The Human Milk Oligosaccharide 2'-Fucosyllactose Quenches Campylobacter jejuni-Induced Inflammation in Human Epithelial Cells HEp-2 and HT-29 and in Mouse Intestinal Mucosa.

BACKGROUND: Campylobacter jejuni causes diarrhea worldwide; young children are most susceptible. Binding of virulent C. jejuni to the intestinal mucosa is inhibited ex vivo by α1,2-fucosylated carbohydrate moieties, including human milk oligosaccharides (HMOSs). OBJECTIVE: The simplest α1,2-fucosylated HMOS structure, 2'-fucosyllactose (2'-FL), can be predominant at ≤5 g/L milk. Although 2'-FL inhibits C. jejuni binding ex vivo and in vivo, the effects of 2'FL on the cell invasion central to C. jejuni pathogenesis have not been tested. Clinical isolates of C. jejuni infect humans, birds, and ferrets, limiting studies on its mammalian pathobiology. METHODS: Human epithelial cells HEp-2 and HT-29 infected with the virulent C. jejuni strain 81-176 human isolate were treated with 5 g 2'-FL/L, and the degree of infection and inflammatory response was measured. Four-week-old male wild-type C57BL/6 mice were fed antibiotics to reduce their intestinal microbiota and were inoculated with C. jejuni strain 81-176. The sensitivity of the resulting acute transient enteric infection and immune response to inhibition by 2'-FL ingestion was tested. RESULTS: In HEp-2 and HT-29 cells, 2'-FL attenuated 80% of C. jejuni invasion (P < 0.05) and suppressed the release of mucosal proinflammatory signals of interleukin (IL) 8 by 60-70%, IL-1ß by 80-90%, and the neutrophil chemoattractant macrophage inflammatory protein 2 (MIP-2) by 50% (P < 0.05). Ingestion of 2'-FL by mice reduced C. jejuni colonization by 80%, weight loss by 5%, histologic features of intestinal inflammation by 50-70%, and induction of inflammatory signaling molecules of the acute-phase mucosal immune response by 50-60% (P < 0.05). This acute model did not induce IL-17 (adaptive T cell response), a chronic response. CONCLUSIONS: In human cells in vitro (HEp-2, HT-29) and in a mouse infection model that recapitulated key pathologic features of C. jejuni clinical disease, 2'-FL inhibited pathogenesis and its sequelae. These data strongly support the hypothesis that 2'-FL represents a new class of oral agent for prevention, and potentially for treatment, of specific enteric infectious diseases.

Human Milk Oligosaccharides and Synthetic Galactosyloligosaccharides Contain 3'-, 4-, and 6'-Galactosyllactose and Attenuate Inflammation in Human T84, NCM-460, and H4 Cells and Intestinal Tissue Ex Vivo.

BACKGROUND: The immature intestinal mucosa responds excessively to inflammatory insult, but human milk protects infants from intestinal inflammation. The ability of galactosyllactoses [galactosyloligosaccharides (GOS)], newly found in human milk oligosaccharides (HMOS), to suppress inflammation was not known. OBJECTIVE: The objective was to test whether GOS can directly attenuate inflammation and to explore the components of immune signaling modulated by GOS. METHODS: Galactosyllactose composition was measured in sequential human milk samples from days 1 through 21 of lactation and in random colostrum samples from 38 mothers. Immature [human normal fetal intestinal epithelial cell (H4)] and mature [human metastatic colonic epithelial cell (T84) and human normal colon mucosal epithelial cell (NCM-460)] enterocyte cell lines were treated with the pro-inflammatory molecules tumor necrosis factor-α (TNF-α) or interleukin-1ß (IL-1ß) or infected with Salmonella or Listeria. The inflammatory response was measured as induction of IL-8, monocyte chemoattractant protein 1 (MCP-1), or macrophage inflammatory protein-3α (MIP-3α) protein by ELISA and mRNA by quantitative reverse transcriptase-polymerase chain reaction. The ability of HMOS or synthetic GOS to attenuate this inflammation was tested in vitro and in immature human intestinal tissue ex vivo. RESULTS: The 3 galactosyllactoses (3'-GL, 4-GL, and 6'-GL) expressed in colostrum rapidly declined over early lactation (P < 0.05). In H4 cells, HMOS attenuated TNF-α- and IL-1ß-induced expression of IL-8, MIP-3α, and MCP-1 to 48-51% and pathogen-induced IL-8 and MCP-1 to 26-30% of positive controls (P < 0.001). GOS reduced TNF-α- and IL-1ß-induced inflammatory responses to 25-26% and pathogen-induced IL-8 and MCP-1 to 36-39% of positive controls (P < 0.001). GOS and HMOS mitigated nuclear translocation of nuclear transcription factor κB (NF-κB) p65. HMOS quenched the inflammatory response to Salmonella infection by immature human intestinal tissue ex vivo to 26% and by GOS to 50% of infected controls (P < 0.01). CONCLUSION: Galactosyllactose attenuated NF-κB inflammatory signaling in human intestinal epithelial cells and in human immature intestine. Thus, galactosyllactoses are strong physiologic anti-inflammatory agents in human colostrum and early milk, contributing to innate immune modulation. The potential clinical utility of galactosyllactose warrants investigation.

