Fusarium toxins in cereals grown in Switzerland.

770 cereal samples of Swiss origin which were collected in various feed mills and cereal collection centres in the years 2000 - 2002 were assayed for Deoxynivalenol (DON) and zearalenone (ZEA). 137 samples were also assayed for T-2 toxin. The prevalence of DON and ZEA contamination was higher in cereals harvested in the rainy summer 2002 than in the previous years. T-2 toxin levels exceeding 100 µg/kg were found only in three oats samples. High levels ofFusarium toxins do not frequently occur in Swiss cereals.

Endothelial cell seeding improves patency of synthetic vascular grafts: manual versus automatized method.

Lack of an endothelial surface is the most important variable causing the relatively poor patency of synthetic bypass grafts. This study was designed to investigate the effect of endothelial cell seeding on small-diameter Dacron grafts seeded with microvascular endothelial cells from omentum, and to evaluate two methods (manual vs automatized) for one-stage seeding in a canine carotid artery model. In 30 mongrel dogs microvascular endothelial cells were harvested from omentum, either by a manual or an automatized method, and seeded onto 6-mm internal diameter Dacron prostheses prior to the graft interposition into the common carotid arteries. Non-seeded Dacron grafts were used as control grafts. All dogs received dipyridamole (75 mg/day) and acetylsalicylic acid (325 mg/day) for 4 weeks. The prostheses were explanted between 2 and 26 weeks after insertion. The results were assessed by patency, angiography, light and scanning electron microscopy, transmission electron microscopy, and morphometry. Endothelial cell seeding improved the patency rate significantly, regardless of the seeding methods used. The overall actuarial patency rates at 5, 12, and 26 weeks were 98%, 94% and 94%, respectively, for the seeded Dacron grafts, and 92%, 62% and 54%, respectively, for the non-seeded grafts. The automatized method yielded more endothelial cells per gram of omental tissue than the manual method (P = 0.0002), but there was no difference (P = 0.34) between the seeding densities per square centimeter of the graft surface. The harvesting and seeding by the automatized method took 55 min for the whole procedure, 20 min less than the manual method. We concluded that one-stage endothelial cell seeding with omental microvascular endothelial cells improved the patency of small-diameter Dacron grafts in a canine model. The automatized method obtained excellent results comparable to the manual procedure, and also reduced the time necessary for the cell seeding.

Seeding with omental cells prevents late neointimal hyperplasia in small-diameter Dacron grafts.

BACKGROUND: The influence of complete endothelialization of a prosthetic graft on development of late neointimal hyperplasia is unknown. This study was designed to investigate the effect of complete coverage with endothelial-like cells on late neointimal hyperplasia in small-diameter Dacron grafts seeded with omental cells in a canine model. METHODS AND RESULTS: Four-mm-ID Dacron grafts were seeded with cells from omentum and implanted in the carotid arteries in 24 mongrel dogs. Each dog received one seeded and one nonseeded graft. The graft patencies were assessed by angiography at 1, 5, 12, 26, and 52 weeks after surgery. The prostheses were explanted at 5, 12, 26, and 52 weeks after surgery and underwent microscopic studies. The actuarial patency rates at 1, 5, 12, 26, and 52 weeks were 100%, 95%, 95%, 95% and 95% for seeded grafts and 100%, 86%, 49%, 40%, and 13% for nonseeded grafts, respectively. The seeded grafts exhibited a uniform endothelial-like luminal monolayer without the development of late neointimal proliferation or anastomotic neointimal hyperplasia. Neointimal tissue thickness increased up to 6 months; no additional progression of the subendothelial tissue thickness was observed, in fact there was an insignificant decrease. CONCLUSIONS: Seeding with omental cells prevents development of late neointimal hyperplasia of small diameter prosthetic vascular grafts in a canine model.

Superior late patency of small-diameter Dacron grafts seeded with omental microvascular cells: an experimental study.

