Optimal sutures for use in the abdomen: an evaluation based on the formation of adhesions and abscesses.

PURPOSE: This study explored the optimal suture materials for use in the peritoneal cavity based on the formation of adhesions and abscesses under clean and contaminated conditions. METHODS: The parietal peritoneum and muscle layer of rats were incised. The incision was followed by interrupted suturing in the clean group. A suspension of E. coli (1.0 × 10(6)) plus Bacteroides fragilis (1.0 × 10(5)) was sprayed onto the incision in the contaminated group, followed by interrupted suturing. Four types of sutures were used: nonabsorbable multifilament silk, absorbable multifilament Polyglactin 910 (Vicryl(®)), absorbable monofilament Polydioxanone (PDS(®)), and Poliglecaprone 25 (Monocryl(®)). The rats were killed at 2, 4 or 8 weeks after the surgery. RESULTS: The incidence of adhesions in the clean group was low with Polyglactin 910. The incidence of adhesions was 96 % or higher regardless of the suture type in the contaminated group. The incidence of severe adhesions was low with Polyglactin 910 and Poliglecaprone 25 and significantly higher with Polydioxanone in the contaminated group. The incidence of abscess formation around the silk was significantly higher than the other three types of sutures in the contaminated group. CONCLUSION: Polyglactin 910 was less likely to form adhesions than the other three types of sutures under both conditions, suggesting that Polyglactin 910 may be the optimal type of suture to use in the peritoneal cavity.

Two-component polyethylene glycol surgical sealant influence on intraperitoneal infection in a refined rodent model.

OBJECTIVE: This study determined the influence of a 2-component polyethylene glycol surgical sealant (Coseal) as an adhesion prevention device on sepsis-related mortality and/or systemic bacterial translocation to the spleen. STUDY DESIGN: A bacterial inoculum and telemetry probe were implanted in 50 treated and 49 untreated rats. Telemetry probes monitored core-body temperature to determine time of death. Spleens were collected on day 3 for quantitative bacteriology of Escherichia coli and Bacteroides fragilis. RESULTS: Median survival time and mortality of treated rats (37.0 hours, 54.0%) were noninferior to untreated rats (47.5 hours, 55.1%). Median E coli titers in treated rats (2.24 log colony forming units/spleen) were significantly less than untreated rats (4.32 log colony forming units/spleen). B fragilis titers were not different. CONCLUSION: This study demonstrates intraperitoneal administration of a 2-component polyethylene glycol surgical sealant as an adhesion prevention device does not alter time to death or sepsis-related mortality and/or systemic bacterial translocation to the spleen.

Porphyromonas gingivalis heat shock protein vaccine reduces the alveolar bone loss induced by multiple periodontopathogenic bacteria.

OBJECTIVES: Heat shock protein (HSP) can be utilized as a vaccine to cross-protect against multiple pathogenic species. The present study was performed to evaluate Porphyromonas gingivalis heat shock protein 60 (HSP60) as a vaccine candidate to inhibit multiple bacteria-induced alveolar bone loss. MATERIAL AND METHODS: Recombinant P. gingivalis HSP60 was produced and purified from P. gingivalis GroEL gene. Rats were immunized with P. gingivalis HSP60, and experimental alveolar bone loss was induced by infection with multiple periodontopathogenic bacteria. RESULTS: There was a very strong inverse relationship between postimmune anti-P. gingivalis HSP immunoglobulin G (IgG) levels and the amount of alveolar bone loss induced by either P. gingivalis or multiple bacterial infection (p=0.007). Polymerase chain reaction data indicated that the vaccine successfully eradicated the multiple pathogenic species. CONCLUSIONS: We concluded that P. gingivalis HSP60 could potentially be developed as a vaccine to inhibit periodontal disease induced by multiple pathogenic bacteria.

Zwitterionic polysaccharides stimulate T cells with no preferential V beta usage and promote anergy, resulting in protection against experimental abscess formation.

Zwitterionic polysaccharides (Zps) from pathogenic bacteria, such as Bacteroides fragilis, are virulence factors responsible for abscess formation associated with intra-abdominal sepsis. The underlying cellular mechanism for abscess formation requires T cell activation. Conversely, abscess formation can be prevented by prophylactic s.c. injection of purified Zps alone, a process also dependent on T cells. Hence, the modulatory role of T cells in abscess formation was investigated. We show that Zps interact directly with T cells with fast association/dissociation kinetics. Vbeta repertoire analysis using RT-PCR demonstrates that Zps have broad Vbeta usage. Zps-specific hybridomas responded to a variety of other Zps, but not to a nonzwitterionic polysaccharide, indicating cross-reactivity between different Zps. Furthermore, Zps-reactive T cell hybridomas could effectively transfer protection against abscess formation. Analysis of the proliferative capacity of T cells recovered from Zps-treated animals revealed that these T cells are anergic to subsequent stimulation by the different Zps or to alloantigens in an MLR. This anergic response was relieved by addition of IL-2. Taken together, the data show that this class of polysaccharides interacts directly with T cells in a nonbiased manner to elicit an IL-2-dependent anergic response that confers protection against abscess formation.

