Ronald L. Cisneros

Ultrastructural Evidence for Piecemeal and Anaphylactic Degranulation of Human Gut Mucosal Mast Cells in vivo

One hundred and seventeen coded intestinal biopsies were examined by electron microscopy and evaluated for morphological evidence of mast cell and basophil secretion in situ. Sixty percent of the biopsies had evidence of secretion. Mast cell secretion was evident in control biopsies, many of which were obtained from uninvolved tissues of patients with inflammatory bowel disease. Biopsies of inflamed continent pouches from ulcerative colitis (UC) patients showed more mast cell secretion than noninflamed UC pouch biopsies. This evidence of mast cell secretion supports recent work that documents high constitutive levels of histamine in jejunal fluids of Crohns disease patients and suggests a proinflammatory role for mast cells in inflammation associated with pouchitis.

Evidence for T Cell-dependent Immunity to Bacteroides fragilis in an Intraabdominal Abscess Model

It has been shown that active immunization of rats with the capsular polysaccharide of Bacteroides fragilis protects these animals against abscess development following intraperitoneal challenge with this species. Passive transfer of hyperimmune globulin from immunized animals to nonimmune recipients provided protection against B. fragilis bacteremia in challenged animals, but did not confer protection against abscess development. On the other hand, adoptive transfer of spleen cells from immunized animals to nonimmunized recipients resulted in protection against abscesses following challenge with B. fragilis. These data suggested that a T cell-dependent immune response was involved in protection against abscess development after immunization with B. fragilis capsular antigen. To determine the possible role of cell-mediated immunity prompted by the capsular antigen, inbred congenitally athymic OLA/Rnu rats and their phenotypically normal littermates were actively immunized. Despite the development of high titers of anti-B. fragilis capsular antibody, 100% of actively immunized athymic rats developed abscesses, as did 100% of unimmunized athymic control rats. However, no phenotypically normal littermate control rats that were actively immunized developed abscesses, while 100% of phenotypically normal unimmunized rats developed abscesses. Additional studies showed that adoptive transfer of T cell-enriched spleen cell preparations from Wistar/Lewis rats immunized with the capsular polysaccharide to nonimmune recipients also resulted in protection against B. fragilis-induced abscesses. We conclude that the protection afforded by immunization with B. fragilis capsule against intraabdominal abscesses caused by that organism is T cell-mediated and does not require the presence of serum antibody.

Ultrastructural identification of exocytosis of granules from human gut eosinophils in vivo.

Twenty-two percent of 117 biopsies of human intestinal tissues had ultrastructural images of classical regulated secretion from eosinophils in vivo i.e. eosinophil granule extrusion (EGE). Replicate intestinal biopsies that were positive for bacteria had EGE more often than not (p < 0.05); 77% of the isolates were Staphylococci. Some of the intestinal biopsies also had damaged nerves; all that had EGE and damaged enteric nerves also had positive bacterial cultures. The EGE that we observed could not account for all enteric nerve damage, suggesting multifactorial mechanisms for nerve damage in gut tissues. Among the possibilities are release of neurotoxic eosinophil granule proteins by an alternate secretory route, i.e., piecemeal degranulation, direct toxicity of tissue invasive bacteria and/or damaged nerves of unknown etiology such as those that are regularly present in uninvolved tissues of patients with Crohns disease.

Human Gut Mucosal Mast Cells: Ultrastructural Observations and Anatomic Variation in Mast Cell-Nerve Associations in vivo

One hundred and seventeen coded intestinal biopsy specimens were examined by electron microscopy. All surgical biopsies were obtained from uninvolved sites of patients with two inflammatory bowel diseases (ulcerative colitis or Crohns disease) and from patients with preneoplastic and neoplastic diseases (adenocarcinoma, rectal polyp, familial polyposis). Biopsy sites included normal ileum, colon, and rectum as well as conventional ileostomies and continent pouches constructed from the ileum. The data reported here describe the ultrastructural anatomy of human gastrointestinal tract mucosal mast cells in vivo and their anatomic associations with enteric nerves.

Axonal necrosis of enteric autonomic nerves in continent ileal pouches. Possible implications for pathogenesis of Crohn's disease.

