Deficiency in X-linked inhibitor of apoptosis protein promotes susceptibility to microbial triggers of intestinal inflammation.

Inflammatory bowel disease (IBD) is characterized by inappropriate immune responses to the microbiota in genetically susceptible hosts, but little is known about the pathways that link individual genetic alterations to microbiota-dependent inflammation. Here, we demonstrated that the loss of X-linked inhibitor of apoptosis protein (XIAP), a gene associated with Mendelian IBD, rendered Paneth cells sensitive to microbiota-, tumor necrosis factor (TNF)­, receptor-interacting protein kinase 1 (RIPK1)­, and RIPK3-dependent cell death. This was associated with deficiency in Paneth cell­derived antimicrobial peptides and alterations in the stratification and composition of the microbiota. Loss of XIAP was not sufficient to elicit intestinal inflammation but provided susceptibility to pathobionts able to promote granulomatous ileitis, which could be prevented by administration of a Paneth cell­derived antimicrobial peptide. These data reveal a pathway critical for host-microbial cross-talk, which is required for intestinal homeostasis and the prevention of inflammation and which is amenable to therapeutic targeting.

Active ß-Catenin Signaling in the Small Intestine of Humans During Infancy.

BACKGROUND: Wnt-ß-catenin signaling is essential for homeostasis of intestinal stem cells in mice and is thought to promote intestinal crypt fission. AIMS: The aim of this study was to investigate Wnt-ß-catenin signaling in intestinal crypts of human infants. METHODS: Duodenal biopsies from nine infants (mean, range 0.9 years, 0.3-2 years) and 11 adults (mean, range 43 years, 34-71 years) were collected endoscopically. Active ß-catenin signaling was assessed by cytoplasmic and nuclear ß-catenin, nuclear c-Myc, and cytoplasmic Axin-2 expression in the base of crypts. Tissues were stained by an immunoperoxidase staining technique and quantified as pixel energy using cumulative signal analysis. Data were expressed as mean ± SD and significance assessed by Student's t test. RESULTS: Crypt fission was significantly higher in infants compared to adults (16 ± 8.6% versus 0.7 ± 0.6%, respectively, p < 0.0001). Expression of cytoplasmic and nuclear ß-catenin was 1.8-fold (p < 0.0001) and 2.9-fold (p < 0.0001) higher in infants, respectively, while cytoplasmic Axin-2 was 3.1-fold (p < 0.0001) increased in infants. c-Myc expression was not significantly different between infants and adults. Expression was absent in Paneth cells but present in the transit amplifying zone of crypts. Crypt base columnar cells, which were intercalated between Paneth cells, expressed c-Myc. CONCLUSIONS: Wnt-ß-catenin signaling was active in crypt base columnar cells (i.e., intestinal stem cells) in human infants. This signaling could promote crypt fission during infancy. Wnt-ß-catenin signaling likely acts in concert with other pathways to promote postnatal growth.

Recipient single nucleotide polymorphisms in Paneth cell antimicrobial peptide genes and acute graft-versus-host disease: analysis of BMT CTN-0201 and -0901 samples.

Host genetics shape the gut microbiota, and gut dysbiosis increases the risk of acute graft-versus-host disease (aGVHD). Paneth cells and microbiota have interactions that contribute to immune regulation. α-defensin-5 (HD5) and regenerating islet-derived protein 3 alpha (Reg3A) are the most abundant Paneth cell antimicrobial peptides (AMPs). We hypothesized that single nucleotide polymorphisms (SNPs) in the genes for HD5 (DEFA5) and Reg3A (REG3A) predict aGVHD risk. We analysed pre-transplant recipient peripheral blood mononuclear cell samples from randomized Blood and Marrow Transplant Clinical Trials Network (BMT CTN) studies 0201 (94 patients with bone marrow and 93 with peripheral blood grafts) and 0901 (86 patients with myeloablative and 77 with reduced-intensity conditioning; all using peripheral blood grafts). In multivariable analysis (with a SNP × graft source interaction term in CTN-0201 and a SNP × conditioning intensity term in CTN-0901), DEFA5 rs4415345 and rs4610776 were associated with altered incidence of aGVHD grade II-IV [rs4415345 G vs. C: hazard ratio (HR) 0·58, 95% confidence interval (95% CI) 0·37-0·92, P = 0·02; rs4610776 T vs. A: HR 1·53, 95% CI 1·01-2·32, P = 0·05] in CTN-0201, but not CTN-0901, suggesting a stronger effect in bone marrow allografts. REG3A SNP was not associated with aGVHD. Host genetics may influence aGVHD risk by modulating Paneth cell function.

