(33/1076) Integrin alpha(2)beta(1) (VLA-2) is a principal receptor used by neutrophils for locomotion in extravascular tissue.
Cell adhesion molecules are critically involved in the multistep process of leukocyte recruitment in inflammation. The specific receptors used by polymorphonuclear leukocytes (PMN) for locomotion in extravascular tissue have as yet not been identified. By means of immunofluorescence flow cytometry and laser scanning confocal microscopy, this study demonstrated that surface expression of the alpha(2)beta(1) (VLA-2) integrin, though absent on blood PMN, is induced in extravasated PMN collected from human skin blister chambers, and rat PMN accumulated in the peritoneal cavity after chemotactic stimulation. Intravital time-lapse videomicroscopy was used to investigate chemoattractant-induced PMN locomotion in the rat mesentery in vivo. Local administration of function-blocking monoclonal antibody or peptide recognizing the alpha(2)beta(1) integrin reduced PMN migration velocity in the extravascular tissue by 73% +/- 3% and 70% +/- 10%, respectively (means +/- SD). The distance f-met-leu-phe peptide (fMLP)-stimulated human PMN migrated in a collagen gel in vitro was markedly reduced by treatment with anti-alpha(2) mAbs or peptide, whereas no effect was observed with antibodies or peptides recognizing the alpha(4)beta(1) or alpha(5)beta(1) integrins. Further evidence for a critical role of expression of alpha(2)beta(1) integrin in PMN locomotion in extravascular tissue was obtained in the mouse air pouch model of acute inflammation where chemoattractant-induced PMN recruitment was substantially inhibited by local anti-alpha(2) mAb treatment. Thus, expression of alpha(2)beta(1) integrin on extravasated PMN has been identified and a novel role of this receptor in regulating the extravascular phase of leukocyte trafficking in inflammation has been formulated. (Blood. 2000;95:1804-1809) (+info)
(34/1076) Efficacy of trisacryl gelatin microspheres versus polyvinyl alcohol particles in the preoperative embolization of meningiomas.
BACKGROUND AND PURPOSE: Trisacryl gelatin microspheres are a new, commercially available nonabsorbable embolic agent. The purpose of this study was to evaluate their efficacy in the preoperative embolization of meningiomas as compared with polyvinyl alcohol (PVA) particles of various sizes. METHODS: In 30 consecutive patients, trisacryl gelatin microspheres (150-300 microm) were used for the preoperative superselective embolization of meningiomas (group 1). Thirty other consecutive patients had embolization with PVA particles of 45 to 150 microm (n = 15, group 2) and of 150 to 250 microm (n = 15, group 3). Extent of devascularization, intraoperative blood loss, blood transfusion, and hemostasis at the time of surgery were recorded for every patient. The inflammatory reaction, the extent of necrotic areas, and the most distal intravascular location of the embolic agent (arterial, arteriolar, precapillary, capillary) were recorded. RESULTS: There was no significant difference in the extent of angiographic devascularization among the groups. Intraoperative blood loss differed significantly between groups 1 and 2 and groups 1 and 3, but not between groups 2 and 3. The trisacryl gelatin microspheres were located more distally in tumor vessels than were the PVA particles of either size. The extent of intratumoral necrosis was not significantly different between the two embolic agents. In all groups there was a mild inflammatory tissue reaction in the vicinity of the embolic agent. CONCLUSION: Trisacryl gelatin microspheres may be effective in the preoperative embolization of meningiomas, producing significantly less blood loss at surgery than seen with PVA particles of either size, possibly because of the significantly more distal vascular penetration of the microspheres. (+info)
(35/1076) Microfabrication of an analog of the basal lamina: biocompatible membranes with complex topographies.
