematocrit, 25-35%). Operative mortality was 22% (2/9) with one death from excessive bleeding and the other from cerebral air embolism. All other animals recovered and were clinically normal 3 weeks after surgery. The authors conclude that successful mitral valve replacement is possible in the large dog.

Technique and postoperative management for successful cardiopulmonary bypass and open-heart surgery in dogs.

Twenty-six healthy mixed-breed dogs (25 to 35 kg) underwent hypothermic (27 C) cardiopulmonary bypass. The heart was arrested with cold (4 C) cardioplegic solution, and left ventriculostomy performed. Postoperative mortality was 11.5% (3/26). Two deaths were attributable to ventricular fibrillation the night after surgery, and one death 2 weeks later was the result of pulmonary embolization. All other dogs recovered promptly and were well at follow-up evaluation 6 weeks later. The most important considerations were (1) the surgical approach, ie, combining left lateral thoracotomy with cannulation of the right atrium and left femoral artery for cardiopulmonary bypass (CPB) (avoiding median sternotomy and aortic arch perfusion), (2) the adherence to strict criteria for CPB perfusion, consisting of blood flow of at least 2.2 L/m2/min, PCV no less than 25%, gas flow through the oxygenator (97% O2, 3% CO2) of at least 3.5 L/min, maintenance of a mean arterial blood pressure greater than 60 mm of Hg, and heparinization to maintain activated clotting time over 480 seconds, (3) the use of dipyridamole infusion to preserve platelets during CPB, resulting in decreased postoperative blood loss and (4) the monitoring of cardiac, respiratory, renal, and neurologic functions before, during, and after the operation, with particular emphasis on fluid balance and electrolytes. We concluded that a high success rate is possible for open-heart surgery in the dog requiring cardiopulmonary bypass, but only through meticulous surgical technique and the combined application of many monitoring techniques, with timely intervention to correct serious departures from homeostasis.

Construction of a large scale membrane reactor system with different compartments for cells, medium and product.

A flat membrane reactor system has been designed where different membranes separate cells from medium and cells from product, respectively. By the use of this system the product can be enriched and partially purified within the reactor. Like other membrane systems perfect protection from mechanical stress of surface adherent as well as suspension type-cells is achieved. The scale up of the design is possible in a wide range. The prototype construction corresponds to a conventional reactor of appr. 300 l and contains a membrane area of 25 m2. Beside the three chamber operation mode it is possible to operate the system as a two chamber reactor. With slightly modified gaskets the reactor resembles a new type of tube reactor, where the cells can be refed on their way through the 50 m long tube.