Thomas H. Ermak

Uptake and transport of copolymer biodegradable microspheres by rabbit Peyer's patch M cells

In this study, we demonstrate the role of M cells in uptake of poly(D-L-lactic-co-glycolic acid) (PLGA) microspheres and transport into rabbit Peyers patches. Microspheres 1 to 10 μm in diameter composed of 50:50 lactic acid:glycolic acid were instilled into in-testinal segments containing jejunal or ileal Peyers patches, and uptake by M cells was examined by electron microscopy. PLGA microspheres visualized as electron-lucent, spherical particles were taken up by M cells by pseudopod-like extensions of the M cell apical membrane and translocated to the pocket region containing mononuclear leukocytes within 60 min. These results indicate that PLGA microspheres can be directed to M cell apical surfaces for delivery to immunocompetent cells in gut-associated lymphoid tissues.

Immunization with recombinant Helicobacter pylori urease decreases colonization levels following experimental infection of rhesus monkeys

Rhesus monkeys, naturally colonized with H. pylori as indicated by culture and histology were immunized with either 40 mg recombinant H. pylori urease administered orally together with 25 microg Escherichia coli heat-labile enterotoxin (LT) or immunized with LT alone. An initial 6 doses were administered over an 8 week period. All five vaccinated monkeys had a greater than two-fold rise in urease-specific serum IgG and IgA level and urease-specific salivary IgA was induced in 3 of 5 vaccinated animals after 6 or 7 doses of vaccine. Vaccination had no measurable therapeutic effect on H. pylori colonization. H. pylori was eradicated from these monkeys with a course of antimicrobials plus omeprazole, a 7th vaccine dose was given (10 months after the 6th dose) and they were rechallenged with H. pylori. Necropsy was performed 23 weeks after rechallenge and H. pylori colonization was determined by histological examination of 12 individual gastric sites. A significant reduction in colonization (p < or = 0.0001; Friedmans analysis of variance) was found in the vaccinated animals. Histopathologic examination of necropsy tissues also revealed a trend towards reduced gastritis and epithelial alterations in the vaccinated group compared to animals receiving LT alone. This study provides the first evidence for effective vaccination of nonhuman primates against H. pylori, and preliminary evidence that a reduction in bacterial density attributable to immunization may lessen gastric inflammation.

Structural specializations for antigen uptake and processing in the digestive tract

ConclusionsEpithelial adaptations for antigen uptake and lymphoid organ cytoarchitecture differ according to the characteristics of specific sites throughout the mucosa but all facilitate antigen uptake. Whether surrounding mucosa is stratified squamous epithelium as in the oropharynx or columnar epithelium as in the intestine, organized lymphoid tissue in each area displays surface specializations which reduce local barriers and facilitate the approach and uptake of microorganisms, particles and macromolecules. In lymphoid tissue in each of these sites cellular defense mechanisms are arrayed for local containment, antigen processing and initiation of immune responses.

Zymogen granules of pancreas decrease in size in response to feeding

SummaryIn the first descriptions of pancreatic enzyme secretion about 100 years ago, it was noticed that zymogen granules became smaller and disappeared from the apical region of acinar cells after feeding. We have repeated these experiments and characterized changes in granule size by quantitative electron microscopy 90 min after feeding previously fasted rats. In fasted animals, granules occupied the apical portion of the cell, had an average number of 45±3 granules per cell section (±SE), and measured 0.85±0.15 μm in diameter (±SD). After feeding, the number and size of granules decreased. Individual samples showed either a decrease in size alone or a decrease in both size and number, but in no case did they show a reduction in granule number alone. The mean diameter of granules decreased to 0.65±0.15 μm (±SD) or about a 55% reduction in average granule volume as compared to controls (0.32 vs. 0.14 μm3). The size distributions were unimodal and normal in both fasted and fed rats; however, in fed animals, the distribution was shifted to lower values (diameter range 0.40–1.40 μm for fasted rats vs. 0.10–1.30 μm for fed rats). The number of granules decreased to an average of 29±2 granules per cell section (±SE) after feeding, and, on the average, samples with the most granules had larger ones than samples with the fewest granules. The present results support the original observations on live rabbit pancreas that individual granules decrease in size in response to feeding. We suggest that these size changes reflect the loss of proteins across the granule membrane as proposed by the equilibrium hypothesis for digestive enzyme secretion.

