Evaluation of the LigaSure(™) Vessel Sealing System for bowel transection and intestinal anastomosis-an experimental study in a porcine model.

BACKGROUND: The purpose of the present study is to assess the value of the LigaSure™ Vessel Sealing System (LVSS) as a means for bowel transection and intestinal anastomosis. METHODS: We compared the LVSS for (1) transecting bowel and (2) creation of an intestinal anastomosis with standard methods such as stapler (S) and hand-sewn (HS) in a porcine model. For each study arm, i.e., bowel transection and anastomosis creation, both the small bowel and colon were examined. In total, ten transections and ten anastomoses were performed for each. Burst and anastomotic leak pressures were compared. RESULTS: In the study arm 1, LVSS achieved lowest burst pressures in both small bowel (LVSS 39.8 ± 3.6 mmHg, S 81.9 ± 3.9, HS 111.9 ± 14.7 mmHg, p < 0.0001) and colon transections (LVSS 21.5 ± 2.6 mmHg, S 79.5 ± 4.9, HS 91.0 ± 5.2 mmHg, p < 0.0001). There was no difference in burst pressures between S and HS in both small bowel and colon transections. In the study arm 2, LVSS showed the lowest anastomotic leak pressures for small bowel (LVSS 26.4 ± 2.6 mmHg, S 52.1 ± 6.2, HS 87.4 ± 7.0 mmHg, p < 0.0001) and colonic anastomoses (LVSS 16.9 ± 1.3 mmHg, S 55.9 ± 4.3, HS 74.4 ± 4.4 mmHg, p < 0.0001). Furthermore, small bowel and colonic anastomoses using S demonstrated significantly lower leak pressures than HS anastomosis p < 0.001 and p = 0.004, respectively. CONCLUSIONS: The LVSS achieves significantly lower burst pressures and anastomotic leak pressures for bowel transection and intestinal anastomosis than S and HS techniques. However, due to the achieved pressure levels of 39.8 ± 3.6 mmHg, LVSS appears to be a sufficient stand-alone method for bowel transection. Whether it can be used to perform intestinal anastomosis warrants further research in a survival model.

Comparison of two different transection techniques in liver surgery-an experimental study in a porcine model.

AIMS: Postoperative morbidity and mortality after liver resection is closely related to the degree of intraoperative blood loss; the majority of which occurs during transection of the liver parenchyma. Many approaches and devices have therefore been developed to limit bleeding, but none has yet achieved perfect results up to now. The aim of this standardized chronic animal study was to compare the safety and efficacy of the LigaSure™ Vessel Sealing System (LVSS) with the stapler technique, which is one of the modern techniques for transecting the parenchyma in liver surgery. METHODS: Sixteen pigs underwent a left liver resection (LLR). Eight pigs received a LLR by means of an Endo GIA, whereas the other eight pigs underwent liver parenchymal transection followed by simultaneous sealing by the LVSS. The operating time, transection time, blood loss during transection, and time of hemostasis were measured on the day of LLR (postoperative day 0/POD 0). Animals were re-explored on postoperative day 7 (POD 7) and the transection surface of remnant liver was observed for fluid collection (hematoma, biloma, and abscess), necrosis, and other pathologies. A biopsy was taken from the area of transection for histopathological examination. RESULTS: All animals survived until POD 7. Operating time and transection time of the liver parenchyma on POD 0 was significantly shorter in the stapler group. There was no significant difference between the two groups in terms of blood loss during transection, time of hemostasis and number of sutures for hemostasis on POD 0, morbidity rate, as well as the histopathological examination on POD 7. Furthermore, the material costs were significantly higher in the stapler group than in the LVSS group. CONCLUSION: In this standardized chronic animal study concerning transection of the parenchyma in liver surgery, LVSS seems not only to be safe, but also comparable with the stapler technique in terms of morbidity and mortality. Additionally, LVSS significantly reduces material costs. However, the transection time is significantly longer for LVSS than for the stapler resection technique.