Donkey milk consumption exerts anti-inflammatory properties by normalizing antimicrobial peptides levels in Paneth's cells in a model of ileitis in mice.

PURPOSE: In this study, we showed the beneficial effects of donkey milk (DM) on inflammatory damages, endogenous antimicrobial peptides levels and fecal microbiota profile in a mice model of Crohn's disease. Nowadays, new strategies of microbiome manipulations are on the light involving specific diets to induce and/or to maintain clinical remission. Interest of DM is explained by its high levels of antimicrobial peptides which confer it anti-inflammatory properties. METHODS: C57BL/6 mice were orally administered with or without indomethacin for 5 days and co-treated with vehicle, DM or heated DM during 7 days. Intestinal length and macroscopic damage scores (MDSs) were determined; ileal samples were taken off for microscopic damage (MD), lysozyme immunostaining and mRNA α-defensin assessments. Ileal luminal content and fecal pellets were collected for lysozyme enzymatic activity and lipocalin-2 (LCN-2) evaluations. Fecal microbiota profiles were compared using a real-time quantitative PCR-based analysis. RESULTS: Administration of indomethacin caused an ileitis in mice characterized by (1) a decrease in body weight and intestinal length, (2) a significant increase in MDS, MD and LCN-2, (3) a reduction in both α-defensin mRNA expression and lysozyme levels in Paneth's cells reflected by a decrease in lysozyme activity in feces, and (4) a global change in relative abundance of targeted microbial communities. DM treatment significantly reduced almost of all these ileitis damages, whereas heated DM has no impact on ileitis. CONCLUSIONS: DM consumption exerts anti-inflammatory properties in mice by restoring the endogenous levels of antimicrobial peptides which contribute in turn to reduce microbiota imbalance.

Pro-inflammatory and pro-apoptotic properties of Human Defensin 5.

Defensins are cationic antimicrobial peptides that play an important role in innate immunity by primarily acting against microbes. Their antimicrobial properties have been widely studied and are well understood. Defensins contribute to regulation of host immunity also. Their effects on cells of the host however are less well understood. Here, we report on the pro-inflammatory and apoptotic properties of Human Defensin 5, the major antimicrobial peptide of ileal Paneth cells. We find that HD-5 up-regulates expression of genes involved in cell survival and inflammation in a NF-kB-dependent fashion in epithelial cells. Further, we find that HD-5 has pro-apoptotic effects on intestinal epithelial cells as well as primary CD4+ T cells.

[Structure and function of enteric alpha defensins in norm and pathology].

This review summarizes currently available data on enteric alpha defensins structure, their functions in the innate and adaptive immunity systems and the role in development of intestinal illnesses.

Identification of a cKit(+) colonic crypt base secretory cell that supports Lgr5(+) stem cells in mice.