Correlation of extracellular matrix components with the cytoarchitecture of mouse Peyer's patches.

SummaryThe distribution patterns of extracellular matrix elements were determined to ascertain whether they play a role in the localization of lymphocytes in discrete T-cell, B-cell and dome antigen-processing domains within Peyers patches. Antibodies against collagen types I, III and IV, laminin and fibronectin were applied to cryosections of mouse Peyers patches and localized by direct or indirect immunoperoxidase methods. T-cell domains were identified with a monoclonal antibody against Thy-1.2. Labeled reticular fibers in distinctive patterns were more numerous in parafollicular and dome areas than within follicles. Germinal centers contained few such fibers. In parafollicular areas, fibers were oriented predominantly toward follicle domes; their distribution corresponded to T-cell zones and lymphocyte traffic areas, with their orientation being parallel to the migration pathways of lymphocytes from high endothelial venules to the antigen-processing domes. Subepithelial and subendothelial basal laminae were immunopositive for type-IV collagen, laminin and fibronectin. The dome subepithelial basal lamina had pore-like discontinuities through which lymphocytes migrated to and from the epithelium. The correspondence of the distribution patterns of extracellular matrix to specific functional domains of Peyers patches suggests that this matrix provides a structural framework for lymphocyte migration and localization.

Sterilizing immunity against experimental Helicobacter pylori infection is challenge-strain dependent.

The development of a murine model of Helicobacter pylori infection through serial in vivo passage of candidate strains has enabled a quantitative assessment of vaccine efficacy. In this study we compare infection with and protection against challenge from both CagA(+) type I, and CagA(-) type II in vivo adapted isolates. In vivo passage of a type II H. pylori isolate resulted in a highly infectious strain (X47-2AL), capable of reproducibly infecting mice to high density (10(7) CFU/g of gastric tissue). Similarly adapted type I strains were found to colonize mice at a significantly lower level (10(4)-10(5) CFU/g tissue). Mucosal immunization with recombinant urease (rUre) significantly protected animals against both types. Protection against X47-2AL was characterized by a > or =100-fold (or 2 log) reduction in bacterial density. However, the presence of a residual infection highlighted the inability to achieve sterilizing immunity against this strain. The level of protection appeared independent of challenge dose, and was stable for up to 6 months, all animals exhibiting a low-level residual infection that did not recrudesce with time. Similarly immunized mice challenged with isolates representing the residual infection were also protected, confirming that they did not represent a sub-population of H. pylori that could escape immunity. Immunization and challenge studies with type I adapted-isolates, demonstrated a similar 2-3 log reduction in the bacterial burden, but that in this instance resulted in sterilizing immunity. These results suggest varied specificity for the murine host by different Helicobacter strains that can influence the outcome of both infection and immunity.

Large decrease in zymogen granule size in the postnatal rat pancreas

The volume density of zymogen granules in rat pancreas decreased dramatically after birth, from about 45% of cytoplasmic volume at birth to 23% by 1 week, and then gradually fell to about 18% by 3 weeks. The decrease in volume density was primarily due to a decrease in granule size from an average of 1.5 μm (range 0.7 to 2.8 μm) in the newborn to about 0.85 μm (range 0.5–1.4 μm) at 3 weeks. This corresponds to a six-fold reduction in granule volume. The distribution of granule diameters was unimodal, relatively normal (lognormal), and maintained this configuration as size decreased over time. The size distribution in the newborn was rather broad and narrowed substantially during the following 3 weeks as it shifted to a lower size range. The condensing vacuole size distribution at birth was within the lower half of the zymogen granule distribution. This and other data suggest that granules prenatally are not formed solely by a reduction in size from condensing vacuoles but increase in size. There was a linear relationship between the mean zymogen granule diameter and the standard deviation around the mean for the different time intervals. This suggests that the same controlling factors produce the broad range of large granules seen in the newborn as the narrow range of much smaller granules seen at 3 weeks. The present data, as well as evidence from other sources, indicate that the zymogen granule is a single structural entity whose size varies as a continuous function with changing physiological state.

Oral immunization with recombinant Helicobacter pylori urease confers long-lasting immunity against Helicobacter felis infection.

