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Non-invasive measurement of colonic blood flow distribution using laser Doppler imaging.

BACKGROUND: This study tested a prototype laser Doppler scanner for the measurement of human colonic blood flow. METHODS: Blood flow distribution was assessed in human colon during operation in six controls and in six patients with inflammatory bowel disease undergoing colectomy. Image processing software analysed several hundred reading points, expressing average flow in perfusion units. RESULTS: Blood flow in the colon was not significantly lower in the control group than in patients with inflammatory bowel disease (mean(s.e.m.) 297.8(24.5) versus 347.2(59.0) perfusion units, P = 0.12). Intestinal colonic mural blood supply was demonstrated up to a distance of 6 cm and ischaemia demarcation lines were identified before the onset of visible changes. CONCLUSIONS: This prototype laser Doppler flowscanner overcomes the previous limitations of laser Doppler flowmeters and may have many clinical and research applications.

Adult↗

A key role for type 1 pili in enterobacterial communicability.

Up to 80% of faecal Escherichia coli strains are able to produce type 1 pili. These filamentous bacterial surface organelles, which mediate mannose-sensitive attachment to mammalian epithelial cells, are also conserved throughout the Enterobacteriaceae. As a potential explanation for their prevalence among intestinal isolates of enteric bacteria, it has been widely speculated that type 1 pili are important for adherence to the host's intestinal mucosa. However, conclusive evidence for this idea is lacking, and there are reasonable grounds for doubting such an effect. Permanent interruption of type 1 piliation in previously pil+ E. coli (by directed mutagenesis of pilA, the gene coding for the major structural subunit of type 1 pili) does not diminish the density of intestinal colonization in individual animals. Rather, as we demonstrate here, this lesion results in a dramatic decrease in transmission of E. coli K1 from experimentally colonized neonatal rats to their littermates. The enhanced communicability associated with type 1 piliation suggests a heretofore unrecognized explanation for the prevalence of type 1 pili among intestinal E. coli; one that does not necessarily require the direct action of these organelles at the intestinal mucosa.

Animals↗

Plasmid-mediated surface fibrillae of Yersinia pseudotuberculosis and Yersinia enterocolitica: relationship to the outer membrane protein YOP1 and possible importance for pathogenesis.

The cell surface properties of Yersinia pseudotuberculosis and Yersinia enterocolitica mutants, constructed by insertional inactivation of genes located on the 40- to 50-megadalton virulence plasmid, were examined. Electron microscopy revealed an absolute correlation between expression of four plasmid-dependent, temperature-inducible properties related to the bacterial surface: (i) a fibrillar matrix covering the outer membrane, (ii) outer membrane protein YOP1, (iii) spontaneous autoagglutination, and (iv) mannose-resistant hemagglutination of guinea pig erythrocytes. Immunoelectron microscopy indicated that YOP1 is a structural component of the fibrillae. Experiments demonstrating inhibition of hemagglutination by anti-YOP1 monoclonal antibody suggested a potential role for YOP1 in adhesion. Insertional inactivation of the gene coding for YOP1, with resultant loss of the ability to express fibrillae, led to a significant reduction in the capacity of Y. enterocolitica, but not Y. pseudotuberculosis, to colonize the ileum of orogastrically infected mice. In both Y. enterocolitica and Y. pseudotuberculosis, inactivation of the genes coding for Ca2+ dependency reduced the ability to maintain intestinal colonization, regardless of the ability to express fibrillae. Both surface fibrillae and Ca2+ dependency seem to reflect pathogenic determinants which are required for the establishment of Y. enterocolitica infection. In Y. pseudotuberculosis, however, no clinical significance of the fibrillae has so far been defined.

Agglutinins↗

Diffuse annular fusiform adenocarcinoma in a dog.

Canine colonic intestinal adenocarcinoma typically presents as rectal polypoid or annular stenotic masses causing clinical signs consistent with large bowel disease. This report discusses an unusual case of intestinal adenocarcinoma in an 11-year-old, neutered male German shepherd dog presented for evaluation of anorexia, profuse watery diarrhea, and weight loss. In this dog, colonic adenocarcinoma diffusely infiltrated the entire large bowel and caused an annular fusiform lesion, as confirmed by endoscopic biopsies and postmortem examination. Other unique features included a paucity of desmoplasia associated with the neoplastic lesion and widespread metastasis to regional lymph nodes, lung, and prostate.

Adenocarcinoma↗

[CK20 expression in the gastrointestinal tract of the embryo and fetus].

