Patricia Estrella

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.

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.

Ultrastructural localization of major basic protein in the human eosinophil lineage in vitro

We examined the ultrastructural localization of (a) a secondary granule matrix protein--eosinophil peroxidase (EPO)--by cytochemistry, (b) a secondary granule core protein (major basic protein, MBP) by immunogold labeling, and (c) a primary granule protein (the Charcot-Leyden crystal protein, CLC protein) by immunogold labeling in eosinophilic myelocytes (EMs) and mature, activated eosinophils that differentiated from umbilical cord blood progenitors cultured in the presence of recombinant human interleukin-5 (rhIL-5). These studies provide the first substructural localization of MBP to condensing cores of immature secondary granules of EMs, as well as identification of unicompartmental, MBP-rich secondary granules that are devoid of matrix compartments and EPO content and are not primary granules by virtue of their lack of CLC protein. These granules occur in quantity in IL-5-activated mature human eosinophils, which have previously been shown to actively transport EPO from the matrix compartments of their secondary granules to the extracellular milieu in smooth membrane-bound cytoplasmic vesicles, a secretory process termed piecemeal degranulation, whereby eosinophils progressively empty cytoplasmic granules of their contents in the absence of classical granule extrusion.

Analysis of Mast Cell Activation Using Diamine Oxidase-Gold Enzyme-Affinity Ultrastructural Cytochemistry

We review a new technique--diamine oxidase (DAO)-gold ultrastructural enzyme-affinity labeling--which we developed to localize histamine in subcellular sites of mast cells. The DAO-gold method showed that isolated human lung mast cells contained abundant histamine in their cytoplasmic granules, a conclusion which was verified by a large number of specificity controls. We also studied mast-cell-rich eyelid lesions which developed in interleukin-4 transgenic mice. The DAO-gold method demonstrated histamine in the electron-dense granules of mast cells in these lesions, but little or no histamine was detected in the swollen, empty granules of mast cells undergoing piecemeal degranulation. This new enzyme-affinity-gold method has permitted the first ultrastructural localization of histamine in subcellular sites of routinely prepared electron microscopy samples. The method has also permitted the first morphological studies of histamine secretion in vivo and has demonstrated that such secretion can be associated with the ultrastructural changes of piecemeal degranulation.