BACKGROUND & AIMS: Paneth cells contribute to the small intestinal niche of Lgr5(+) stem cells. Although the colon also contains Lgr5(+) stem cells, it does not contain Paneth cells. We investigated the existence of colonic Paneth-like cells that have a distinct transcriptional signature and support Lgr5(+) stem cells. METHODS: We used multicolor fluorescence-activated cell sorting to isolate different subregions of colon crypts, based on known markers, from dissociated colonic epithelium of mice. We performed multiplexed single-cell gene expression analysis with quantitative reverse transcriptase polymerase chain reaction followed by hierarchical clustering analysis to characterize distinct cell types. We used immunostaining and fluorescence-activated cell sorting analyses with in vivo administration of a Notch inhibitor and in vitro organoid cultures to characterize different cell types. RESULTS: Multicolor fluorescence-activated cell sorting could isolate distinct regions of colonic crypts. Four major epithelial subtypes or transcriptional states were revealed by gene expression analysis of selected populations of single cells. One of these, the goblet cells, contained a distinct cKit/CD117(+) crypt base subpopulation that expressed Dll1, Dll4, and epidermal growth factor, similar to Paneth cells, which were also marked by cKit. In the colon, cKit(+) goblet cells were interdigitated with Lgr5(+) stem cells. In vivo, this colonic cKit(+) population was regulated by Notch signaling; administration of a γ-secretase inhibitor to mice increased the number of cKit(+) cells. When isolated from mouse colon, cKit(+) cells promoted formation of organoids from Lgr5(+) stem cells, which expressed Kitl/stem cell factor, the ligand for cKit. When organoids were depleted of cKit(+) cells using a toxin-conjugated antibody, organoid formation decreased. CONCLUSIONS: cKit marks small intestinal Paneth cells and a subset of colonic goblet cells that are regulated by Notch signaling and support Lgr5(+) stem cells.

Evaluation of amoebicidal potential of Paneth cell cryptdin-2 against Entamoeba histolytica.

BACKGROUND: Amoebiasis is a major public health problem in tropical and subtropical countries. Currently, metronidazole is the gold choice medication for the treatment of this disease. However, reports have indicated towards the possibility of development of metronidazole-resistance in Entamoeba strains in near future. In view of the emergence of this possibility, in addition to the associated side effects and mutagenic ability of the currently available anti-amoebic drugs, there is a need to explore newer therapeutics against this disease. In this context, the present study evaluated the amoebicidal potential of cryptdin-2 against E. histolytica. METHODS/PRINCIPAL FINDINGS: In the present study, cryptdin-2 exhibited potent in-vitro amoebicidal activity against E. histolytica in a concentration dependent manner at a minimum amoebicidal concentration (MAC) of 4 mg/L. Scanning electron microscopy as well as phase contrast microscopic investigations of cryptdin-2 treated trophozoites revealed that the peptide was able to induce significant morphological alterations in terms of membrane wrinkling, leakage of the cytoplasmic contents and damaged plasma membrane suggesting a possible membrane dependent amoebicidal activity. N-phenyl napthylamine (NPN) uptake assay in presence of sulethal, lethal as well as twice the lethal concentrations further confirmed the membrane-dependent mode of action of cryptdin-2 and suggested that the peptide could permeabilize the plasma membrane of E. histolytica. It was also found that cryptdin-2 interfered with DNA, RNA as well as protein synthesis of E. histolytica exerting the highest effect against DNA synthesis. Thus, the macromolecular synthesis studies correlated well with the observations of membrane permeabilization studies. SIGNIFICANCE/CONCLUSIONS: The amoebicidal efficacy of cryptdin-2 suggests that it may be exploited as a promising option to combat amoebiasis or, at least, may act as an adjunct to metronidazole and/or other available anti-amoebic drugs.

Biosynthesis and antimicrobial evaluation of backbone-cyclized α-defensins.

