Helminth-induced alterations of the gut microbiota exacerbate bacterial colitis.

Infection with the intestinal helminth parasite Heligmosomoides polygyrus exacerbates the colitis caused by the bacterial enteropathogen Citrobacter rodentium. To clarify the underlying mechanism, we analyzed fecal microbiota composition of control and helminth-infected mice and evaluated the functional role of compositional differences by microbiota transplantation experiments. Our results showed that infection of Balb/c mice with H. polygyrus resulted in significant changes in the composition of the gut microbiota, characterized by a marked increase in the abundance of Bacteroidetes and decreases in Firmicutes and Lactobacillales. Recipients of the gut microbiota from helminth-infected wide-type, but not STAT6-deficient, Balb/c donors had increased fecal pathogen shedding and significant worsening of Citrobacter-induced colitis compared to recipients of microbiota from control donors. Recipients of helminth-altered microbiota also displayed increased regulatory T cells and IL-10 expression. Depletion of CD4+CD25+ T cells and neutralization of IL-10 in recipients of helminth-altered microbiota led to reduced stool C. rodentium numbers and attenuated colitis. These results indicate that alteration of the gut microbiota is a significant contributor to the H. polygyrus-induced exacerbation of C. rodentium colitis. The helminth-induced alteration of the microbiota is Th2-dependent and acts by promoting regulatory T cells that suppress protective responses to bacterial enteropathogens.

The role of gut microbiota in programming the immune phenotype.

The human fetus lives in a germ-free intrauterine environment and enters the outside world containing microorganisms from several sources, resulting in gut colonization. Full-term, vaginally born infants are completely colonized with a diverse array of bacterial families in clusters (Phyla) and species (>1000) by the first year of life. Colonizing bacteria communicating with the gut epithelium and underlying lymphoid tissues ('bacterial-epithelial crosstalk') result in a functional immune phenotype and no expression of disease (immune homeostasis). Appropriate colonization is influenced by the prebiotic effect of breast milk oligosaccharides. Adequate colonization results in an innate and adaptive mucosal immune phenotype via communication between molecular patterns on colonizing bacteria and pattern-recognition receptors (e.g., toll-like receptors) on epithelial and lymphoid cells. This ontogeny affects the immune system's capacity to develop oral tolerance to innocuous bacteria and benign antigens. Inadequate intestinal colonization with premature delivery, delivery by Cesarean section and excessive use of perinatal antibiotics results in the absence of adequate bacterial-epithelial crosstalk and an increased incidence of immune-mediated diseases [e.g., asthma, allergy in general and necrotizing enterocolitis (NEC)]. Fortunately, infants with inadequate intestinal colonization can be restored to a bacterial balance with the intake of probiotics. This has been shown to prevent debilitating diseases such as NEC. Thus, understanding the role of gut microbiota in programming of the immune phenotype may be important in preventing disease expression in later childhood and adulthood.

Immune factors in breast milk and the development of atopic disease.

Breast-feeding provides protection against infections and contains numerous factors that modulate and promote the development of the infant immune system. These factors include secretory IgA, antimicrobial proteins like CD14, cytokines, and fatty acids. Studies examining the role of breast-feeding in the development of allergic disease in infants demonstrate potentially protective as well as neutral or nonprotective effects, likely due to the heterogeneity in their study design. In this overview, we explore the potential role of immune factors in the breast milk, as well as selected probiotics, in the development of allergy.

Clinical evidence for immunomodulatory effects of probiotic bacteria.

Close, tightly orchestrated interactions between the intestinal epithelium and the mucosa-associated immune system are critical for normal intestinal absorptive and immunological functions. Recent data indicate that commensal intestinal microbiota represents a major modulator of intestinal homeostasis. This review analyzes the process of intestinal colonization and the interaction of microbiota with the intestinal epithelium and mucosal immune system, with special reference to the first years of extrauterine life. Dysregulation of the symbiotic interaction between intestinal microbiota and the mucosa may result in a pathological condition with potential clinical repercussions. Based on the concept that there is a beneficial and symbiotic relation between the host and endogenous microbiota, strategies aimed at directly modulating intestinal microbiota with regard to disease prevention or treatment have been developed. One strategy involves administering viable probiotic bacteria. Clinical evidence for the beneficial effect of probiotics in the prevention and/or treatment of necrotizing enterocolitis, infectious and antibiotic-associated diarrhea, allergic diseases, and inflammatory bowel disorders is reviewed herein.

Pathophysiology of intestinal food allergy.