Glucocorticoids and microbiota regulate ontogeny of intestinal fucosyltransferase 2 requisite for gut homeostasis.

At weaning, the intestinal mucosa surface glycans change from predominantly sialylated to fucosylated. Intestinal adaptation from milk to solid food is regulated by intrinsic and extrinsic factors. The contribution by glucocorticoid, an intrinsic factor, and colonization by microbiota, an extrinsic factor, was measured as the induction of α1,2/3-fucosyltransferase and sucrase-isomaltase (SI) activity and gene expression in conventionally raised, germ-free, and bacteria-depleted mice. In conventionally raised mice, cortisone acetate (CA) precociously accelerated SI gene expression up to 3 weeks and fut2 to 4 weeks of age. In germ-free mice, CA treatment induces only SI expression but not fucosyltransferase. In post-weaning bacteria-deficient (germ-free and bacteria-depleted) mice, fut2 expression remains at low suckling levels. In microbiota deficient mice, intestinal fut2 (but not fut1, fut4 or fut7) was induced only by adult microbiota, but not immature microbiota or CA. Fut2 induction could also be restored by colonization by Bacteroides fragilis, but not by a B. fragilis mutant unable to utilize fucose. Restoration of fut2 expression (by either microbiota or B. fragilis) in bacteria-depleted mice is necessary for recovery from dextran sulfate sodium-induced mucosal injury. Thus, glucocorticoids and microbes regulate distinct aspects of gut ontogeny: CA precociously accelerates SI expression and, only in colonized mice, fut2 early expression. The adult microbiota is required for the fut2 induction responsible for the highly fucosylated adult gut phenotype and is necessary for recovery from intestinal injury. Fut2-dependent recovery from inflammation may explain the high incidence of inflammatory disease (Crohn's and necrotizing enterocolitis) in populations with mutant FUT2 polymorphic alleles.

TGF-ß2 induces maturation of immature human intestinal epithelial cells and inhibits inflammatory cytokine responses induced via the NF-κB pathway.

OBJECTIVES: Breast milk transforming growth factor (TGF)-ß2 is associated with healthy immune maturation and reduced risk of immune-mediated disease in infants. We sought to investigate whether conditioning with TGF-ß2 may result in a more mature immune responder phenotype in immature human intestinal epithelial cells (IECs). METHODS: Primary human fetal IECs (hFIECs) and the human fetal small intestinal epithelial cell line (H4 cells) were conditioned with breast milk levels of TGF-ß2, and an inflammatory response was subsequently induced. Inflammatory cytokine secretion and mRNA expression were measured by enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction, respectively. Alterations in activation of inflammatory signaling pathways were detected from IECs by immunoblotting and immunofluorescence. The effects of TGF-ß2 conditioning on gene expression patterns in hFIECs were assessed by cDNA microarray analysis and quantitative PCR. RESULTS: Conditioning with TGF-ß2 significantly attenuated subsequent interleukin (IL)-1ß-, TNF-α-, and poly I:C-induced IL-8 and IL-6 responses in immature human IECs. Conditioning with TGF-ß2 inhibited IL-1ß-induced IκB-α degradation and NF-κB p65 nuclear translocation, which may partially result from TGF-ß2-induced changes in the expression of genes in the NF-κB signaling pathway detected by cDNA microarray and qPCR. CONCLUSIONS: Conditioning with TGF-ß2 attenuates the subsequent inflammatory cytokine response in immature human IECs by inhibiting signaling in the NF-κB pathway. The immunomodulatory potential of breast milk may in part be mediated by TGF-ß2, which may provide a novel means of supporting intestinal immune maturation in neonates.