CD4+ T cells mediate abscess formation in intra-abdominal sepsis by an IL-17-dependent mechanism.

Abscess formation associated with intra-abdominal sepsis causes severe morbidity and can be fatal. Previous studies have implicated T cells in the pathogenesis of abscess formation, and we have recently shown that CD4(+) T cells activated in vitro by zwitterionic capsular polysaccharides from abscess-inducing bacteria such as Staphylococcus aureus and Bacteroides fragilis initiate this host response when transferred to naive rats. In this study, we show that mice deficient in alphabetaTCR-bearing T cells or CD4(+) T cells fail to develop abscesses following challenge with B. fragilis or abscess-inducing zwitterionic polysaccharides, compared with CD8(-/-) or wild-type animals. Transfer of CD4(+) T cells from wild-type mice to alphabetaTCR(-/-) animals reconstituted this ability. The induction of abscesses required T cell costimulation via the CD28-B7 pathway, and T cell transfer experiments with STAT4(-/-) and STAT6(-/-) mice demonstrated that this host response is dependent on STAT4 signaling. Significantly higher levels of IL-17, a proinflammatory cytokine produced almost exclusively by activated CD4(+) T cells, were associated with abscess formation in Th2-impaired (STAT6(-/-)) mice, while STAT4(-/-) mice had significantly lower levels of this cytokine than control animals. The formation of abscesses was preceded by an increase in the number of activated CD4(+) T cells in the peritoneal cavity 24 h following bacterial challenge. Confocal laser-scanning microscopy analysis revealed that CD4(+) T cells comprise the abscess wall in these animals and produce IL-17 at this site. Administration of a neutralizing Ab specific for IL-17 prevented abscess formation following bacterial challenge in mice. These data delineate the specific T cell response necessary for the development of intra-abdominal abscesses and underscore the role of IL-17 in this disease process.

Alteration of V beta usage and cytokine production of CD4+ TCR beta beta homodimer T cells by elimination of Bacteroides vulgatus prevents colitis in TCR alpha-chain-deficient mice.

A major pathogenic factor for the development of inflammatory bowel disease (IBD) is the breakdown of the intestinal homeostasis between the host immune system and the luminal microenvironment. To assess the potential influence of luminal Ags on the development of IBD, we fed TCR alpha(-/-) mice an elemental diet (ED). ED-fed TCR alpha(-/-) mice showed no pathologic features of IBD, and their aberrant mucosal B cell responses were suppressed. Similar numbers of CD4(+), TCR betabeta homodimer T cells (betabeta T cells) were developed in the colonic mucosa of ED-fed mice; however, Th2-type cytokine productions were lower than those seen in diseased regular diet (RD)-fed mice. The higher cytokine production in diseased RD-fed mice could be attributed to the high incidence of Bacteroides vulgatus (recovered in 80% of these mice), which can induce Th2-type responses of colonic CD4(+), betabeta T cells. In contrast, ED-fed TCR alpha(-/-) mice exhibited a diversification of Vbeta usage of betabetaT cell populations from the dominant Vbeta8 one associated with B. vulgatus in cecal flora to Vbeta6, Vbeta11, and Vbeta14. Rectal administration of disease-free ED-fed mice with B. vulgatus resulted in the development of Th2-type CD4(+), betabeta T cell-induced colitis. These findings suggest that the ED-induced alteration of intestinal microenvironments such as the enteric flora prevented the development of IBD in TCR alpha(-/-) mice via the immunologic quiescence of CD4(+), betabeta T cells.

Effect of molecular size on the ability of zwitterionic polysaccharides to stimulate cellular immunity.