The gastrointestinal tract plays an important role in the mucosal immune response. While acting as a conduit allowing the transfer of nutrients from the intestinal lumen to the systemic circulation, it also protects against invasion by microbes and other antigens by the induction of an immune response. A downregulation of these immune responses to nonharmful antigenic substances is termed oral tolerance. A breakdown or underdevelopment of oral tolerance may therefore lead to the development of food allergy. Adverse immunologic reactions to food may be a consequence of both IgE- and non-IgE-mediated mechanisms. Although genetic factors play a major role in the development of allergic disease, other factors involved in an immature mucosal immune response have been implicated. Non-IgE-mediated allergic responses tend to involve a T cell-mediated delayed hypersensitivity reaction, and released cytokines act as determinants of the immune response. The "hygiene hypothesis" proposes that a reduction in infections in early infancy predisposes to allergic responses. Early childhood infections promote the induction of a T-helper type 1 response that protects against the development of allergy, which is predominantly a T-helper type 2 response. The role of B cells and T cells in the development of food allergy is incompletely under- stood, but advances in the evaluation and characterization of food allergens has opened exciting new avenues in this study.

Cadaverine prevents the escape of Shigella flexneri from the phagolysosome: a connection between bacterial dissemination and neutrophil transepithelial signaling.

Shigella flexneri causes bacillary dysentery in humans by invading epithelial cells of the colon, which is characterized by an acute polymorphonuclear leukocyte (PMNL)-rich inflammation. Our recent studies demonstrated that cadaverine, a polyamine, specifically acts to abrogate transepithelial signaling to PMNL induced by S. flexneri. Here, insight is provided into the cellular mechanisms by which cadaverine attenuates the ability of Shigella species to induce PMNL signaling. It was found that cadaverine retards the lysis of the Shigella species-containing vacuole, suggesting that a blockade is established, in which the pathogen is prevented from adequately interacting with the cytoskeleton. Furthermore, an IcsA mutant of S. flexneri that cannot interact with the cytoskeleton and spreads intercellularly fails to induce transmigration of PMNL. Results indicate that cadaverine-induced compartmentalization of Shigella species to the phagolysosome might be a protective response of the host that directly contributes to the diminished ability of PMNL to transmigrate across model intestinal epithelia.

An in vitro evaluation of the accuracy of the root ZX in the presence of various irrigants.

The purpose of this study was to evaluate the accuracy of the Root ZX in vitro in the presence of a variety of endodontic irrigants. The in vitro model, described by Donnelly, consisted of refrigerated gelatin made with 0.9% sodium chloride instead of water. The following irrigants were tested: 2% lidocaine with 1:100,000 epinephrine, 5.25% sodium hypochlorite, RC Prep, liquid EDTA, 3% hydrogen peroxide, and Peridex. A total of 30 extracted, single-rooted teeth were used. The experimental measurements in the presence of the various irrigants were compared with the actual canal lengths. The present data indicate that the Root ZX electronic apex locator reliably measured canal lengths to within 0.31 mm and that there was virtually no difference in the length determination as a function of the seven irrigants used. These results strongly support the concept that the Root ZX is a useful, versatile, and accurate device for the determination of canal lengths over a wide range of irrigants commonly used in the practice of endodontics.

Effect of ultrasonic vibration and various sealer and cement combinations on titanium post removal.

The purpose of this study was to determine the effect of ultrasonic vibration on the force required to remove prefabricated posts. Ninety-six extracted human canines were divided into eight groups, which were prepared and obturated with gutta-percha and either a eugenol-containing (Roth's 801 Elite) or eugenol-free (AH26) sealer. Titanium #6 Parapost XH posts were cemented with either zinc phosphate or Panavia 21 resin cement. One half of the sample was subjected to ultrasonic vibration for 16 min at the post-dentin interface, whereas the other half received no vibration (controls). Each combination of sealer, cement, and vibration status was subjected to tensile load to failure using an Instron testing machine. Posts cemented in teeth obturated with gutta-percha and AH26 sealer demonstrated significantly greater resistance to dislodgement, compared with teeth obturated with gutta-percha and Roth's 801 Elite sealer. There was no statistical difference in retention of posts cemented with either zinc phosphate or Panavia 21, regardless of the sealer used. Additionally ultrasonic vibration increased post retention for both cements.

PH changes at the surface of root dentin after intracanal dressing with calcium oxide and calcium hydroxide.