Breast milk-transforming growth factor-ß2 specifically attenuates IL-1ß-induced inflammatory responses in the immature human intestine via an SMAD6- and ERK-dependent mechanism.

BACKGROUND: Breast milk is known to protect the infant against infectious and immuno-inflammatory diseases, but the mechanisms of this protection are poorly understood. OBJECTIVES: We hypothesized that transforming growth factor-ß2 (TGF-ß2), an immunoregulatory cytokine abundant in breast milk, may have a direct anti-inflammatory effect on immature human intestinal epithelial cells (IECs). METHODS: Human fetal ileal organ culture, primary human fetal IECs, and the human fetal small intestinal epithelial cell line H4 were stimulated with interleukin 1ß (IL-1ß) with or without TGF-ß2. Pro-inflammatory cytokine secretion and mRNA expression were measured by ELISA and quantitative real-time polymerase chain reaction, respectively. Alterations in ERK signalling were detected from IECs by immunoblotting and in fetal ileal tissue culture by immunohistochemistry. SMAD6 knockdown was performed by transfecting the cells with SMAD6 siRNA. RESULTS: TGF-ß2 significantly attenuated IL-1ß-induced pro-inflammatory cytokine production in fetal intestinal organ culture and the cell culture models. In addition, TGF-ß2 reduced the IL-1ß-induced IL-8 and IL-6 mRNA response in H4 cells. TGF-ß2 markedly inhibited IL-1ß-induced phosphorylation of ERK, which was necessary for the cytokine response. The inhibitory effect of TGF-ß2 on IL-1ß-induced cytokine production was completely abrogated by SMAD6 siRNA knockdown. CONCLUSIONS: TGF-ß2 attenuates IL-1ß-induced pro-inflammatory cytokine production in immature human IECs by inhibiting ERK signalling. The anti-inflammatory effect of TGF-ß2 is dependent on SMAD6. Breast milk TGF-ß2 may provide the neonate with important immunoregulatory support. TGF-ß2 might provide a novel means to improve intestinal immunophysiology in premature neonates.

Exploring the interplay of barrier function and leukocyte recruitment in intestinal inflammation by targeting fucosyltransferase VII and trefoil factor 3.

Intestinal mucosal integrity is dependent on epithelial function and a regulated immune response to injury. Fucosyltransferase VII (Fuc-TVII) is an essential enzyme required for the expression of the functional ligand for E- and P-selectin. Trefoil factor 3 (TFF3) is involved in both protecting the intestinal epithelium against injury as well as aiding in wound repair following injury. The aim of the present study was to assess the interplay between barrier function and leukocyte recruitment in intestinal inflammation. More specifically, we aimed to examine how targeted disruption of Fuc-TVII either in wild-type or TFF3(-/-) mice would alter their susceptibility to colonic injury. TFF3 and Fuc-TVII double-knockout mice (TFF3/Fuc-TVII(-/-) mice) were generated by mating TFF3(-/-) and Fuc-TVII(-/-) mice. Colitis was induced by administration of dextran sodium sulfate (DSS) (2.5% wt/vol) in the drinking water. Changes in baseline body weight, diarrhea, and fecal blood were assessed daily. Upon euthanasia, extents of colonic inflammation were assessed macroscopically, microscopically, and through quantification of myeloperoxidase (MPO) activity. Colonic lymphocyte subpopulations were assessed at 6 days after administration of DSS by flow cytometry and immunohistochemistry. No baseline intestinal inflammation was found in TFF3/Fuc-TVII(-/-), TFF3(-/-), Fuc-TVII(-/-), or wild-type mice. Loss of Fuc-TVII resulted in a reduction in disease severity whereas TFF3(-/-) mice were markedly more susceptible to DSS-induced colitis. Remarkably, the loss of Fuc-TVII in TFF3(-/-) mice markedly decreased the severity of DSS-induced colitis as evidenced by reduced weight loss, diarrhea, decreased colonic MPO levels and improved survival. Furthermore, the loss of TFF3 resulted in increased severity of spontaneous colitis in IL-2/beta-microglobulin-deficient mice. These studies highlight the importance of the interplay between factors involved in the innate immune response, mucosal barrier function, and genes involved in regulating leukocyte recruitment and other aspects of the immune response.