The purpose of the study was to investigate the effect of omental microvascular cell seeding on the patency of small-diameter Dacron prostheses usable for coronary artery bypass grafting. In a canine carotid artery model, each dog (n = 64) received one seeded and one similar nonseeded Dacron prosthesis (internal diameter = 4 or 6 mm). Enzymatically harvested omental microvascular cells (omentum = 27.6 +/- 5.9 g [+/- the standard deviation]; range, 17 to 50 g) were seeded prior to implantation. The seeding density was 1.91 +/- 0.26 [+/- the standard error] x 10(6) cells/cm2 of graft surface. Dipyridamole (75 mg/d) and acetylsalicylic acid (325 mg/d) were administered orally for 4 weeks postoperatively. The prostheses were explanted between 2 and 52 weeks after placement. The results were assessed by angiography; light, scanning electron, and transmission electron microscopy; and morphometry. The seeded grafts developed a uniform luminal monolayer of endothelial cells with minimal platelet or cellular deposition. These grafts also had a significantly higher overall patency rate and significantly larger thrombus-free surface areas than the nonseeded grafts. The overall actuarial patency rates at 1 week, 5, 12, 26, and 52 weeks were 100%, 98%, 93%, 93%, and 93%, respectively, for seeded Dacron grafts and 100%, 91%, 61%, 54%, and 18%, respectively, for nonseeded grafts. The patency rates of Dacron grafts usable for coronary artery bypass grafting are significantly improved by seeding with omental microvascular cells in a canine model.

Long-term results of tissue development and cell differentiation on Dacron prostheses seeded with microvascular cells in dogs.

PURPOSE: The purpose of this study was to investigate in vivo the long-term development, differentiation, and proliferation of the subendothelial tissue on Dacron prostheses seeded with microvascular cells (MVC). METHODS: Autologous MVC from omental adipose tissue were seeded on 4 mm Dacron prostheses and the prostheses interposed in the carotid arteries of mongrel dogs for 5, 13, and 26 weeks. RESULTS: Light and electron microscopic evaluation of patent seeded prostheses demonstrated an almost complete monolayer of endothelial cells and well-organized subendothelial tissue, whereas patent control prostheses were mainly covered by red and white thrombi, which were partially replaced by organized tissue with increased implantation time. The measurements of the thickness of the luminal cell layer in seeded and control grafts showed no statistically significant increase between 5 and 26 weeks of implantation. The subendothelial tissue of seeded prostheses demonstrated a time-dependent maturation of highly synthesizing myofibroblasts embedded in a collagen matrix to cells with features of smooth muscle cells located in a collagen-elastin matrix. In control grafts examined after 26 weeks the spontaneous endothelialization was accompanied by a delayed or incomplete maturation of subendothelial tissue. CONCLUSIONS: Our study indicates that MVC seeded onto Dacron prostheses are able to generate a vascular wall that does not continue to proliferate after prolonged implantation and that increasingly resembles the wall of a normal artery in cell differentiation and intercellular matrix.

[Long-term results in the dog carotid artery with small lumen vascular prostheses with microvascular endothelial cells]. / Langzeitresultate in der Hundekarotis mit kleinlumigen Gefässprothesen mit mikrovaskulären Endothelzellen.

In this study we evaluated the long-term results of microvascular endothelial cell seeding of small diameter Dacron grafts with omentally derived cells in a canine model. 6 cm long and 4 mm I.D. seeded Dacron prostheses were implanted end-to-end in the carotid position in 12 dogs for 6 to 12 months. Microvascular endothelial cells were enzymatically harvested from omentum prior to implantation and seeded onto Dacron grafts with a seeding density of 1.5 x 10(6) cells/cm2 of the graft. The antiplatelet therapy (Aspirin, Dipyridamol) was administered for 4 weeks postoperatively. All seeded grafts were patent throughout the study. The thrombus-free surface area for seeded grafts was 99.6 +/- 0.8% and 99.6 +/- 0.9% at 6 months and one year, respectively. Scanning electron microscopy revealed a confluent endothelial layer. We concluded that endothelial cell seeding of smaller-diameter prosthetic vascular grafts with omentally derived endothelial cells obtained excellent long-term patency rate in the canine model.

Immediate shear stress resistance of endothelial cell monolayers seeded in vitro on fibrin glue-coated ePTFE prostheses.