Adenovirally transferred p16INK4/CDKN2 and p53 genes cooperate to induce apoptotic tumor cell death.

Repression of cell cycle progression by tumor suppressors might provide a means for tumor therapy. Here we demonstrate that ectopic overexpression of the p16INK4/CDKN2 tumor suppressor from an adenovirus vector in various cell lines results in block of cell division and, subsequently, in a gradual reduction of the levels of the product of retinoblastoma susceptibility gene, pRb. Overexpression of p53 and p16INK4/CDKN2, but not p53 on its own, induces apoptotic death only in tumor cells. Simultaneous adenoviral transfer of p16 and p53 genes leads to inhibition of tumor growth in nude mice. These results suggest that combined delivery of two cooperating genes like p16 and p53 could be the basis for the development of a new strategy for cancer gene therapy.

Lipopolysaccharide-mediated transcriptional activation of the human tissue factor gene in THP-1 monocytic cells requires both activator protein 1 and nuclear factor kappa B binding sites.

Lipopolysaccharide (LPS) activation of cells of monocytic lineage leads to rapid and transient expression of a set of inflammatory gene products, including tissue factor (TF). This transmembrane receptor is the major cellular initiator of the blood coagulation cascades, and induced expression of TF is postulated to play a role in inflammation. Functional studies using transfected THP-1 monocytic cells revealed the presence of a 56-bp LPS response element (LRE) within the TF promoter that conferred LPS responsiveness to a heterologous promoter. LPS stimulation of these cells activated proteins that bound to nucleotide sequences within the LRE resembling consensus binding sites for activator protein 1 (AP-1) and nuclear factor kappa B (NF-kappa B). Induction of the TF gene may represent a prototypic example of gene activation in monocytic cells by assembly of transcription factor complexes, and may clarify the role of AP-1 and NF-kappa B in the regulation of other LPS-responsive genes.

Effectiveness of ozone against endodontopathogenic microorganisms in a root canal biofilm model.

AIM: To assess the antimicrobial efficacy of aqueous (1.25-20 microg mL(-1)) and gaseous ozone (1-53 g m(-3)) as an alternative antiseptic against endodontic pathogens in suspension and a biofilm model. METHODOLOGY: Enterococcus faecalis, Candida albicans, Peptostreptococcus micros and Pseudomonas aeruginosa were grown in planctonic culture or in mono-species biofilms in root canals for 3 weeks. Cultures were exposed to ozone, sodium hypochlorite (NaOCl; 5.25%, 2.25%), chlorhexidine digluconate (CHX; 2%), hydrogen peroxide (H(2)O(2); 3%) and phosphate buffered saline (control) for 1 min and the remaining colony forming units counted. Ozone gas was applied to the biofilms in two experimental settings, resembling canal areas either difficult (setting 1) or easy (setting 2) to reach. Time-course experiments up to 10 min were included. To compare the tested samples, data were analysed by one-way anova. RESULTS: Concentrations of gaseous ozone down to 1 g m(-3) almost and aqueous ozone down to 5 microg mL(-1) completely eliminated the suspended microorganisms as did NaOCl and CHX. Hydrogen peroxide and lower aqueous ozone concentrations were less effective. Aqueous and gaseous ozone were dose- and strain-dependently effective against the biofilm microorganisms. Total elimination was achieved by high-concentrated ozone gas (setting 2) and by NaOCl after 1 min or a lower gas concentration (4 g m(-3)) after at least 2.5 min. High-concentrated aqueous ozone (20 microg mL(-1)) and CHX almost completely eliminated the biofilm cells, whilst H(2)O(2) was less effective. CONCLUSION: High-concentrated gaseous and aqueous ozone was dose-, strain- and time-dependently effective against the tested microorganisms in suspension and the biofilm test model.

Enhanced VDUP-1 gene expression by PPARgamma agonist induces apoptosis in human macrophage.