Bacterial symbionts induce a FUT2-dependent fucosylated niche on colonic epithelium via ERK and JNK signaling.

The intestinal epithelium of the adult gut supports a complex, dynamic microbial ecosystem and expresses highly fucosylated glycans on its surface. Uncolonized gut contains little fucosylated glycan. The transition toward adult colonization, such as during recovery from germ-free status or from antibiotic treatment, increased expression of fucosylated epitopes in the colonic epithelium. This increase in fucosylation is accompanied by induction of fut2 mRNA expression and alpha1,2/3-fucosyltransferase activity. Colonization stimulates ERK and JNK signal transduction pathways, resulting in activation of transcription factors ATF2 and c-Jun, respectively. This increases transcription of fut2 mRNA and expression of alpha1,2/3-fucosyltransferase activity, resulting in a highly fucosylated intestinal mucosa characteristic of the adult mammalian gut. Blocking the ERK and JNK signaling cascade inhibits the ability of colonization to induce elevated fut2 mRNA and fucosyltransferase activity in the mature colon. Thus pioneer-mutualist symbiotic bacteria may utilize the ERK and JNK signaling cascade to induce the high degree of fucosylation characteristic of adult mammalian colon, and we speculate that this fucosylation facilitates colonization by adult microbiota.

Secreted probiotic factors ameliorate enteropathogenic infection in zinc-deficient human Caco-2 and T84 cell lines.

Zinc deficiency enhances infectious diarrhea whereas probiotics may inhibit pathogen enterocyte invasion. The effect of probiotics on zinc-deficient versus normal human intestinal epithelium (Caco-2 and T-84) with regard to invasion and subsequent inflammatory response by Salmonella typhimurium was determined. Cells were infected with pathogens and preincubated with media conditioned by several lactobacilli or Bifidobacterium bifidum 12. Pathogen invasion was quantified, inflammation was determined by IL-8 secretion, and MAP kinase activation in the epithelium was analyzed. Probiotic inhibiting factors were partially characterized based on physicochemical properties. Zinc deficiency allowed for greater pathogen invasion and enhanced IL-8 secretion. Probiotic conditioned media reduced activation of proinflammatory signaling via the ERK and p38 pathway. Probiotic factors reverse increased susceptibility of zinc-deficient enterocytes to S. typhimurium invasion, suggesting an additive protective effect of probiotics in zinc deficiency. Probiotic conditioned media but not bacteria inhibited pathogen invasion and IL-8 production in zinc deficient enterocytes. Probiotic inhibitory factors are stable to treatment with proteases, deoxyribonucleases (DNAses), ribonucleases (RNAse), strong acid, and heat.

Regulation of intestinal ontogeny: effect of glucocorticoids and luminal microbes on galactosyltransferase and trehalase induction in mice.

Intestinal maturation can be influenced by intrinsic factors (glucocorticoid hormones) and by extrinsic factors (resident microflora); their relative roles in ontogeny of mouse intestinal trehalase expression, a marker of general gut development, and of beta1,4-galactosyltransferase (beta GT), a marker of glycosyltransferase development, were investigated. In conventional (CONV) mice, beta GT and trehalase gene expression rapidly increased to adult levels by the fourth postnatal week. In germ-free (GF) mice, beta GT expression remained at initial low levels and was rapidly induced on reintroduction of luminal microbes of the adult gut but not of microbes characteristic of the suckling gut. Similar developmental patterns were observed for colonic galactosyl beta1,4-linked glycoconjugates, products of beta GT activity. These results indicate an essential role for microbes in the ontogeny of beta GT. In both CONV and GF mice, cartisone acetate (CA) precociously accelerated the ontogeny of beta GT and trehalase until maturation of the gut occurred (day 22). In the mature gut of CONV mice, both beta GT and trehalase are elevated and insensitive to CA; in GF mature mice, the expression of beta GT remains low, whereas the expression of trehalase was at mature levels, regardless of CA treatment. These changes in enzyme activity were accompanied by parallel changes in mRNA, implying transcriptional regulation. Thus both microbes and cortisone regulate gut ontogeny, but only suckling gut responds to CA, an intrinsic factor, whereas adult gut beta GT expression remains sensitive to microflora, an extrinsic factor. However, induction of the adult pattern of glycosyltransferase expression in mature gut requires colonization by microflora typical of adult gut, suggesting an essential role for intestinal colonization in the ontogeny of normal intestinal mucosal cell surface glycoconjugate receptors.

