SUMMARY1. We have investigated the endothelial actomyosin system with particular emphasis on its possible role in actively opening a paracellular route for permeability.2. Actin and myosin comprised 16% of total endothelial protein with a molar actin/myosin ratio of 16-2 which is close to the actin/myosin ratio of muscle (studies on freshly isolated pig pulmonary arterial endothelial cells, PAEC).3. By immunocytochemistry at the light and electron microscope levels the bulk of actin and myosin was colocalized in close vicinity to the intercellular clefts of both micro-and macrovascular endothelial cells in situ and in vitro.4. Calcium-ionophore-induced rise in permeability of human umbilical venous endothelial cells (HUVEC) and PAEC monolayers grown on filters in a two-chamber permeability system was caused by opening of intercellular gaps. Gap formation depended on the rise in intracellular Ca2+ and could be blocked by the calmodulinbinding drugs trifluperazine (TFP) and W7.5. In skinned monolayers of cultured PAEC and in isolated sheets of HUVEC gap formation was shown to require ATP and occurred only when free myosin binding sites were available on endothelial actin filaments (experiments with myosin subfragment 1 modified by N-ethylmaleimide, S1-NEM).6. These experiments suggest that actin and myosin in endothelial cells play a central role in regulating the width of the intercellular clefts, thereby controlling the paracellular pathway of vascular permeability.

Acute graft-versus-host disease (aGVHD) often limits feasibility and outcome of allogeneic bone marrow transplantation. Current pathophysiologic concepts of aGVHD involve conditioning regimens, donor-derived T cells, proinflammatory cytokines, and bacterial lipopolysaccharide (LPS) as a major trigger for aGVHD. LPS derives mostly from gram-negative bacteria and can enter circulation through the impaired mucosal barrier after the conditioning regimen. Probiotic microorganisms have been shown to alter the composition of the intestinal microflora and thereby mediate anti-inflammatory effects. We hypothesized that modifying the enteric flora using the probiotic microorganism Lactobacillus rhamnosus GG, would ameliorate aGVHD. Here we show that oral administration of Lactobacillus rhamnosus GG before and after transplantation results in improved survival and reduced aGVHD. Furthermore, subculturing of mesenteric lymph node tissue revealed a reduced translocation of enteric bacteria. Our findings suggest that alteration of the intestinal microflora plays an important role in the initiation of experimental aGVHD. IntroductionAcute graft-versus-host disease (aGVHD) remains one of the major obstacles in allogeneic bone marrow transplantation (BMT). Despite the development of potent immunosuppressive drugs and reduction of conditioning regimens, a high percentage of patients develop aGVHD, resulting in a high mortality after transplantation. Bacterial lipopolysaccharide (LPS) is considered a major player in the development of aGVHD. 1-3 Hence, bowel decontamination using broad-spectrum antibiotics prior to transplantation has been introduced as standard practice to date. [4][5][6] In experimental studies, the proinflammatory potency of LPS varies from bacterial species to species. However, the clinical impact of different intestinal microorganisms on aGVHD is still unclear.Chronic inflammatory bowel disease (IBD) appears to be the result of a genetically determined unbalanced immune response to ubiquitous luminal bacterial antigens. 7 Probiotic bacteria, mainly lactobacilli and bifidobacteria, are defined as living microbes, which confer benefits to the host. There is increasing evidence that probiotic therapy is effective in the treatment of IBD. 8 However, the mechanisms by which these bacteria mediate their effects are still unclear. It has been shown that probiotic bacteria have a temporary impact on the composition of the intestinal flora, but a direct influence on the immune system has also been suggested. [9][10][11] To address the impact of modulation of the bowel flora on aGVHD, we used a well-described murine transplantation model in which aGVHD is induced across a haploidentical major histocompatibility complex (MHC) mismatch. The model is characterized by severe damage of the bowel mucosa, high-serum LPS levels after transplantation, and strong release of proinflammatory cytokines. 12-14 Study designMice, BMT, assessment of GVHD, and treatment protocols C57BL/6 and B6D2F1 mice (8 to 12 weeks old) were purchas...

Effects of Staphylococcus aureus alpha-toxin and Pseudomonas aeruginosa cytotoxin on the permeability of an endothelial monolayer were studied. Porcine pulmonary artery endothelial cells were grown on a polycarbonate membrane, mounted in a chamber, and exposed to a continuous hydrostatic pressure of 10 cmH2O. On application of this trans-endothelial pressure, endothelial monolayer became "sealed," i.e., the filtration rate for water decreased and the reflection coefficient for albumin increased, reaching a plateau after 1-2 h. Sealed monolayer had a hydraulic conductivity of 2.1 X 10(-6) cm.s-1.cmH2O and an albumin reflection coefficient of 0.73. Permeability of the monolayer was increased on addition of an excess of EDTA and reversed on readdition of calcium. Within 60-90 min after addition of 1 microgram/ml alpha-toxin, the filtration rate increased 75-fold, and the albumin reflection coefficient dropped to 0.20. These changes in permeability were accompanied by cell retraction and formation of large intercellular gaps between endothelial cells. Effects of alpha-toxin were abolished by preincubation with neutralizing antibodies and by inhibitors of calmodulin function. Pseudomonas aeruginosa cytotoxin (25 and 50 micrograms/ml) also increased the permeability of the endothelial monolayer, but it was only about one-third as effective as alpha-toxin.