The fate and phenotype of lesion macrophages is regulated by cellular oxidative stress. Thioredoxin-1 (Trx-1) plays a major role in the regulation of cellular redox balance, with resultant effects on gene expression and cellular responses including cell growth and death. Trx-1 activity is inhibited by interaction with vitamin D-upregulated protein-1 (VDUP-1). Peroxisome proliferator-activated receptor gamma (PPARgamma) is expressed by human monocyte-derived macrophages (HMDM) and PPARgamma agonism has been reported to decrease expression of inflammatory genes and to promote apoptosis of these cells. To determine whether VDUP-1 may be involved in regulating the effects of PPARgamma agonists in macrophages, we investigated the effect of a synthetic PPARgamma agonist (GW929) on the expression of VDUP-1 in HMDM. GW929 concentration-dependently increased HMDM expression of VDUP-1 (mRNA and protein). Transfection of different fragments of the VDUP-1 promoter as well as gel shift analysis revealed the presence of functional PPARgamma response elements (PPRE) in the promoter. Under conditions in which PPAR agonism altered levels of VDUP-1, caspase-3 activity, and macrophage apoptosis were also elevated. The results suggest that PPARgamma activation stimulates apoptosis in human macrophages by altering the cellular redox balance via regulation of VDUP-1.

Invasion front-specific overexpression of tissue inhibitor of metalloproteinase-1 in liver metastases from colorectal cancer.

BACKGROUND: Matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), have been implicated in invasion and metastasis. The distribution of MMPs and TIMPs in the invasion front of liver metastases from colorectal cancer were investigated in order to understand their potential role in invasiveness. MATERIALS AND METHODS: Freshly frozen material of colorectal metastases of the liver was microdissected into four separate compartments, namely pure liver, liver invasion, tumour invasion and pure tumour. RNA was isolated and analyzed on Affymetrix microarrays. Expression of TIMP-1 was confirmed by quantitative polymerase chain reaction in 10 colorectal liver metastases. Cellular localisation of TIMP-1 was examined by immunohistochemistry. RESULTS: Affymetrix microarray data revealed that several MMP and TIMP genes including MMP-2, -3, -7, -9, -11, -12, -14, -15, -16, -19 and -24, and TIMP-1, -2 and -3 were generally up-regulated in both invasion front compartments. Among these genes, TIMP-1 showed the highest expression. The qPCR results indicated an average 15-fold upregulation of TIMP-1 in the liver invasive front and an average 13-fold up-regulation in the tumor invasive front, each compared to normal liver tissue. Immunohistochemistry revealed expression of TIMP-1 in tumour epithelia as well as in host tissue cells, including fibroblastic cells. CONCLUSION: Our data indicate that tumour invasion in colorectal liver metastasis is associated with increased TIMP-1 RNA and protein levels in both tumour and host cells.

Interferon-gamma inhibits macrophage apolipoprotein E production by posttranslational mechanisms.

Macrophage-derived apolipoprotein (apo) E and multimers of a synthetic apo E-peptide display monokine-like functions by inhibiting mitogen- or antigen-driven lymphocyte proliferation. This study demonstrated how the target lymphocyte itself can modulate macrophage apo E production. The lymphokine interferon-gamma (IFN) dramatically inhibited the accumulation of apo E in the supernatant of human monocytic THP-1 cells when present during phorbol myristate acetate-induced differentiation. A similar effect was observed when IFN was added to differentiated THP-1 cells. Treatment with IFN did not change the steady-state levels of apo E mRNA. Furthermore, in the presence of IFN no increased degradation or increased uptake of extracellular apo E was detected. Pulse-chase experiments indicated that IFN reduced the accumulation of extracellular apo E and increased the degradation of intracellular apo E. The inhibitory effect of IFN on apo E production also was observed in human monocyte-derived macrophages. Thus, our data demonstrated that IFN inhibited macrophage apo E production by posttranslational mechanisms. This represents a previously uncharacterized immunoregulatory interaction and lends further support to a relationship between lipid metabolism and the immune system.