Defensins are antimicrobial peptides that are important in the innate immune defense of mammals. Upon stimulation by bacterial antigens, enteric α-defensins are secreted into the intestinal lumen where they have potent microbicidal activities. Cryptdin-4 (Crp4) is an α-defensin expressed in Paneth cells of the mouse small intestine and the most bactericidal of the known cryptdin isoforms. The structure of Crp4 consists of a triple-stranded antiparallel ß-sheet but lacks three amino acids between the fourth and fifth cysteine residues, making them distinct from other α-defensins. The structure also reveals that the α-amino and C-terminal carboxylic groups are in the proximity of each other (d ≈ 3 Å) in the folded structure. We present here the biosynthesis of backbone-cyclized Crp4 using a modified protein splicing unit or intein. Our data show that cyclized Crp4 can be biosynthesized by using this approach both in vitro and in vivo, although the expression yield was significantly lower when the protein was produced inside the cell. The resulting cyclic defensins retained the native α-defensin fold and showed equivalent or better microbicidal activities against several Gram-positive and Gram-negative bacteria when compared to native Crp4. No detectable hemolytic activity against human red blood cells was observed for either native Crp4 or its cyclized variants. Moreover, both forms of Crp4 also showed high stability to degradation when incubated with human serum. Altogether, these results indicate the potential for backbone-cyclized defensins in the development of novel peptide-based antimicrobial compounds.

Activation of REG family proteins in colitis.

AIMS: To do a genome-wide gene expression study of active and inactive ulcerative colitis and Crohn's disease (inflammatory bowel disease--IBD) and examine the most differentially expressed genes. As the study showed an extreme upregulation of all regenerating islet-derived genes (REG proteins) in active IBD, we further studied the expression of REGs on protein level in active and inactive IBD, as well as in non-IBD (pseudomembranous) colitis. METHODS: Microarray analysis was done on a total of 100 pinch biopsy samples from healthy controls and patients with Crohn's disease or ulcerative colitis. Tissue samples from IBD and pseudomembranous colitis were examined with routine histology and immunohistochemical analysis for REGIα, REGIV, DEFA6, and serotonin. RESULTS: REG mRNAs were up to 83 times overexpressed in diseased mucosa compared with mucosa from healthy individuals. REGIα and REGIV were overexpressed at immunohistochemistry and located to different mucosal cell types. REGIα was expressed in basal half of crypts, REGIV in mid and outer parts of crypts and in surface epithelium and seems to be stored in, and secreted from, goblets. Pseudomembranous colitis samples showed similar staining patterns, and some IBD samples stained REG positive without inflammation on routine histology. CONCLUSIONS: All REG family mRNAs are upregulated in IBD. REGIα and REGIV have different cellular localization, possibly reflecting different biological functions. REG protein expression also in pseudomembranous colitis shows that REG family proteins are regulated in inflammatory injury and repair, not specifically for IBD as previously thought.

CD1d expression in paneth cells and rat exocrine pancreas revealed by novel monoclonal antibodies which differentially affect NKT cell activation.

BACKGROUND: CD1d is a nonpolymorphic MHC class I-like molecule which presents nonpeptide ligands, e.g. glycolipids, to NKT cells. These cells are known to have multiple effects on innate and adaptive immune responses and on the development of pathological conditions. In order to analyze CD1d expression and function in the rat, the first rat CD1d-specific monoclonal antibodies (mAbs) were generated. METHODOLOGY/PRINCIPAL FINDINGS: Two mAbs, WTH-1 and WTH-2, were generated which bound equally well to cell surface-expressed rat and mouse CD1d. Their non-overlapping epitopes were mapped to the CD1d heavy chain. Flow cytometry and immunohistological analyses revealed a nearly identical degree and pattern of CD1d expression for hematopoieitic cells of both species. Notable is also the detection of CD1d protein in mouse and rat Paneth cells as well as the extremely high CD1d expression in acinar exocrine cells of the rat pancreas and the expression of CD4 on rat marginal zone B cells. Both mAbs blocked α-galactosylceramide recognition by primary rat and mouse NKT cells. Interestingly, the two mAbs differed in their impact on the activation of various autoreactive T cell hybridomas, including the XV19.2 hybridoma whose activation was enhanced by the WTH-1 mAb. CONCLUSIONS/SIGNIFICANCE: The two novel monoclonal antibodies described in this study, allowed the analysis of CD1d expression and CD1d-restricted T cell responses in the rat for the first time. Moreover, they provided new insights into mechanisms of CD1d-restricted antigen recognition. While CD1d expression by hematopoietic cells of mice and rats was extremely similar, CD1d protein was detected at not yet described sites of non-lymphatic tissues such as the rat exocrine pancreas and Paneth cells. The latter is of special relevance given the recently reported defects of Paneth cells in CD1d(-/-) mice, which resulted in an altered composition of the gut flora.