Glucocorticoid responsiveness in developing human intestine: possible role in prevention of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a major inflammatory disease of the premature human intestine that can be prevented by glucocorticoids if given prenatally before the 34th wk of gestation. This observation suggests that a finite period of steroid responsiveness exists as has been demonstrated in animal models. Human intestinal xenografts were used to determine whether a glucocorticoid responsive period exists in the developing human intestine. Developmental responsiveness was measured by lactase activity and inflammatory responsiveness by IL-8, IL-6, and monocyte chemotactic protein-1 (MCP-1) induction after an endogenous (IL-1 beta) or exogenous (LPS) proinflammatory stimulus, respectively. Functional development of ileal xenografts were monitored for 30 wk posttransplantation, and the lactase activity recapitulated that predicted by in utero development. Cortisone acetate accelerated the ontogeny of lactase at 20 wk (immature) but the effect was lost by 30 wk (mature) posttransplant. Concomitant with accelerated maturation, the IL-8 response to both IL-1 beta and LPS was significantly dampened (from 6- to 3-fold) by glucocorticoid pretreatment in the immature but not mature xenografts. The induction of IL-8 was reflected at the level of IL-8 mRNA, suggesting transcriptional regulation. The excessive activation of IL-8 in the immature gut was mediated by a prolonged activation of ERK and p38 kinases and nuclear translocation of NF-kappa B due to low levels of I kappa B. Steroid pretreatment in immature intestine dampens activation of all three signaling pathways in response to proinflammatory stimuli. Therefore, accelerating intestinal maturation by glucocorticoids within the responsive period by accelerating functional and inflammatory maturation may provide an effective preventive therapy for NEC.

Normal and glucocorticoid-induced development of the human small intestinal xenograft.

The aim of this study was to determine whether intestinal xenografts could recapitulate human in utero development by using disaccharidases as markers. Twenty-week-old fetal intestine was transplanted into immunocompromised mice and was followed. At 20-wk of gestation, the fetal human intestine was morphologically developed with high sucrase and trehalase but had low lactase activities. By 9-wk posttransplantation, jejunal xenografts were morphologically and functionally developed and were then monitored for

The role of indigenous microflora in the development of murine intestinal fucosyl- and sialyltransferases.

Most enteric bacteria use intestinal brushborder glycoconjugates as their target host cell receptors. It has been postulated that resident microbes regulate specific glycosyltransferases that are responsible for synthesizing brushborder glycoconjugates. To investigate this hypothesis, we measured glycosyltransferase enzyme activities in intestine from different regions of maturing conventional (CONV), germ-free (GF), and ex-germ-free (XGF) mice and compared them to general enzyme markers of gut development, for example, disaccharidases. High alpha2,3/6-Sialyltransferase (ST) activity and low alpha1,2-fucosyltransferase (FT) activities were detected from duodenum to colon in suckling CONV mice, but the relative levels of these activities reversed during the third postnatal wk, rapidly reaching adult levels by the fourth wk. These age-related enzyme changes were significantly attenuated in GF mice, maintaining an immature pattern well past 3 wk. Introduction of gut microflora in GF mice rapidly initiated maturation of glycosyltransferase activity but had no significant affect on developmental programming of dissacharidases. Therefore, in mice, intestinal glycosyltransferase activities are under tissue and developmental control and microflora play a major role in their specific ontogeny but not in overall development. These findings may help explain the regional specificity of commensal bacteria and of enteric pathogens and may also relate age-related changes in microflora to susceptibility to enteropathogens.

Region-specific ontogeny of alpha-2,6-sialyltransferase during normal and cortisone-induced maturation in mouse intestine.