Activated transcription factor nuclear factor-kappa B is present in the atherosclerotic lesion.

Nuclear factor-kappa B (NF-kappaB)/Rel transcription factors play an important role in the inducible regulation of a variety of genes involved in the inflammatory and proliferative responses of cells. The present study was designed to elucidate the implication of NF-kappaB/Rel in the pathogenesis of atherosclerosis. Activation of the dimeric NF-kappaB complex is regulated at a posttranslational level and requires the release of the inhibitor protein IkappaB. The newly developed mAb alpha-p65mAb recognizes the IkappaB binding region on the p65 (RelA) DNA binding subunit and therefore selectively reacts with p65 in activated NF-kappaB. Using immunofluorescence and immunohistochemical techniques, activated NF-kappaB was detected in the fibrotic-thickened intima/media and atheromatous areas of the atherosclerotic lesion. Activation of NF-kappaB was identified in smooth muscle cells, macrophages, and endothelial cells. Little or no activated NF-kappaB was detected in vessels lacking atherosclerosis. Electrophoretic mobility shift assays and colocalization of activated NF-kappaB with NF-kappaB target gene expression suggest functional implications for this transcription factor in the atherosclerotic lesion. This study demonstrates the presence of activated NF-kappaB in human atherosclerotic tissue for the first time. Atherosclerosis, characterized by features of chronic inflammation and proliferative processes, may be a paradigm for the involvement of NF-kappaB/Rel in chronic inflammatory disease.

Effect of aqueous ozone on the NF-kappaB system.

Ozone has been proposed as an alternative oral antiseptic in dentistry, due to its antimicrobial power reported for gaseous and aqueous forms, the latter showing a high biocompatibility with mammalian cells. New therapeutic strategies for the treatment of periodontal disease and apical periodontitis should consider not only antibacterial effects, but also their influence on the host immune response. Therefore, our aim was to investigate the effect of aqueous ozone on the NF-kappaB system, a paradigm for inflammation-associated signaling/transcription. We showed that NF-kappaB activity in oral cells stimulated with TNF, and in periodontal ligament tissue from root surfaces of periodontally damaged teeth, was inhibited following incubation with ozonized medium. Under this treatment, IkappaBalpha proteolysis, cytokine expression, and kappaB-dependent transcription were prevented. Specific ozonized amino acids were shown to represent major inhibitory components of ozonized medium. In summary, our study establishes a condition under which aqueous ozone exerts inhibitory effects on the NF-kappaB system, suggesting that it has an anti-inflammatory capacity.

Tissue factor mRNA in THP-1 monocytic cells is regulated at both transcriptional and posttranscriptional levels in response to lipopolysaccharide.

Tissue factor (TF) is transiently expressed in human monocytes exposed to the inflammatory agonist bacterial lipopolysaccharide (LPS). Since TF is the major cellular initiator of the coagulation protease cascades, it is inferred that its expression within the vasculature is strictly regulated. In this study, we investigated mechanisms which control TF mRNA expression in the human monocytic cell line THP-1. LPS induced a rapid and transient accumulation of the mature 2.2-kb TF mRNA, which was maximal at 2 h. After stimulation, the rate of transcription of the TF gene was increased (3.3 +/- 1.3)fold. In addition, we observed a significant change in TF mRNA stability: at 1 h after LPS stimulation, TF mRNA was stable during a 60-min period and had a half-life of greater than 120 min, whereas at 2 h, the half-life had declined to 25 +/- 5 min. Furthermore, a larger (3.4-kb) TF RNA species was induced in these cells; the size of this species and data from selective hybridizations with intron-specific probes are consistent with the presence of an unspliced copy of intron 1. These results demonstrate that the LPS-induced accumulation of TF mRNA levels in these monocytic cells is accomplished by both transcriptional and posttranscriptional control mechanisms.