A microfabrication approach was used to produce novel analogs of the basal lamina with complex topographic features. A test pattern of ridges and channels with length scales (40 to 310 micrometer) similar to the invaginations found in a native basal lamina was laser machined into the surface of a polyimide master chip. Negative replicates of the chip were produced using polydimethylsiloxane silicone elastomer and these replicates were used as templates for the production of thin ( approximately 21 micrometer) membranes of collagen or gelatin. The resulting membranes had a complex topography of ridges and channels that recapitulated the features of the master chip. To demonstrate their utility, these complex membranes were laminated to type I collagen sponges and their surfaces were seeded with cultured human epidermal keratinocytes to form a skin equivalent. The keratinocytes formed a differentiated and stratified epidermis that conformed to the features of the microfabricated membrane. The topography of the membrane influenced the differentiation of the keratinocytes because stratification was enhanced in the deeper channels. Membrane topography also controlled the gross surface features of the skin equivalent; infolds of the epidermis increased as channel depth increased. These novel microfabricated analogs of the basal lamina will help to elucidate the influence of topography on epithelial cell proliferation and differentiation and should have applications in the tissue engineering of skin equivalents as well as other basal lamina-containing tissues. (+info)
(36/1076) Viral vector delivery in solid-state vehicles: gene expression in a murine prostate cancer model.
BACKGROUND: Although there are increasingly more clinical trials involving gene therapy, efficient gene transfer remains a major hurdle to success. To enhance the efficiency of delivery of viral vectors in gene therapy protocols, we evaluated the effect of various matrices to act as a vehicle for recombinant virus during intratumoral injection. METHODS: The ability of several vehicles (catgut spacer, polyglycolic acid, chromic catgut, and gelatin sponge matrix) to deliver the canarypox virus ALVAC to the cells of the murine prostate cancer cell line RM-1 was studied in vitro and in vivo. ALVAC recombinants encoding the murine cytokines interleukin 2 (IL-2), interleukin 12 (IL-12), and tumor necrosis factor-alpha (TNF-alpha) were used to assess enhancement of antitumor activity after intratumoral inoculation. Confirmatory experiments were conducted by use of another mouse prostate cancer cell line, RM-11, and a mouse bladder cancer cell line, MB-49. All statistical tests were two-sided. RESULTS: The gelatin sponge matrix proved to be the most effective solid-state vehicle for delivering viral vectors to cells in culture. In addition, this matrix statistically significantly enhanced expression of ALVAC-delivered reporter genes in tumor models when compared with fluid-phase delivery of virus (P =.037 for the RM-1 model and P =.03 for the MB-49 model). Statistically significant growth inhibition of established tumors was observed when a combination of the three recombinant ALVAC viruses expressing IL-2, IL-12, and TNF-alpha was delivered with the matrix in comparison with 1) fluid-phase intratumoral injection of the ALVAC recombinants, 2) no treatment, or 3) treatment with parental ALVAC (all P<.05). CONCLUSIONS: Viral vector delivery in a solid-state vehicle resulted in improved recombinant gene expression in vivo and translated to greater inhibition of tumor growth in an immunotherapy protocol for heterotopic tumor nodules. The efficient delivery of reporter genes described herein may prove useful in many solid tumor gene therapy protocols. (+info)
(37/1076) Stimulation of integrin-mediated cell contractility by fibronectin polymerization.
Ligation of integrins with extracellular matrix molecules induces the clustering of actin and actin-binding proteins to focal adhesions, which serves to mechanically couple the matrix with the cytoskeleton. During wound healing and development, matrix deposition and remodeling may impart additional tensile forces that modulate integrin-mediated cell functions, including cell migration and proliferation. We have utilized the ability of cells to contract floating collagen gels to determine the effect of fibronectin polymerization on mechanical tension generation by cells. Our data indicate that fibronectin polymerization promotes cell spreading in collagen gels and stimulates cell contractility by a Rho-dependent mechanism. Fibronectin-stimulated contractility was dependent on integrin ligation; however, integrin ligation by fibronectin fragments was not sufficient to induce either tension generation or cell spreading. Furthermore, treatment of cells with polyvalent RGD peptides or pre-polymerized fibronectin did not stimulate cell contractility. Fibronectin-induced contractility was blocked by agents that inhibit fibronectin polymerization, suggesting that the process of fibronectin polymerization is critical in triggering cytoskeletal tension generation. These data indicate that Rho-mediated cell contractility is regulated by the process of fibronectin polymerization and suggest a novel mechanism by which extracellular matrix fibronectin regulates cytoskeletal organization and cell function. (+info)
(38/1076) The domains of human fibronectin mediating the binding of alpha antigen, the most immunopotent antigen of mycobacteria that induces protective immunity against mycobacterial infection.