Regional differences in the ontogeny of mouse intestinal alpha-2,6-sialyltransferase activities (alpha-2,6-ST) and the influence of cortisone acetate (CA) on this expression were determined. High ST activity and alpha-2,6-ST mRNA levels were detected in immature small and large intestine, with activity increasing distally from the duodenum. As the mice matured, ST activity (predominantly alpha-2,6-ST) in the small intestine decreased rapidly to adult levels by the fourth postnatal week. CA precociously accelerated this region-specific ontogenic decline. A similar decline of ST mRNA levels reflected ST activity in the small, but not the large, intestine. Small intestinal sialyl alpha-2,6-linked glycoconjugates displayed similar developmental and CA induced-precocious declines when probed using Sambucus nigra agglutinin (SNA) lectin. SNA labeling demonstrated age-dependent diminished sialyl alpha2,6 glycoconjugate expression in goblet cells in the small (but not large) intestine, but no such regional specificity was apparent in microvillus membrane. This suggests differential regulation of sialyl alpha-2,6 glycoconjugates in absorptive vs. globlet cells. These age-dependent and region-specific differences in sialyl alpha-2,6 glycoconjugates may be mediated in part by altered alpha-2,6-ST gene expression regulated by trophic factors such as glucocorticoids.

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.

Inflammation in the developing human intestine: A possible pathophysiologic contribution to necrotizing enterocolitis.

Necrotizing enterocolitis (NEC), a major cause of morbidity and mortality in premature infants, occurs after the introduction of oral feedings in conjunction with initial bacterial colonization of the gut and is hypothesized to be due to an immature (inappropriate) enterocyte response to bacterial stimuli. To test this hypothesis, we compared the enterocyte IL-8 response to inflammatory stimuli [lipopolysaccharide (LPS) and IL-1beta] in immature vs. mature human small intestine. Initial in vitro studies comparing confluent Caco-2 cells, a model for mature human enterocytes, with a primary human fetal intestinal cell line (H4 cells) demonstrated that after inflammatory stimulation fetal cells secreted more IL-8 (LPS, 8-fold; IL-1beta, 20-fold) than Caco-2 cells. IL-8 mRNA activity in fetal compared to Caco-2 cells was proportionately increased by the same magnitude with both stimuli. To validate the in vitro observations, small intestinal organ cultures from fetuses vs. older children were exposed to LPS and IL-1beta. Again in human organ cultures from fetuses compared to older children, IL-8 secretion was greater (LPS, 2.5-fold; IL-1beta, 200-fold) and mRNA activity after stimulation was comparably higher, suggesting that increased transcription of the IL-8 gene may account for the excessive response. Using immunohistochemical staining to identify the cellular source of IL-8, activity was noted predominantly in villous and crypt epithelium but also in a few immunoresponsive lymphoid cells. The observation that immature human enterocytes react with excessive pro-inflammatory cytokine production after inflammatory stimulation may help in part explain why prematures exposed to initial colonizing bacteria develop necrotizing enterocolitis.

Rat trehalase: cDNA cloning and mRNA expression in adult rat tissues and during intestinal ontogeny.

A partial rat trehalase cDNA has been cloned and used to examine trehalase mRNA expression. Northern blotting with total RNA from 11 adult rat tissues showed a trehalase transcript only in small intestine, where it was abundant in proximal regions but declined steeply toward the ileum. During development, trehalase mRNA was not detectable in jejunum until postnatal day 19 and then increased markedly through day 25. Modest levels in trehalase mRNA were induced precociously by administration of dexamethasone, with increasing responsiveness evident between the first and second postnatal weeks. In contrast, analysis of sucrase-isomaltase mRNA on the same blots showed maximal induction at both ages. In adrenalectomized animals, the ontogenic increase of trehalase mRNA began as usual but proceeded more slowly than in control animals. Overall, trehalase mRNA expression in the rat displayed both similarities and differences compared with rabbit. Moreover, the differences revealed in glucocorticoid responsiveness of trehalase mRNA and sucrase-isomaltase mRNA suggest that the actions of these hormones on the developing intestine may be more complex than previously recognized.

Expression of a constitutively active Ca2+/calmodulin-dependent kinase in Aspergillus nidulans spores prevents germination and entry into the cell cycle.