The shear stress resistance of endothelial cells (EC) previously seeded onto ePTFE grafts was assessed by morphometric determination of the number of cells per cm2 of graft surface before and after exposure of 6 h of arterial blood flow interposed in the canine femoral artery. Autologous venous endothelial cells (AVEC) were harvested from the extrajugular veins of five dogs. The AVEC were cultured in vitro and seeded at a density of 150 x 10(3) cells per cm2 onto 4 mm ID ePTFE grafts precoated with fibrin glue and human fibronectin. Subsequently, the AVEC monolayers on the grafts were cultured for 8 days using a perfusion system and then implanted end-to-end in the femoral artery. All grafts remained patent (5/5). Scanning electron microscopy demonstrated complete, thrombus-free monolayers of AVEC after 6 h of arterial blood flow. The cell densities were 124 +/- 14 and 129 +/- 7 x 10(3) cells per cm2 respectively before and after implantation. It is concluded that in vitro lining of 4 mm ePTFE vascular prostheses is feasible and results in EC monolayers on the graft surface which are shear stress resistant and athrombogenic.

[Effect of hemodynamic conditions and prosthesis structure on endothelialization of clinical and experimental small lumen vascular prostheses]. / Einfluss der hämodynamischen Bedingungen und der Prothesenstruktur auf die Endothelialisierung von klinischen und experimentellen kleinlumigen Gefässprothesen.

Seeding of small-diameter vascular prostheses (ID less than or equal to 6 mm) with autologous microvascular cells (AMVC) results in a complete endothelial cell layer on the luminal surface. The purpose of this study was to examine the influence of the blood flow velocity (due to 4 or 6 mm ID) and the structure of inner graft surface (crimped, uncrimped) on the endothelialization. AMVC were harvested from omental adipose tissue (mean: 0.56 X 10(6) cells/g tissue) from 10 mongrel dogs (mean: 27.9 kg). During preclotting, the 4 mm uncrimped and the 6 mm crimped double velour Dacron prostheses (Meadox Medicals, Inc.) were seeded with 1.0 X 10(6) cells/cm2 graft surface. Grafts were implanted into the carotid arteries (N = 5 in each group). The animals received antiplatelet therapy. After five weeks, all seeded prostheses were patent. The thrombus free surface (TFS) of seeded prostheses was 99.9% (4 mm) and 90.5% (6 mm). Scanning electron microscopy revealed an athrombogenic layer of endothelial cells on a smooth surface. -It is concluded that in canine experiments endothelialization of 4 and 6 mm grafts after seeding with AMVC is not affected by blood flow velocity or graft structure.

[Endothelialization of 6 mm vascular prostheses with microvascular endothelial cells from fatty tissue of the omentum]. / Endothelialisierung von 6-mm-Gefässprothesen mit mikrovaskulären Endothelzellen aus dem Fettgewebe des Omentums.

The seeding of 6 mm Dacron prostheses with microvascular endothelial cells from omental adipose tissue leads to endothelialized prostheses, 5 weeks later with a complete coverage of functional and thrombus free endothelium. The method is ready for clinical trials.

In situ cannulation, microgrid follow-up and low-density plating provide first passage endothelial cell masscultures for in vitro lining.

A rapid and reliable harvest and culture technique was developed to provide a sufficient number of autologous endothelial cells for the confluent in vitro lining of cardiovascular prostheses. Enzymatic endothelial cell detachment was achieved by the in situ application of collagenase to short vessel segments. This harvest technique resulted in a complete lack of contaminating smooth muscle cells in all of 124 cultures from nonhuman primates and 13 cultures from human adults. The use of a microgrid technique enabled the daily in situ quantification of available endothelial cells. To assess ideal plating densities after passage the population doubling time was continuously related to the cell density. Surprisingly, a low plating density of 1.5 X 10(3) endothelial cells/cm2 achieved 43% shorter cell cycles than the usual plating density of 1.0 X 10(4) endothelial cells/cm2. Moreover, low density plating enabled mass cultures after one single cell passage, thereby reducing the cell damaging effect of trypsin. When the growth characteristics of endothelial cells from five anatomically different vessel sites were compared, the external jugular vein--which would be easily accessible and dispensable in each patient--proved to be an excellent source for endothelial cell cultures. By applying in situ administration of collagenase, low density plating and microgrid follow-up to adult human saphenous vein endothelial cells, 14,000,000 first passage endothelial cells--sufficient for the in vitro lining of long vascular prostheses--were obtained 26.2 days after harvest. (95% confidence interval:22.3 to 32.2 days).