Response of crypt paneth cells in the small intestine following total-body gamma-irradiation.

Ionizing irradiation causes damage and functional failure of irradiation-sensitive systems and tissues such as small intestine. The molecular mechanisms underlying inflammatory and adaptive responses to acute irradiation damage are poorly understood. Using a mouse model of total-body γ-irradiation, we assessed the irradiation response of crypt host-defense Paneth cells by measuring alpha-defensin 4 (AD4) expression and correlated the gathered data with activation of the caspase-1/IL-1ß inflammatory signaling cascade. The irradiation injury was produced in CD2F1 mice exposed to 9.25 Gy γ-radiation. This dose resulted in 85-100 percent mortality at the 15(th) day post-irradiation. Small intestine tissue samples were collected at the 7th day post-irradiation. Assessment of irradiation-associated pro-inflammatory alterations in small intestine tissue and expression of AD4 in Paneth cells was conducted using confocal immunofluorescence imaging, transmission electron microscopy (TEM), light microscopy, and immunoblotting techniques. The small intestine analysis revealed an increase in the precursor form of IL-1ß, the activated form of IL-1ß, and the activated form of caspase-1 (p10 CASP-1) at the 7(th) day post-irradiation. Immunoprecipitation analysis showed increased interaction between IL-1ß and p10 CASP-1 after irradiation. This effect was observed in the irradiated small intestine and CD15-positive Paneth cells using confocal imaging techniques. The pro-inflammatory alterations in Paneth cells were accompanied by increases in AD4 mRNA and its 8 kD peptide product. Paneth cell secretory activity was observed at the sites of bacterial translocation in the crypt lumens. These data suggest that Paneth cells can contribute to small intestine inflammatory remodeling during the post-irradiation period.

Intelectin: a novel lipid raft-associated protein in the enterocyte brush border.

Intelectin is a mammalian Ca2+-dependent, D-galactosyl-specific lectin expressed in Paneth and goblet cells of the small intestine and proposed to serve a protective role in the innate immune response to parasite infection. In addition, it is structurally identical to the intestinal lactoferrin receptor known to reside in the enterocyte brush border. To clarify this apparent discrepancy with regard to localization, the aim of this work was to study the cellular and subcellular distribution of small intestinal intelectin by immunofluorescence and immunogold electron microscopy. Secretory granules of lysozyme-positive Paneth cells in the bottom of the crypts as well as goblet cells along the crypt-villus axis were intensively labeled with intelectin antibodies, but quantitatively, the major site of intelectin deposition was the enterocyte brush border. This membrane is organized in stable glycolipid-based lipid raft microdomains, and like the divalent lectin galectin-4, intelectin was enriched in microvillar "superrafts", i.e., membranes that resist solubilization with Triton X-100 at 37 degrees C. This strategic localization suggests that the trimeric intelectin, like galectin-4, serves as an organizer and stabilizer of the brush border membrane, preventing loss of digestive enzymes to the gut lumen and protecting the glycolipid microdomains from pathogens.

Adenomyoma with goblet and Paneth cells of the ileum.