Recombinant Helicobacter pylori urease (rUre) has been shown to confer protection against challenge with Helicobacter felis in mice. The purpose of the present study was to examine duration of the immune response and long-term protective efficacy of immunization with rUre. Swiss Webster mice were orally immunized four times at weekly intervals with 100 microg rUre plus 5 microg heat-labile enterotoxin of Escherichia coli (LT) adjuvant, or with LT only. At 4, 10, 20 or 40 weeks post immunization, 25 rUre-immunized mice and control mice were challenged with H. felis and sacrificed at 2 or 10 weeks post-challenge. H. felis infection was assessed by gastric urease assay and by histology. Anti-H. pylori urease specific antibody levels were measured in serum and saliva both pre- and post-challenge. Over the 40 week time period, the infection rates in rUre-immunized mice were significantly lower than those in controls (p < 0.05) as assessed by gastric urease activity. Protection ranged from 79 100% at 2 weeks post-challenge and 63-78% at 10 weeks post-challenge. Gastric bacterial density in rUre-immunized mice was significantly lower than that of controls (p < 0.03) as determined by histologic assessment. Anti-urease antibody levels remained elevated in the serum and mucosal compartments at 39 weeks following immunization. This study shows that immunization with rUre plus LT results in long-lasting protective immunity against challenge with H. felis.

Internal organization of the zymogen granule: formation of reticular structures in Vitro

Previous studies on the chemical and physical properties of isolated zymogen granules have shown that their contents, including some 10 to 20 different digestive enzymes, exist as an ordered solid-state array. In the present ultrastructural study, we have looked for related morphologic organization by examining (1) zymogen granules partially or fully depleted of their contents as a result of dilution in large suspension volumes; (2) previously released granule contents reaggregated with granule membrane; and (3) granules suspended in a detergent. In all of these circumstances, a reticular meshwork was observed that was comprised of electron-dense strands of variable thickness. In granular form, the reticulum outlines spherical and polygonal spaces and is attached at discrete points to the internal surface of the granule membrane. In reaggregated material, the strands form an open reticulum attached to membrane fragments. As a result of dilution, reticulated granules were observed ranging from almost filled to virtually empty (frequently containing a residual filamentous reticulum). The reticular spaces produced in granules by increasing the suspension volume were formed throughout the granule and not simply from the surface inward. The maintenance of the reticular structure did not appear to depend upon either enclosure by the granule membrane or the presence of intragranular hydrophobic bonding since Triton X treatment left a membraneless, electron-dense, reticulated structure. Reticular structures similar to those reported here have been observed by others in zymogen granules in situ.

Treatment of murine lupus with monoclonal antibody to L3T4. I. Effects on the distribution and function of lymphocyte subsets and on the histopathology of autoimmune disease.

Monoclonal antibodies (MoAb) to L3T4 have been used successfully to suppress autoimmunity in murine models for several human autoimmune diseases. To clarify the immunologic and clinical consequences of treatment with anti-L3T4, we examined the effects of chronic administration of anti-L3T4 on the composition of lymphoid organs, the function of lymphocytes, and the histopathology of autoimmune disease in lupus-prone NZB/NZW F1 (B/W) mice. Weekly treatment with anti-L3T4 (2 mg/mouse) from age 5 to 8 months depleted L3T4+ cells from the spleen and lymph nodes, and prevented the development of splenomegaly and lymphadenopathy. The MoAb bound to target cells in the thymus and modulated their expression of the L3T4 antigen but, in contrast to its effect in extrathymic sites, anti-L3T4 did not deplete the target population from the thymus. In fact, after 3 months of therapy, mice that had been treated with anti-L3T4 had much larger thymuses than control mice that had been treated with saline, suggesting that treatment with anti-L3T4 prevented the thymic atrophy that occurs spontaneously in murine lupus. Despite depleting L3T4+ cells from the spleen, treatment with anti-L3T4 did not diminish the response of splenic lymphocytes to T and B cell mitogens, and it augmented splenic natural killer (NK) cell activity. Finally, treatment with anti-L3T4 decreased the diverse histopathologic manifestations of murine lupus. It dramatically reduced glomerular immunoglobulin and complement deposition and diminished lymphocytic infiltration and vasculitis in the kidneys. Treatment also reduced extrarenal immunopathology, including focal hepatitis and salivary gland infiltration. These observations have implications regarding the use of CD4 MoAb in people with autoimmune diseases.