This study was designed to compare pH changes at the cervical and apical surfaces of root dentin after canal obturation with calcium oxide or calcium hydroxide pastes. The effect of the exposure to CO2 on the pH at the root surface also was assessed for both materials. Thirty-six extracted human canines were selected and instrumented. Wells 1 mm deep x 1.5 mm in diameter were drilled in the cervical and apical regions of the buccal root surface. The teeth were randomly divided into two groups and obturated with either calcium hydroxide or calcium oxide. pH was measured in the cervical and apical wells at varying time intervals until 48 days posttreatment. After pH measurement on day 48 the vials were flushed with CO2 and the pH measured again at days 53 and 68. The results indicated a similar pattern of pH increase after treatment with either calcium oxide or calcium hydroxide. pH dropped significantly after exposure to CO2 in both groups. This study demonstrated that hydroxyl ions produced when calcium oxide reacts with water diffuse through the dentinal tubules to the surface of the root in a manner similar to hydroxyl ions from calcium hydroxide.

Developmental regulation of intestinal epithelial hydrolase activity in human fetal jejunal xenografts maintained in severe-combined immunodeficient mice.

Intestinal epithelial brush border hydrolases are important and sensitive enzyme markers of gastrointestinal development and function. Little is know about the mechanisms that regulate the induction of these enzymes during human fetal development, as these events occur primarily in utero. The present work used ectopically grafted human fetal jejunal xenografts (median age,13.3 wk of gestation), maintained in severe-combined immunodeficient mice, to study the differential expression of five different hydrolases after 10 wk of xenotransplantation. The spatio-temporal distribution of brush border alkaline phosphatase, aminopeptidase-N, alpha-glucosidase, lactase-phlorizin hydrolase, and dipeptidyl peptidase IV enzyme activities were measured quantitatively using scanning microdensitometry along the crypt-villus axes of fetal, xenograft, and pediatric (median age, 34 mo) biopsies. Ectopic grafting of fetal jejunum closely recapitulated the development of these enzymes in utero, with alkaline phosphatase, aminopeptidase-N, alpha-glucosidase, and dipeptidyl peptidase IV enzyme activities closely matching the spatio-temporal distribution and levels recorded in pediatric duodenal biopsies. Lactase-phlorizin hydrolase was the only enzyme not to reach values recorded in pediatric brush border membranes, although activities were significantly (5.6-fold) higher than in pretransplanted fetal bowel. Human jejunal xenografts therefore demonstrate an appropriate developmental induction of brush border hydrolase activity and may represent a useful model to study trans-acting factors that promote human epithelial differentiation and function in vivo. Characterization of such agents may be of potential therapeutic use in the treatment of diseases associated with gastrointestinal immaturity, notably necrotizing enterocolitis.

Hypothesis: inappropriate colonization of the premature intestine can cause neonatal necrotizing enterocolitis.

Neonatal necrotizing enterocolitis (NEC) is a major cause of morbidity in preterm infants. We hypothesize that the intestinal injury in this disease is a consequence of synergy among three of the major risk factors for NEC: prematurity, enteral feeding, and bacterial colonization. Together these factors result in an exaggerated inflammatory response, leading to ischemic bowel necrosis. Human milk may decrease the incidence of NEC by decreasing pathogenic bacterial colonization, promoting growth of nonpathogenic flora, promoting maturation of the intestinal barrier, and ameliorating the proinflammatory response.

Pathologic and physiologic interactions of bacteria with the gastrointestinal epithelium.

Communication between microorganisms and the gastrointestinal epithelium, ie, bacterial-epithelial "crosstalk," is examined. Because most basic research on the molecular interaction of bacteria with the gut epithelium relates to pathogen-enterocyte interaction, crosstalk with pathologic bacterial is considered in detail. Through their interactions with the intestinal epithelium, pathogens can modify epithelium function to enhance their penetration across the epithelial barrier and to exploit mucosal host defenses for their own benefit. Three representative pathogens are used to illustrate the various adaptive techniques used to colonize and penetrate the mucosal barrier. Salmonella enterica typhimurium interacts with the physiologic receptor for epidermal growth factor to co-opt the receptor's signal transduction mechanisms. Enteropathic Escherichia coli secretes a receptor (type III secretion) into the microvillus surface of enterocytes that disrupts the microvillus and alters its actin structure to form a dome-like anchoring site. Shigella flexneri is used to illustrate how pathogens use the follicular epithelial cell (M cell), the physiologic conduit for antigens to reach gut associated-lymphoid tissues, for penetration of the epithelial barrier. Shigella organisms attached to M cells use their endocytotic properties to enter the cell. Once inside the cell, the organism lyses the endocytic vacuole and co-opts actin and myosin to form a propelling tail for further penetration of the epithelium through the basolateral surface. Probiotics can protect the intestine by competing with pathogens for attachment, strengthening tight junctions between enterocytes, and enhancing the mucosal immune response to pathogens. However, additional molecular studies are needed to define more precisely the mechanism of probiotic-epithelial crosstalk.