A case of ileal adenomyoma with goblet and Paneth cells is reported. A 75-year-old man died of ruptured hepatocellular carcinoma. As an incidental finding at autopsy, a 9 x 7 x 6 mm(3)-sized nodule was found in the ileal wall. Histologically, the lesion occupied the submucosa and muscularis propria, and consisted of glandular structures of various sizes and interlacing smooth muscle bundles surrounding the glandular elements. Goblet cells and Paneth cells were interspersed in the glandular element. Immunohistochemically, the glandular element was positive for cytokeratin (CK) 7 and negative for CK 20. This is the first reported case of adenomyoma of the gastrointestinal tract that contained Paneth cells. The result of the immunohistochemical staining favored the heterotopic pancreas theory concerning its pathogenesis. The appearance of goblet and Paneth cells might be the result of metaplasia.

Paneth cell carcinoma of the ampulla of Vater.

We describe a Paneth cell carcinoma arising within the ampulla of Vater in a 64-year-old man. The phenotype of virtually all neoplastic cells was consistent with that of Paneth cells, based on routine morphology and their strong positive immunostaining for lysozyme. Additional widespread positive immunostaining for carcinoembryonic antigen and CA 19.9 supports a totipotential cell as the origin of such neoplastic cells. This case, therefore, represents a true Paneth cell carcinoma, as opposed to inclusion of occasional neoplastic Paneth cells into a poorly differentiated adenocarcinoma. This pattern of differentiation is rare, and predictions regarding its ultimate biological behavior and malignant potential must be guarded.

The role of Paneth cells and their antimicrobial peptides in innate host defense.

The intestinal epithelium is the largest surface area that is exposed to various pathogens in the environment, however, in contrast to the colon the number of bacteria that colonize the small intestine is extremely low. Paneth cells, one of four major epithelial cell lineages in the small intestine, reside at the base of the crypts and have apically oriented secretory granules. These granules contain high levels of antimicrobial peptides that belong to the alpha-defensin family. Paneth cells secrete these microbicidal granules that contain alpha-defensins when exposed ex vivo to bacteria or their antigens, and recent evidence reveals that antimicrobial peptides, particularly alpha-defensins, that are present in Paneth cells contribute to intestinal innate host defense.

Crohn's disease and the NOD2 gene: a role for paneth cells.

BACKGROUND & AIMS: The NOD2 gene, which is strongly associated with susceptibility to Crohn's disease (CD) of the terminal ileum, interacts with bacterial lipopolysaccharide (LPS), inducing cellular activation. However, the mechanisms by which NOD2 mutations cause terminal ileitis are unknown, and NOD2 is expressed most highly by peripheral blood monocytes, which are distributed ubiquitously and readily respond to LPS via cell-surface receptors. Paneth cells on the other hand, are most numerous in the terminal ileum, are critically important in enteric antibacterial defense, and respond to LPS through as yet undefined pathways. We therefore determined if these specialized intestinal epithelial cells also expressed the NOD2 gene. METHODS: In situ hybridization, immunohistochemistry, and laser-capture microdissection were used to determine RNA and protein expression in tissue sections, and real-time reverse-transcription polymerase chain reaction (RT-PCR) was used to quantitate gene expression in intestinal epithelial cells and peripheral blood mononuclear cells. RESULTS: NOD2 was detected readily in monocytes, but not in mature macrophages in the lamina propria or within granulomas, and levels declined as monocytes differentiated into macrophages in vitro, so that Caco-2 cells expressed more NOD2 mRNA than macrophages. NOD2 mRNA was enriched in crypts compared with villi, and in situ, Paneth cells were the most prominent cells expressing NOD2 in normal and CD-affected intestinal tissue, where they also strongly expressed tumor necrosis factor alpha (TNFalpha) RNA. CONCLUSIONS: The NOD2 gene product is most abundant in Paneth cells in the terminal ileum, which could therefore play a critical and hitherto unrecognized role in the pathogenesis of NOD2-associated CD.

Isolation and characterization of rabbit single chain antibodies to human Reg Ialpha protein.