The large-molecular-sized zwitterionic capsular polysaccharide of the anaerobe Bacteroides fragilis NCTC 9343, designated polysaccharide (PS) A, stimulates T cell proliferation in vitro and induces T cell-dependent protection against abscess formation in vivo. In the present study, we utilized a modification of a recently developed ozonolytic method for depolymerizing polysaccharides to examine the influence of the molecular size of PS A on cell-mediated immunity. Ozonolysis successfully depolymerized PS A into structurally intact fragments. PS A with average molecular sizes of 129.0 (native), 77.8, 46.9, and 17.1 kDa stimulated CD4+-cell proliferation in vitro to the same degree, whereas the 5.0-kDa fragment was much less stimulatory than the control 129.0-kDa PS A. Rats treated with 129.0-kDa, 46.9-kDa, and 17.1-kDa PS A molecules, but not those treated with the 5.0-kDa molecule, were protected against intraabdominal abscesses induced by challenge with viable B. fragilis. These results demonstrate that a zwitterionic polysaccharide as small as 22 repeating units (88 monosaccharides) elicits a T cell-dependent immune response. These findings clearly distinguish zwitterionic T cell-dependent polysaccharides from T cell-independent polysaccharides and give evidence of the existence of a novel mechanism for a polysaccharide-induced immune response.

Vaccination and periodontitis: myth or reality.

In most industrialised countries approximately 15% of the population has enhanced risk for moderate to severe periodontitis. The disease is caused by infection by gram-negative, anaerobic bacteria including Porphyromonas gingivalis and Bacteroides forsythus. There is evidence that P. gingivalis is a key pathogen. Using ligature-induced periodontitis in the non-human primate Macaca fascicularis as a model, we immunised 10 animals using intact killed P. gingivalis and SAF-M adjuvant and 10 controls using adjuvant only. The vaccine, containing 250 microg protein/ml, was injected subcutaneously in the neck and into the deltoid muscle (0.5 ml each site) at baseline and weeks 3, 6, and 16, and the mandibular posterior teeth ligated at week 16. At weeks 30 and 36 changes in alveolar bone, measured using digital subtraction radiography, were used as the outcome measure. Even though periodontitis in humans and in this animal model is a polymicrobial disease, immunisation with a vaccine containing a single bacterial species, P. gingivalis, induced protection. Of all the P. gingivalis components that have been studied, the cysteine proteases have the greatest potential as vaccine antigens. In a pilot study using the same protocol, we have shown that porphypain-2 purified from P. gingivalis is effective in inducing protection. Although opsonisation and bacterial cell killing may be involved in protection, other mechanisms such as antibody mediated reduction of levels of inflammatory mediators such as PGE2 and neutralisation of virulence factors may be important. In neither the whole cell vaccine nor the purified cysteine protease vaccine studies were signs of toxicity observed. In light of the increasing evidence that periodontitis significantly increases risk for potentially fatal diseases such as coronary heart disease, stroke and complications from diabetes mellitus a successful vaccine for periodontitis could have health benefits far exceeding the prevention of periodontitis.

Protection against experimental intraabdominal sepsis by two polysaccharide immunomodulators.

Two immunomodulating polysaccharides, poly-(1-6)-beta-glucotriosyl-(1-3)-beta-glucopyranose (PGG)-glucan and Bacteroides fragilis polysaccharide A (PS A), were evaluated for the prevention of mortality and abscess formation associated with experimental intraabdominal sepsis. Prophylactic treatment with a combination of these compounds significantly reduced mortality (8% vs. 44% in the saline-treated control group) and the incidence of abscesses (30% vs. 100% in the saline-treated control group) after challenge with rat cecal contents. These compounds were also effective when administered therapeutically after bacterial contamination of the peritoneal cavity. PS A treatment conferred long-term protection against abscess formation and resulted in significantly fewer total aerobes and anaerobes in the peritoneal fluid of animals challenged with cecal contents. These data demonstrate the usefulness of two immunomodulatory polysaccharides in preventing experimental intraabdominal sepsis in the absence of antimicrobial therapy and may represent a new adjunct to antibiotic regimens currently used to prevent clinical cases of this disease.

Effect of adjuvants on antibody responses of sheep immunised with recombinant pili from Dichelobacter nodosus.

OBJECTIVE: To compare the effects of two oil emulsion adjuvants (incomplete Freunds adjuvant and a proprietary oil adjuvant), DEAE-dextran, L-tyrosine particles and Quil A on the humoral immune responses of sheep immunised with recombinant pili of Dichelobacter Nodosus (strain A). PROCEDURE: Antibody titres were studied for up to 32 weeks and were measured by bacterial agglutination and ELISA. The relative avidity of antibodies for pili was determined and the incidence and severity of adverse reactions at the site of injection of vaccines were recorded. RESULTS: The oil emulsion adjuvants and Quil A were more effective than either DEAE-dextran or L-tyrosine at stimulating antibodies in sheep. The incidence and severity of adverse reactions was lower in sheep which received vaccines containing either Quil A or DEAE-dextran than in sheep which received vaccines containing oil emulsion adjuvants. L-tyrosine had no adverse effects. CONCLUSION: Quil A was as effective as oil adjuvants at stimulating high levels of antibodies against recombinant pili in sheep and had the significant advantage of being less irritant after subcutaneous injection.