Evidence for an innate immune response in the immature human intestine: toll-like receptors on fetal enterocytes.

The intestinal epithelium is an active participant in the mucosal immune response against luminal pathogens. Microorganisms and their cell wall products, i.e. lipopolysaccharide (LPS), can stimulate the enterocyte to produce an innate immune response with the increased production of IL-8 via an activation of the transcription factor NFkappaB. The innate response mechanism, however, has not been understood until the recent description of a family of human toll-like receptors (hTLR) on immune cells that interact with LPS and modulate the IL-8 response via an intracellular signal transduction pathway similar to that of the IL-1 receptor family. Accordingly, in this study we have sought to determine the constitutive and regulated expression of hTLR on a nonmalignant human fetal primary small intestinal cell line (H4 cells) and on small intestinal samples of ileum from human fetuses (age 18-21 wk). Specimens were examined by reverse-transcription PCR, Western blot analysis, and immunofluorescence for hTLR2 and hTLR4 mRNA and protein and to determine whether their expression was regulated by LPS or by an endogenous inflammatory stimulus, IL-1beta. hTLR2 and hTLR4 were expressed constitutively on H4 cells and on human fetal small intestinal enterocytes, predominantly on the basolateral surface of crypt enterocytes. Inflammatory stimuli appeared to regulate hTLR transcription (IL-1beta increased both hTLR2 and hTLR4 whereas LPS decreased hTLR4) and possibly translation (qualitative observations). The presence of hTLR on human fetal enterocyte suggests a mechanism for the innate immune response to pathogens and could provide the basis for further study of the accentuated inflammatory response in age-dependent gastrointestinal diseases such as necrotizing enterocolitis.

Post retention: the effect of sequence of post-space preparation, cementation time, and different sealers.

Many endodontic sealers contain constituents that have been shown to inhibit the polymerization of resin cements. This may be important when prefabricated posts are cemented at the same appointment as root canal obturation. This study evaluated the effects of cementing posts with a resin cement immediately or at a delayed time period after obturation using Roth's 801 Elite Grade or AH26 sealer cements. The contribution of mechanical post-space preparation was also assessed as a critical variable. One hundred twenty extracted canines were randomly divided into eight experimental groups. The variables evaluated were the order of post preparation (either before or after obturation), the type of sealer used, and the time of post cementation. All teeth received a stainless steel #6 Parapost XP cemented with a resin cement, Panavia 21. Each experimental group underwent tensile testing for retention using an Instron universal testing machine. For both sealers posts cemented in teeth in which the canal was obturated before post-space preparation and thus had sealer-contaminated dentin removed by the space preparation procedure had significantly higher retentive values than those obturated after post-space preparation in which contaminated dentin might remain. Sealer used and time of cementation had no specific effect on retention. Achieving a clean, "freshened" dentinal surface during mechanical post-space preparation seems to be a critical variable for post retention when a resin cement is used.

Epidemiological evaluation of the outcomes of nonsurgical root canal treatment in a large cohort of insured dental patients.

Selected outcomes following initial nonsurgical root canal treatment (NSRCT) procedures were retrospectively assessed using an insurance company database of 110,766 nonsurgical root canal procedures that were completed by endodontists and their referring general dentists. A subset of 44,613 cases, with a minimum required follow-up time of 2 yr, showed incidences of extraction, retreatment and periradicular surgery equal to 5.56%, 2.47%, and 1.41%, respectively. The incidence of subsequent extraction increased with patient age. Teeth that were not restored after root canal therapy were significantly more likely to undergo extraction than restored teeth. Although the practice pattern for endodontists consisted of a significantly higher proportion of molars (48% more; p < 0.001) and a smaller proportion of anterior teeth (43% less; p < 0.001) than general dentists, both groups of providers had comparable rates of untoward events. These data strongly support the hypothesis that the specialist practice provides similar rates of clinical success compared with other providers, even when treating significantly more complex NSRCT cases. Overall, 94.44% of nonsurgical root canal treated teeth remained functional over an average follow-up time of 3.5 yr. These results are an important indication of the benefits of endodontic treatment when provided in an integrated health care delivery system of endodontists and their referring general dentists.