To investigate the role of Reg Ialpha in human inflammatory bowel disease (IBD), we made two phage-displayed single chain variable fragment (scFv) libraries from rabbits immunized with recombinant or native human Reg Ialpha. After one to three rounds of panning, we were able to isolate phage-displaying scFvs, which bound to human Reg Ialpha. Anti-Reg Ialpha scFvs from both libraries showed similar immunoreactivity to different processed forms of the protein. Despite several DNA fingerprint patterns among these clones, their deduced amino acid sequences are highly homologous with 100% identity in the complementarity-determining regions (CDRs) of the variable segment of heavy chain (VH) region and a small variation in the CDR1 of the variable segment of light chain (VL) region. We also expressed and purified soluble myc-tagged or glutathione S-transferase (GST) fusion scFv proteins from bacteria. Immunohistochemical studies using one of our anti-Reg Ialpha scFv antibodies showed prominent staining in the metaplastic Paneth cell population and light staining in the lamina propria. This scFv antibody is now being used for studies of the role of Reg Ialpha in human IBD.

5-HT(2A) receptors: location and functional analysis in intestines of wild-type and 5-HT(2A) knockout mice.

The distribution and function of the 5-hydroxytryptamine (5-HT(2A)) receptor were investigated in the intestines of wild-type (5-HT(2A) +/+) and knockout (5-HT(2A) -/-) mice. In 5-HT(2A) +/+ mice, rats, and guinea pigs, 5-HT(2A) receptor immunoreactivity was found on circular and longitudinal smooth muscle cells, neurons, enterocytes, and Paneth cells. Muscular 5-HT(2A) receptors were concentrated in caveolae; neuronal 5-HT(2A) receptors were found intracellularly and on the plasma membranes of nerve cell bodies and axons. Neuronal 5-HT(2A) immunoreactivity was detected as early as E14 in ganglia, intravillus nerves, and the deep muscle plexus. The 5-HT(2A) -/- colon did not express 5-HT(2A) receptors and did not contract in response to exogenous 5-HT. 5-HT(2A) -/- enterocytes were smaller, Paneth cells fewer, and muscle layers thinner (and showed degeneration) compared with those of 5-HT(2A) +/+ littermates. The 5-HT(2A) receptor may thus be required for the maintenance and/or development of enteric neuroeffectors and other enteric functions, although gastrointestinal and colonic transit times in 5-HT(2A) -/- and +/+ mice did not differ significantly.

Identification of Paneth cells in pyloric glands associated with gastric and intestinal mixed-type intestinal metaplasia of the human stomach.

We have proposed that intestinal metaplasia (IM) of the human stomach be divided into two types on the basis of cell differentiation status: a gastric and intestinal (GI) mixed type and a solely intestinal (I) type. In the GI mixed type, gastric (foveolar epithelial and pyloric gland cells) and intestinal (goblet, intestinal absorptive, and Paneth cells) phenotype cells coexist in the same intestinalized gastric glands in various combinations and degrees. Consequently, intestinalized gastric glands are hybrids. Although we have described the rare appearance of Paneth-like cells in pyloric glands of GI mixed-type IM, the absence of an appropriate Paneth cell marker leaves room for doubt as to their true character. The purpose of this study was to clearly identify Paneth cells in pyloric glands in IM lesions using a new Paneth cell marker, a polyclonal antibody human defensin (HD)-5, raised against HD-5, which is included in granules of Paneth cells. A total of 105 gastric samples (4 biopsy and 101 surgical resected specimens) were examined. In only nine cases (8.6%), the antibody allowed demonstration of Paneth cells in pyloric glands in GI mixed-type IM, confirming our previous finding. Analysis of the proliferative cell (P) zone indicated that a common stem cell might generate both GI phenotype cells by upward and downward migration. No Paneth cells were found above the P zone. The results suggest that the stem cells show abnormal cell differentiation in IM lesions but preserve their normal direction of migration.