Polysaccharide-mediated protection against abscess formation in experimental intra-abdominal sepsis.

Abscess formation is a major complication of intra-abdominal sepsis that causes significant morbidity and mortality. In such cases, Bacteroides fragilis is the predominant anaerobic isolate. In a rat model of intra-abdominal sepsis, the capsular polysaccharide complex (CPC) from B. fragilis promotes abscess formation and when administered sub-cutaneously, protects against this host response by a T cell-dependent immune mechanism. In the present study, the polysaccharide A (PS A) component of CPC protected animals against challenge with live heterologous bacterial species (mixtures of anaerobes and facultative organisms) that are most commonly isolated from intra-abdominal abscesses in humans. Protection against heterologous bacterial challenge was transferred by T cells. Administration of PS A shortly before or even after challenge with B. fragilis protected against this host response. In experiments designed to simulate fecal contamination of the human peritoneal cavity, PS A protected animals against abscess formation induced by a rat cecal contents inoculum. The surprisingly broad protective activity of PS A indicates that this molecule is likely suppressing a nonspecific host tissue reaction that forms in response to a variety of abscess-inducing organisms and that it might be useful in preventing abscess formation associated with intra-abdominal sepsis in the clinical setting.

Prevention of dry socket with clindamycin. A retrospective study.

Clindamycin and other agents were compared for efficacy in preventing the entity "dry socket." A total 765 patients were treated with clindamycin, per os, and 408 patients were treated with other antibiotics or were non-treated controls. All patients underwent surgical removal of impacted mandibular third molars. The incidence of dry socket in untreated control and in non-clindamycin antibiotic-treated patients varied from 15 to 31 percent, while in those patients receiving clindamycin, the incidence was 0.65 percent. The results demonstrate a remarkable effectiveness of clindamycin in reducing the incidence of dry socket following surgical removal of impacted mandibular third molar.

Inadequate levels of metronidazole in subcutaneous fat after standard prophylaxis.

The efficacy of antibiotic prophylaxis depends on appropriate tissue levels of the drug being present at the time of potential wound contamination. Metronidazole concentrations in serum, muscle and subcutaneous fat were measured after a single intravenous dose given at two different intervals before operation. Twenty-six patients undergoing abdominal wall procedures were divided into two groups. Patients in group 1 received metronidazole 500 mg intravenously 2 h before surgery, and those in group 2 were given the drug during induction of anaesthesia. Mean plasma levels of metronidazole at the beginning of the procedure were significantly lower (P = 0.01) in group 1 (7.3 (95 per cent confidence interval 5.7-8.9)) micrograms/ml than in group 2 (12.3 (8.9-15.7)) micrograms/ml although in both cases were above the minimum inhibitory concentration for 90 per cent of Bacteroides fragilis. Similar therapeutic concentrations of metronidazole were achieved in plasma and muscle in both groups at the end of the operation. However, patients in both groups had non-therapeutic concentrations of metronidazole in subcutaneous fat: group 1 0.9 (0.6-1.2) micrograms/mg, group 2 1.2 (0.7-1.7) micrograms/mg at the beginning of operation, and 1.2 (0.8-1.6) and 1.5 (0.9-2.1) micrograms/mg respectively at the end of the procedure. It is concluded that infusion of metronidazole 2 h before surgery or during induction of anaesthesia achieved adequate plasma and muscle levels but failed to achieve therapeutic levels in subcutaneous fat.

Aztreonam vs. gentamicin in experimental peritonitis and intra-abdominal abscess formation.

The activity of gentamicin is known to be decreased in acidic environments, and both the peritoneum during peritonitis and the interior of abscesses have been shown to be acidic and hypoxic. The activity of beta-lactam antibiotics is felt to be relatively less diminished under the same circumstances. We determined that the minimum inhibitory concentration of gentamicin against one pathogenic strain of Escherichia coli increased eight-fold, to 8 mugm/mL, when testing conditions were changed from normoxic and neutral to hypoxic and acidic, whereas the MIC of aztreonam doubled under the same conditions, to 0.25 mugm/mL. In further experiments in a murine model of mixed Escherichia coli/Bacteroides fragilis intra-abdominal abscesses, we demonstrated that a combination of aztreonam and clindamycin was superior to a combination of gentamicin and clindamycin in terms of completely preventing abscess formation (33% vs. 0%) and eliminating Escherichia coli from abscesses that did form (100% vs. 61%).