Effects of gonadectomy on foreign-body tumorigenesis in CBA/H mice.

Sarcomas were induced by sc implantation of unplasticized polyvinylchloride-vinylacetate films in gonadectomized and normal male and female CBA/H mice. Gonadectomy did not demonstrably influence tumor incidence and tumor latencies in males but significantly prolonged tumor latencies in females. The results suggest that estrogen influences the pace of foreign-body tumorigenesis in CBA/H mice.

Carcinogenesis from polymer implants: new aspects from chromosomal and transplantation studies during premalignancy.

Inbred CBA/H and CBA/H-T6 mice received implants of 15 X 22 mm plastic films. Plastic inserts and tissue capsules were cut in thirds at half monthly and monthly intervals. The first portion of the inserts and capsules was left in the original animal. The second portion was separated and individually transplanted into recipients that differed from the original animals with respect to the T6 marker chromosome. The third portion and all tumors which developed in original and recipient animals were examined by karyological, histological, and cultural methods. Film pieces caused tumors in recipient animals up to 9 months after transfer, capsule tissue only up to about 1 month after transfer. Tumors in original and corresponding recipient animals were identical in their chromosomal stemlines and pace of premalignant maturation. The karyotype of the stemline was never discovered among the film-attached cell population because there seemed to be no cell division. This points to the existence of a single, specific premalignant cell clone residing on the film surface in a dormant state of nondivision many months before tumor appearance. At the end, the (pre)malignant cells detached from the film, invaded the capsule tissue, and propagated to produce the tumor within about 4 weeks. The existence of a specific inhibition phenomenon during the premalignant phase is suggested.

Foreign-body tumorigenesis induced by glass and smooth and rough plastic. Comparative study of preneoplastic events.

Foreign-body (FB) tumorigenesis was induced in female CBH/H and CBA/H-T6 mice and their hybrids by sc implantation of about 0.2-mm thick, large (660-720 mm2) or small (210-400 mm2) pieces of glass, smooth-surfaced plastic, or roughened plastic (rigid unplasticized vinyl chloride vinyl acetate copolymer). The tumorigenic process was analyzed in the various implantation groups by the evaluation of tumor incidences and latencies, and by the determination of 1) frequency of originator ("parent") cells, 2) appearance of preneoplastic cells in FB-reactive capsule tissue, 3) expansion of preneoplastic cell clones throughout the tissue capsule, and 4) pace of cellular preneoplastic maturation in terms of time remaining until neoplastic autonomy. Established methods included transfer of preneoplastic FB-reactive tissue capsules to recipient animals (hybrids of CBA/H and CBA/Br or C57BL/10ScSn). Specific preneoplastic events or stages of FB tumorigenesis were affected differently, depending on the size, material, and surface properties of implants.

Foreign-body tumorigenesis: number, distribution, and cell density of preneoplastic clones.

Double 15 times 22-mm plastic films (two films on top of each other) were implanted subcutaneously in CBA/H-T6 mice. After 7.5, 8.5, and 9.5 months the films were removed. Each interior film (next to the abdominal wall), covered by a cell monolayer on one side only, was cut in four 7 times 10-mm segments. Wiping to 5 times 7, 4 times 4, 3 times 3, or 2 times 2 mm reduced the cell areas on three of them. They were then transferred to (CBA/H times CBA/Br)F1 or (CBA/H-Ty times CBA/Br)F1 recipient mice. Tumors arising from transferred film segments were analyzed as to chromosome number and morphology, sarcoma type, degree of anaplasticity, and posttransfer latency. We used these criteria to determine whether tumors originated from the same or different clones. From this information it was estimated that preneoplastic clones were present in limited numbers and that they were either widely disseminated or spatially restricted on the implant surfaces. The extent of clone dissemination appeared to be related to preneoplastic progression.

Foreign-body tumorigenesis: in vitro isolation and expansion of preneoplastic clonal cell populations.