OBJECTIVE Axonal necrosis was first described in samples of small intestine from patients with Crohns disease (A.M. Dvorak et al. Hum Pathol 1980; 11:620-634). Clinically evident inflammation of continent ileal reservoirs (pouches) has clinical features that resemble Crohns disease. Possible similarities in the pathogenesis of Crohns disease and pouchitis were sought using ultrastructural and microbiologic tools to identify damaged enteric nerves and tissue bacteria. METHODS An encoded ultrastructural and microbiologic study of replicate biopsies from 114 samples of human intestine was done. Biopsies from ileum, colon, conventional ileostomy or continent pouch were obtained from patients with ulcerative colitis, Crohns disease, or familial polyposis and grouped into three clinical study groups (control, normal pouch, pouchitis), based on clinical and endoscopic criteria. Biopsies were prepared for electron microscopy with standard methods; replicate biopsy samples were washed extensively before preparing cultures designed to identify aerobic as well as facultative and obligate anaerobic bacteria (Onderdonk et al. J Clin Microbiol 1992; 30:312-317). The ultrastructural diagnosis of damaged enteric nerves was based on previously published criteria for axonal necrosis (A.M. Dvorak and W. Silen. Ann Surg 1985; 201:53-63). Intergroup comparisons were tested for significance using Chi-square analysis. RESULTS The highest incidence of axonal necrosis was present in Crohns disease control biopsies (53%), regardless of whether bacteria were present (or not) in cultures of replicate biopsies. Axonal necrosis also occurred in more ulcerative colitis and familial polyposis biopsies (regardless of biopsy site) that had positive bacterial cultures than in those that did not (p < 0.001). In addition, axonal necrosis was documented in 42% of the pouch biopsies from ulcerative colitis and familial polyposis patients, particularly in those pouches that were found to be inflamed by clinical criteria and that also had positive bacterial cultures of the biopsied tissues. Control biopsies from patients with ulcerative colitis and familial polyposis had significantly less nerve damage than pouch biopsies in the presence of positive cultures (p < 0.01). Among the clinically inflamed pouches biopsied in ulcerative colitis or familial polyposis patients, we found that none had damaged enteric nerves when bacterial cultures were negative (p < 0.005). If the presence of axonal necrosis alone was compared with the presence of undamaged enteric nerves in all biopsies from patients with ulcerative colitis, a highly significant number of ulcerative colitis biopsies with axonal necrosis occurred in pouches (72%) compared with controls (p < 0.001). CONCLUSIONS The ultrastructural finding of axonal necrosis in Crohns disease confirms previous studies. The presence of damaged enteric nerves in patients with pouchitis provides ultrastructural support to the clinical impression of similarities between pouchitis and Crohns disease. The association of damaged nerves and invasive bacteria in pouchitis suggests mechanistic similarities for the pathogenesis of Crohns disease that requires further investigation.

Prophylaxis with the Immunomodulator: PGG Glucan Enhances Antibiotic Efficacy in Rats Infected with Antibiotic-Resistant Bacteria

The emergence of multiple antibiotic-resistant microorganisms has led to a search for alternatives to traditional therapeutic regimens. PGG glucan is a soluble beta-glucan immunomodulator that selectively enhances the microbicidal activities of neutrophils and macrophages without stimulating proinflammatory cytokine production. In the present studies, we examined the ability of PGG glucan to act in concert with antibiotics to decrease mortality in a rat model of intraabdominal sepsis using antibiotic-resistant bacteria as infectious inocula. Results of these studies demonstrated that prophylaxis with PGG glucan in combination with antibiotics provided enhanced protection against lethal challenge with Esherichia coli or Staphylococcus aureus as compared with the use of antibiotics alone.

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.