We have recently shown that alpha antigen (alpha-Ag), the immunodominant antigen of mycobacteria, has a novel fibronectin (FN)-binding motif that is unique among mycobacteria [Naito, Ohara, Matsumoto and Yamada (1998) J. Biol. Chem. 273, 2905-2909]. In this study, we examined the domains of human FN that interacted with alpha-Ag. Fragments of FN generated by either proteolysis or recombinant DNA techniques were compared for their ability to bind to alpha-Ag. Fragments containing either the C-terminal heparin-binding domain or the central cell-binding domain consistently bound to alpha-Ag. The fragment of the C-terminal heparin-binding domain, upon mutation that resulted in the loss of its heparin-binding activity, could not bind with alpha-Ag. These findings suggested that the mutated site, i.e. the main heparin-binding site of FN, was also the principal site for binding to alpha-Ag. The alpha-Ag-binding domains of FN could bind whole mycobacterial bacilli, suggesting that these two domains are important contributors to mycobacterial infection. (+info)
(39/1076) 1alpha,25-dihydroxyvitamin D3 and its analogues down-regulate cell invasion-associated proteases in cultured malignant cells.
Vitamin D and its derivatives (deltanoids) are potent regulators of cell proliferation and differentiation. Targeted production of proteolytic enzymes like serine proteases and metalloproteinases is an important part of the invasive process of cancer cells. Treatment with 1 alpha25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] decreases the invasive properties of breast carcinoma cells. Here we have analyzed the effects of 1alpha,25(OH)2D3 and its synthetic analogues on the secretion and cell surface association of the components of the plasminogen activator (PA) system and on the secretion of certain matrix metalloproteinases (MMPs) and their inhibitors in MDA-MB-231 breast carcinoma cells. Deltanoids were able to decrease the secretion of urokinase PA and tissue-type PA activity in a dose-dependent manner and to increase PA inhibitor 1 secretion, leading to reduced total PA activity. CB1093 was the most potent analogue, effective at concentrations several logarithms lower than 1alpha,25(OH)2D3. Transient transfection of different urokinase PA promoter reporter constructs to HT-1080 fibrosarcoma indicator cells indicated that vitamin D-responsive sequences were located between nucleotides -2350 and -1870 in the 5' region of the promoter. Treatment of MDA-MB-231 cells with 1alpha,25(OH)2D3 or other deltanoids also resulted in decreased MMP-9 levels in association with increased tissue inhibitor of MMP 1 activity. Membrane-type 1-MMP expression or proteolytic processing were not appreciably affected by deltanoids. Vitamin D and its analogues caused a decrease in Matrigel invasion assays of MDA-MB-231 cells. Cancer cell invasion is associated with coordinated secretion of proteolytic enzymes and their inhibitors. Vitamin D and its derivatives can evidently influence invasive processes by two means: (a) decreasing the expression and activity of cell invasion-associated serine proteases and metalloproteinases; and (b) inducing their inhibitors. (+info)
(40/1076) Preparation for regional anaesthesia induces changes in thrombelastography.
The effects of crystalloid and colloid infusions on coagulation measured by thrombelastography (TEG) present a confused picture. The influence of environmental factors may explain the disparity between previous studies. We studied two groups of 20 women presenting at term for elective Caesarean section. In the first group, TEG analysis was performed before and after infusion of Gelofusine 500 ml over 15 min. The second group was treated in the same way except that subjects did not receive fluid. We found significant changes in r and k values in both groups, suggesting enhanced coagulation. As hypercoagulable changes were also seen in the group that did not receive fluid preload, the hypothesis that moderate haemodilution causes hypercoagulability must be questioned. The influence of environmental factors can explain differences reported between in vivo and in vitro studies. (+info)