Late neutrophil infiltration of canine endothelial cell seeded Dacron grafts.

Neutrophil infiltration is known to affect the endothelial monolayer of seeded vascular grafts. The aim of this study was to develop an in vitro system allowing the monitoring of neutrophil (PMN) adherence after graft implantation. Dacron prostheses were seeded with autologous canine microvascular cells from omental adipose tissue and implanted for 35 days. In vitro, mature monolayers of canine homologous venous endothelial cells (CHVENC) were exposed to heparinized whole blood samples taken at days one and four postoperatively, followed by weekly tests. PMNs adherent to the CHVENC were counted per culture area. Results showed the feasibility of PMN monitoring, and demonstrated a late PMN adhesion, reaching its maximum about 20 days after implantation and decreasing to normal values after five weeks. It is concluded that in vitro tests can be used for noninvasive studies of host plasma factors and leukocyte activation.

Internal-mammary coronary artery grafts: is their superiority also due to a basically intact endothelium?

The internal mammary artery (IMA) is a superior conduit for coronary artery revascularization and many factors have been suggested for explanation of this superiority. IMA and saphenous vein grafts have been systematically analysed with scanning electron microscopy (SEM) in a series of 11 patients undergoing coronary artery revascularization. At the time of implantation endothelial damage is almost absent in internal-mammary-artery (IMA) grafts; small areas of exposed subendothelial matrix may be present but are essentially non-thrombogenic as reflected by the lack of clots in these areas. In contrast the endothelium of harvested human saphenous veins (SV) shows large thrombogenic defects with exposed collagenous fibrils. The extent and deepness of the defects deteriorated in the period between removal of the vein and its attachment to the aorta. We conclude that long-term superiority of IMA grafts may also be due to the lack of primary intimal defects.

[Development of a small-lumen vascular prosthesis coated with autologous endothelial cells]. / Entwicklung einer kleinlumigen mit autologen Endothelzellen beschichteten Gefässprothese.

A new compliant prosthesis with a monolayer of autologous endothelial cells (ENC) has been developed. It consists of a porous polyurethane-siloxane-copolymer reinforced by a polyester network to prevent excessive dilatation. On the inner surface an ENC monolayer is established before implantation by a cell culture procedure. The prosthesis displays compliance (13.2 +/- 3.0 x 10(-4) mmHg-1) comparable to native arteries. It is non-kinkable (minimal radius of curvature less than 5 mm). Burst resistance, remaining deformation, cut out force and tensile strength are superior to standard values. ENC-coverage in excess of 95% of the inner surface was produced in vitro using a lining procedure. The monolayer of confluent cells was demonstrated to consist of endothelial cells by their characteristic cobblestone morphology, the expression of factor VIII related antigen and the specific uptake of Dil-Ac-LDL. The unstimulated prostacyclin production was similar both in native veins as well as in lined prostheses. Antithrombogenicity of the endothelial cell lining was demonstrated in 24 h animal implants.

An improved procedure for enzymatic harvesting of highly purified canine venous endothelial cells for experimental small diameter vascular prostheses.

We developed a new device, the vein holder, to improve yield and purity of enzymatic harvests of venous endothelial cells. External jugular veins of mongrel dogs were dissected by a no-touch technique. In vitro length and circumference of the vein segments were decreased to about half of the in situ dimensions. The vein holder enabled mounting of the veins at 80% of their in situ length during endothelial cell harvesting. Trypan blue staining and scanning electron microscopic observations revealed that vein eversion as well as the new vein holder technique successfully removed the endothelium. Endothelial cell harvests by the eversion technique were, however, low and varied in size, viability, and purity. In contrast, the defined handling by the new vein holder technique regularly provided markedly increased amounts of endothelial cells. Most of the cells attached and developed cultures consisting of endothelial cells only, as shown by the uptake of DilAcLDL. Prostacyclin production of confluent cultures was similar to that of native veins. It is concluded that minimal handling, defined mounting, and prevention of overfilling the vein markedly improves endothelial cell harvests, providing greater amounts of viable and purified endothelial cells.