Histologic and microbiologic features of biopsy samples from patients with normal and inflamed pouches

PURPOSE: This study was undertaken to assess the electron microscopic and microbiologic findings in tissue biopsy samples from patients with pouchitis and to compare them with findings in patients with normal pouches, conventional ileostomies, and normal ileum. METHODS: Tissue samples were obtained from 78 patients: 23 patients with normal pouches endoscopically and histologically (Group 1), 12 patients with endoscopic and histologic evidence of inflammation (pouchitis) (Group 2), 14 patients who had either endoscopic or histologic evidence of inflammation but not both (Group 3), 20 patients with conventional ileostomies (Group 4), and 9 patients without ileostomies from whom biopsy samples of normal ileum were obtained (Group 5). RESULTS: The mean total aerobic facultative counts in the biopsy samples from the pouchitis patients were significantly higher when compared with biopsy samples from Groups 4 and 5 (P<0.05). There were no significant differences in the mean anaerobic counts among the five groups. Positive cultures were obtained in 90 percent of patients with pouches compared with 69 percent of patients with conventional ileostomies or normal ileum (P<0.05). Intramural bacteria were observed on electron microscopy in biopsy specimens of 47 percent patients with pouches compared with 14 percent of patients with conventional ileostomies or normal ileum (P<0.05). However, the proportion of patients with positive cultures or intramural bacteria was not increased in the pouchitis group compared with the normal pouch group. CONCLUSION: These data suggest that intramural aerobic facultative bacterial counts are elevated in patients with pouchitis and may play a role in the pathogenesis of pouchitis.

Protection against Lethal Intra-abdominal Sepsis by 1-(3-dimethylaminopropyl)-3-ethylurea

Sodium hyaluronate-carboxymethylcellulose (HA/CMC) formulations are gels that effectively reduce postoperative adhesions in both animals and humans, when placed in the peritoneal or pelvic cavities concomitant with surgical manipulation. However, it has been suggested that the use of these products may increase the risk of peritoneal infection after contamination with intestinal contents during surgery. Using the rat intra-abdominal sepsis model, we found that administration of HA/CMC gels before bacterial challenge did not increase mortality but did significantly protect rats against lethal infection. This effect was dose and time dependent. Protection was conferred not by the HA/CMC gels themselves but by 1-(3-dimethylaminopropyl)-3-ethylurea (EDU), a small molecule released from the gel complex under physiologic conditions. Our results suggest that the protective effect exhibited by EDU is related to down-regulation of T cell-dependent responses and suppression of the proinflammatory-cytokine cascade associated with mortality during the early phase of disease.

Antimicrobial efficacy testing of moxifloxacin during the peritonitis and abscess formation stages of intra-abdominal sepsis: A controlled trial in the rat model

Abstract Background: Moxifloxacin is a fluoroquinolone with a broad range of antimicrobial activity against gram-negative and gram-positive, facultative, and obligately anaerobic bacteria, including certain quinolone-resistant organisms. Objective: Efficacy of moxifloxacin was compared with that of a combination of clindamycin + gentamicin in a rat model of intra-abdominal sepsis, with end points including mortality during the first 2 to 4 days after initiation of sepsis and the incidence of intra-abdominal abscess noted at necropsy. The bacterial challenge was prepared from cecal contents or an enhanced cecal content preparation. Methods: Groups of 20 animals were employed in the first part of the study, which used a cecal content challenge: group 1 served as saline-treated controls; group 2 received moxifloxacin 15 mg once daily; and group 3 received a combination of clindamycin 15 mg TID + gentamicin 2 mg TID. Dosing was determined for all antimicrobial agents based on peak and trough serum levels by prior testing (ie, experiments initiated before this study to determine the appropriate dosage in animals that approximates the levels obtained in humans with the antimicrobial agent under investigation), adjusting doses according to a body surface area/body weight ratio, and comparing these doses and levels with studies in humans. In the second part of the study, moxifloxacin was compared with levofloxacin (both 15 mg once daily) in an enhanced abscess model. Results: The mortality rate was 57.9% in the control group versus 0% in the moxifloxacin group and 5% in the clindamycin + gentamicin group ( P clindamycin + gentamicin group ( P Bacteroides fragilis —enhanced abscess model, the incidence of abscess was significantly lower in the moxifloxacin group compared with both the saline-treated controls (419 [21.1%] vs55 [100%], respectively; P = 0.003) and the levofloxacin group (419 [21.1%] vs1120 [55%], respectively; P = 0.042). Conclusions: This study shows that moxifloxacin is as effective as other established therapeutic regimens in reducing the incidence of both mortality and intra-abdominal abscesses in this animal model. In addition, moxifloxacin is extremely effective in preventing abscesses caused by obligate anaerobes, even in an enhanced model for abscess development, and its efficacy is superior to that of levofloxacin.