The unique gene for Ca2+/calmodulin-dependent protein kinase (CaMK) has been shown to be essential in Aspergillus nidulans. Disruption of the gene prevents entry of spores into the nuclear division cycle. Here we show that expression of a constitutively active form of CaMK also prevents spores from entering the first S phase in response to a germinating stimulus. Expression of the constitutively active kinase induces premature activation of NIMEcyclin B/NIMXcdc2 in G0/G1. As NIMXcdc2 is present in spores, the elevation of maturation promotion factor activity may be secondary to the early production of NIMEcyclin B or post-translation modification of maturation promotion factor. The expression of the constitutively active CaMK also results in the appearance of NIMA kinase activity within 1 h of the germinating signal. These results support the contention that the activities of maturation promotion factor and NIMA are coincidentally regulated in A. nidulans and suggest that the unscheduled appearance of one or both of these activities may be sufficient to prevent A. nidulans spores from entering into DNA synthesis.

Role of Ca++/calmodulin binding proteins in Aspergillus nidulans cell cycle regulation.

The goal of this review is to summarise the current knowledge concerning the targets of Ca++/calmodulin that are essential for cell cycle progression in lower eukaryotes. Emphasis is placed on Aspergillus nidulans since this is the only organism to date shown to posses essential Ca++ dependent calmodulin activated enzymes. Two such enzymes are the calmodulin activated protein phosphatase, calcineurin and the calmodulin dependent protein kinase. These proteins, each the product of a unique gene, are required for progression of quiescent spores into the proliferative cycle and also for execution of the nuclear division cycle in exponentially growing germlings.

Distinguishing normal and glucocorticoid-induced maturation of intestine using bromodeoxyuridine.

Exogenous glucocorticoids administered during the first two postnatal weeks are capable of eliciting precocious maturation of the rat intestine. However, it is not known whether this represents an alternative developmental pathway or is essentially an advancement of normal ontogeny. The goal of the present study was to address this question using the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU), which is known to selectively inhibit differentiation in a number of tissues. Intestinal development was assessed by following changes in sucrase, trehalase, glucoamylase, and lactase activities. The first experiment assessed whether BrdU has any influence on the cellular differentiation that occurs continuously along the crypt-villus axis. After administration of BrdU to suckling and mature animals, there was no effect on lactase and sucrase activities, respectively. Thus BrdU does not inhibit crypt-villus differentiation in either the suckling or mature jejunum. In the second experiment, dexamethasone was used to induce precocious maturation in the rat jejunum on day 10. BrdU treatment significantly inhibited glucocorticoid-induced elevation of sucrase, trehalase, and glucoamylase but had no effect on the lactase activity. In contrast, treatment with BrdU during normal development significantly accelerated the ontogenic rise of sucrase and trehalase as well as the ontogenic decline of lactase. The acceleration of development was also seen in adrenalectomized rats, indicating that it is the glucocorticoid-independent component of normal intestinal ontogeny that is activated by BrdU. The opposite effect of BrdU on glucocorticoid-induced precocious maturation suggests that such maturation involves different molecular mediators than normal ontogeny.

Ontogeny of sucrase-isomaltase gene expression in rat intestine: responsiveness to glucocorticoids.

The goal of our study was to determine how sucrase-isomaltase (SI) gene expression in the rat small intestine is modulated by glucocorticoids during the second and third postnatal weeks. SI mRNA was quantitated by Northern blot and was compared with sucrase activities in the same tissue samples. The role of endogenous glucocorticoids was assessed by measuring SI mRNA and enzyme activity in rat pups adrenalectomized (ADX) on postnatal day 9. ADX pups showed a retarded appearance of sucrase activity compared with sham-operated control pups, and the appearance of SI mRNA paralleled the enzyme activity. To determine the role of exogenous glucocorticoids, a saturating dose of dexamethasone (Dex) was administered daily in three series of experiments starting on days 10, 16, and 18. In the day 10 series, Dex caused precocious appearance of both SI mRNA and sucrase activity. In the day 16 series, Dex accelerated the rate of rise of the two parameters, whereas by day 18 there was no significant effect of Dex. To investigate whether the accelerated rise in the day 16 series was associated with changes in epithelial cell kinetics, the location of SI protein was assessed by immunofluorescence staining. The results indicated that the effect of Dex at this age was due to faster emergence of SI-bearing enterocytes from the intestinal crypts.(ABSTRACT TRUNCATED AT 250 WORDS)