A compliant small-diameter vascular prosthesis lined with functional venous endothelial cells.

The long-term patency of small-diameter vascular grafts is still unsatisfactory. In contrast to native arteries, they are inelastic and lack active antithrombogenicity. To improve long-term patency, a new 4 mm internal diameter prosthesis was developed which is compliant and lined with functional endothelial cells (ENC). The wall of this prosthesis consists of a microporous polyurethane-siloxane copolymer reinforced with a polyester network. It displays compliance (13.2 x 10(-4) mmHg-1) comparable to native arteries, is nonkinkable (minimum radius of curvature = 5 mm), burst resistant, and easily suturable. Using a lining procedure, coverage of prostheses by ENC was in excess of 95%. The ENC populations were found to be highly pure (by factor VIII-related antigen, DilAcLDL uptake) and to produce about 0.3 ng prostacyclin per cm2. In vitro tests of shear stress resistance demonstrated that ENC monolayers on the new elastic prosthesis remain intact for 3 hr in physiologically pulsating culture medium (Vmax = 50 cm/sec). Lined prostheses implanted for 24 hours in mongrel dogs as an arteriovenous shunt demonstrated the antithrombogenicity of the cultured ENC. The results suggest that small-diameter vascular prostheses which are compliant, porous, and actively antithrombogenic are feasible.

[Development of small-diameter vascular prostheses covered with human endothelial cells: Possibilities and limits of in vitro-tests]

There is an increasing demand for small-diameter vascular prostheses for the replacement of arteriosclerotic coronary arteries. They may be replaced by autologous blood vessels, usually parts of the saphenous vein. Prostheses of synthetic materials and an inner diameter of less than 4 to 6 mm are unsatisfactory and, therefore, not implanted for coronary arteries. A substantial improvement is, however, expected for prostheses covered with human autologous endothelial cells. It has to be proved that this new type of vascular prostheses is an adequate replacement for small arteries. Tests of the new prosthesis should comprise cell and tissue compatibility of the synthetic materials as well as normal function of the endothelial cells. The aim of the present paper was to reduce the number of animal experiments in this development by establishing new in vitro tests for endothelial cell compatibility of synthetic materials and for the adherence of endothelial cell on the prosthesis. Physiologically haemodynamic streaming conditions are in vitro produced by self-constructed circulatory systems. First results demonstrate that physiologic shear stress is achieved. Limits and relevance of the in vitro tests are discussed in relation to animal experiments and clinical studies.

On the role of type IX collagen in the extracellular matrix of cartilage: type IX collagen is localized to intersections of collagen fibrils.

The tissue distribution of type II and type IX collagen in 17-d-old chicken embryo was studied by immunofluorescence using polyclonal antibodies against type II collagen and a peptic fragment of type IX collagen (HMW), respectively. Both proteins were found only in cartilage where they were co-distributed. They occurred uniformly throughout the extracellular matrix, i.e., without distinction between pericellular, territorial, and interterritorial matrices. Tissues that undergo endochondral bone formation contained type IX collagen, whereas periosteal and membranous bones were negative. The thin collagenous fibrils in cartilage consisted of type II collagen as determined by immunoelectron microscopy. Type IX collagen was associated with the fibrils but essentially was restricted to intersections of the fibrils. These observations suggested that type IX collagen contributes to the stabilization of the network of thin fibers of the extracellular matrix of cartilage by interactions of its triple helical domains with several fibrils at or close to their intersections.

Neoplastic modulation of extracellular matrix: proteoglycan changes in the rabbit mesentery induced by V2 carcinoma cells.