Foreign-body reactions were induced in coisogenic CBA/H and CBA/H-T6 mice by sc implantation of 15 times 22 times 0.2-mm unplasticized vinyl chloride vinyl acetate copolymer films. At 6 months' post implantation, implants and unopened tissue capsules were transferred to recipient animals of the T6-different partner strain. After another 3 months, part of the film/capsule complex was transferred to (C57BL/10ScSn times CBA/H-T6)F1 mice for tumor development. Capsule-derived and film-attached cells of the other part were separately cultured. Cultures consisting initially of euploid cells were often gradually replaced by different cells with specific aneuploid karyotypes which were identical with, or closely related to, those of the corresponding tumors. The cultured cells implanted in hybrid recipients at different passage numbers frequently gave rise to homologous tumors. Hence, it was possible to prepare in vitro cells with prefixed specific tumor determinants at different stages of preneoplastic maturation.

Foreign-body tumorigenesis by vinyl chloride vinyl acetate copolymer: no evidence for chemical cocarcinogenesis.

We investigated whether vinyl chloride monomers, released from implants of vinyl chloride vinyl acetate copolymer (VCA), exerted cocarcinogenic activity and added thereby to the mechanism of foreign-body (FB) tumorigenesis. CBA/H and CBA/H-T6 mice were used. No evidence was found to indicate that chemical carcinogenic activity partakes in tumorigenesis by VCA implants. Hence it was concluded that VCA plastic is not suitable for the study of the combined process of FB/chemical cocarcinogenesis. Furthermore, experimental results obtained with VCA film implants were representative of FB tumorigenesis in the absence of demonstrable chemical carcinogenic activity.

Foreign-body tumors of mice: strain and sex differences in latency and incidence.

Sarcomas were induced by sc implantation of unplasticized polyvinylchloride acetate films in female and male mice of strains AKR/J, BALB/cJ, BALB/cWat, CBA/H and CBA/H-T6, C3H/HeJ, C57BL/10ScSn, C57BL/6J-bgj, C57BL/cdJ, DBA/-1J l/LnJ, LP/J, SJL/J, X/Gf, 129/J, and hybrids (CBA/H-T6 X AKR/J)F1, (C57BL/10ScSn x CBA/H or CBA/H-T6)F1, (C57BL/6J-bgj x C57BL/6J)F1. The strains and sexes showed marked differences in incidence and mean latency of resulting tumors. Crucial information was provided for the selection of appropriate mouse strains for the study of interrelationships between genotypes, defined somatic properties, and the multifactorial process of foreign-body tumorigenesis.

Immunosuppression studies in foreign body tumorigenesis: no evidence for tumor-specific antigenicity.

Sarcomas were induced in CBA/H mice by sc implantation of 15 X 22 X 0.2-mm polyvinyl chloride vinyl acetate copolymer films. The animals were immunosuppressed with azathoprine, antilymphocyte globulin, or thymectomy. Sarcoma development was not accelerated in comparison to nonimmunosuppressed demonstrated in sarcomas of immunosuppressed mice. It was concluded that foreign body tumorigenesis in mice in neither associated with nor dependent on the emergence of tumor-specific transplantation antigens.

Foreign-body tumorigenesis in mice: DNA synthesis in surface-attached cells during preneoplasia.

(CBA/H X CBA/H-T6)F1 mice were given sc implants of unplasticized vinyl chloride acetate 15 X 22-mm copolymer films. The animals were pulsed with [3H]thymidine at various times during the 15 months following implantation. DNA synthesis occurred in the film-attached cell population, predominantly macrophages, throughout the preneoplastic phase in both females and males. Giant cells with fewer than 10 nuclei were labeled synchronously and asynchronously. No DNA synthesis was detected in giant cells with more than 10 nuclei. Previous studies have shown that phagocytic inactivity and ultrastructural signs of functional dormancy are characteristic for the foreign-body-reactive macrophage. However, this investigation demonstrated that the macrophage was not dormant with respect to DNA synthesis.