Previous studies have shown that the invasion of V2 carcinoma cells in the rabbit mesentery is associated with marked extracellular matrix synthesis leading eventually to an overall increase in mesenteric mass. The purpose of the present study was to investigate the structural and biochemical composition of the extracellular matrix in tumor-free parts of rabbit mesenteries at various stages after intraperitoneal implantation of V2 carcinoma cells. The overall thickness of the tumor-implanted mesenteries increased progressively and peaked at about Day 14, when it was about 8 times greater than the untreated or liver-implanted controls. This was mainly the result of an accumulation of extracellular matrix components. In particular, there was a marked increase in both collagen fibers and proteoglycan granules, as well as filaments, probably hyaluronic acid, as visualized by ruthenium hexammine trichloride. Stereological analysis showed a 6-fold increase in collagen fibers and a significant increase in the density and average diameter of proteoglycan granules. Biochemical analysis revealed a marked elevation in uronic acid content in the tumor-implanted mesenteries. Specifically, they contained 2.6 and 8.6 times the amount of hyaluronic acid and chondroitin sulfate, respectively, than did controls. Furthermore, the relative percentage of chondroitin sulfate was elevated markedly (26 versus 6% in controls). However, the content of heparan or dermatan sulfate did not vary significantly. Stereological analysis of the fibroblasts showed that their absolute number had doubled and that the cell volume of the individual fibroblast had increased markedly. This suggests that the fibroblasts were responsible for the excessive production of the extracellular matrix. These results support the concept that carcinoma cells can modulate their surrounding extracellular environment by stimulating the synthesis of connective tissue in the host mesenchymal cells.

Ultrastructural evidence for contacts between migrating L5222 rat leukemia cells and extracellular matrix components of the rat mesentery.

The nature of interactions between cells migrating through tissues and their structural surroundings are largely unknown. We have therefore examined the ultrastructural relationship between L5222 rat leukemia cells, moving through the loose connective tissue of the mesentery, and components of the extracellular matrix (ECM). Ultrathin tissue sections, fixed in the presence of ruthenium hexammine trichloride (RHT), revealed the following: Constitutents of fibrillar and nonfibrillar elements of the ECM are in contact with the plasma membrane of L5222 cells. Linear nonfibrillar ECM elements contact the plasma membrane at point-like sites, often associated with root-like structures present within the submembraneous microfilament mesh. Aggregates of ECM material are connected to patch-like cell membrane sites, associated with a condensed, plate-like part of the microfilament mesh. Point-like and patch-like contacts are more numerous at the anterior part of polarized migrating L5222 cells than on the posterior end. In round resting leukemia cells they are evenly distributed around the cell periphery. We suggest that the ECM-cell membrane contacts represent tissue adhesion sites. We therefore hypothesize that in migrating cells a coordinate interaction occurs between the contact sites and the continuous microfilament meshwork which results in a simultaneous backward movement of ECM-membrane contacts on the cell body and in a net forward movement of the whole cell. Since Dembo et al. (1981) present a similar mechanism for in vitro locomotion of granulocytes, we assume that blood cell locomotion in vivo and in vitro depends on similar molecular mechanisms: force generation by the cell, transmembraneous linkage between cytoskeletal and ECM elements, and membrane fluidity. The major difference in blood cell locomotion through a three-dimensional tissue or on a plane substratum would then be given by the distribution of contact sites, occurring around the cell periphery or limited to the ventral cell surface, respectively.

Tumor-associated desmoplasia in the rabbit mesentery characterized by morphological, biochemical and cytophotometric methods.

Intraperitoneal implantation of V2 carcinoma cells in the rabbit leads to invasion of the mesentery and to structural tissue alterations which are concomitantly of a destructive and a desmoplastic type. In this report, we describe the desmoplastic changes which are characterized by the increased formation of collagen and of proteoglycans resulting in an increased thickness of the membrane. Biochemical data indicate that the total amount of collagen increases with time after implantation, whereas the relative amount per unit of dry weight, as well as the contributions of type I (15-25%) and type III (6-8%), stay within the same range. The increased synthesis of extracellular matrix is accompanied by a change in the appearance of the fibroblasts which now show the morphologic features of synthesizing cells. Also, an appreciable number have entered the S-phase. We propose that the desmoplastic changes are tumor-associated, since implantation of epithelial cells from normal rabbit liver does not result in similar alteration. Our findings are discussed in view of the role played by tumor and/or host cells in the increased production of extracellular matrix, of possible factor(s) elaborated by the tumor cells, and of the general significance of desmoplastia for tumor spread.