A novel type of cytokeratin, cytokeratin 20 (CK20), was added in 1990 to the classic catalog of human cytokeratins, a heterogeneous group of proteins present in almost all epithelia. In man, the expression of CK20 is almost entirely confined to the gastro-intestinal epithelium, to the urothelium and to Merkel cells. Since only few data are available regarding the expression of CK20 in the developing human intestinal mucosa, we studied CK20 immunoreactivity in fetal and neonatal human gut. Immunoreactivity for CK20 was tested in fetuses and newborns, from the twelfth up to the fortieth week of gestation. In each subject, a specimen from the oesophagus, stomach, small intestine, colon, appendix was studied. Tissue samples were routinely processed and paraffin sections were stained with the CK20-specific antibody IT-Ks 20.8. CK20 immunoreactivity was absent in the oesophageal epithelium and it was unevenly distributed in the gastrointestinal mucosa. Three main patterns of immunoreactivity were observed during normal development: the first, found in the stomach and in the small bowel, is characterized by a progressive increase in CK20 expression during gestation; the second pattern, found in the duodenum, shows a progressive decrease in CK20 expression during gestation; in colon and appendix (third pattern), we did not find significant changes in the degree of immunoreactivity for CK20 during gestation. CK20 is unevenly expressed in developing human intestinal mucosa. The degree of positivity for CK20 appears to be related to the epithelial maturation stage only in gastric and small bowel mucosa. Further studies are needed to verify if the uneven CK20 immunoreactivity in the gastrointestinal tract persists even in adulthood.

Biomarkers↗

[The frequency of staphylococcal colonization of the intestines in children with the manifestations of dysbacteriosis].

In 2100 children of different age groups the microbiocenosis of the large intestine was studied. The study revealed that the colonization of the mucous membrane of the large intestine with staphylococci developed in 30% of children with intestinal dysbacteriosis. Young children were mainly affected (91%). The prevailing species among isolated staphylococci was S. aureus (86%), capable of persistence in the intestine (30.9%). In children non typing S. aureus strains mainly circulated (70%), and among phage-typing strains isolates of phage group III prevailed (70.2%). The colonization of the intestine with coagulase-negative staphylococci was possible (14%). Microecological intestinal disturbances in children of different age groups were characterized by different degrees of changes in normal microflora with the prevalence of opportunistic microorganisms in the microbial picture.

Bacteriophage Typing↗

TcpA pilin sequences and colonization requirements for O1 and O139 vibrio cholerae.

The distribution, characterization and function of the tcpA gene was investigated in Vibrio cholerae O1 strains of the El Tor biotype and in a newly emergent non-O1 strain classified as serogroup O139. The V. cholerae tcpA gene from the classical biotype strain O395 was used as a probe to identify a clone carrying the tcpA gene from the El Tor biotype strain E7946. The sequence of the E7946 tcpA gene revealed that the mature El Tor TcpA pilin has the same number of residues as, and is 82% identical to, TcpA of classical biotype strain O395. The majority of differences in primary structure are either conservative or clustered in a manner such that compensatory changes retain regional amino acid size, polarity and charge. In a functional analysis, the cloned gene was used to construct an El Tor mutant strain containing an insertion in tcpA. This strain exhibited a colonization defect in the infant mouse cholera model similar in magnitude to that previously described for classical biotype tcpA mutants, thus establishing an equivalent role for TCP in intestinal colonization by El Tor biotype strains. The tcpA analysis was further extended to both a prototype El Tor strain from the Peru epidemic and to the first non-O1 strain known to cause epidemic cholera, an O139 V. cholerae isolate from the current widespread Asian epidemic. These strains were shown to carry tcpA with a sequence identical to E7946. These results provide further evidence that the newly emergent non-O1 serogroup O139 strain represents a derivative of an El Tor biotype strain and, despite its different LPS structure, shares common TCP-associated antigens.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

Age-dependent variation in the proportion and number of intestinal lymphocyte subsets, especially natural killer T cells, double-positive CD4+ CD8+ cells and B220+ T cells, in mice.

The age-dependent variation in the proportion and number of lymphocyte subsets was examined at various extrathymic sites, including the liver, small intestine, colon and appendix in mice. In comparison with young mice (4 weeks of age), the number of total lymphocytes yielded by all tested organs was greater in adult (9 weeks) and old (40 weeks) mice. The major lymphocyte subset that expanded with age was interleukin-2 receptor (IL-2R) beta+ CD3int cells (50% of them expressed NK1.1) in the liver, whereas it was CD3+ IL-2Rbeta- NK1.1- cells at all intraepithelial sites in the intestine. Although NK1.1+ CD3+ cells were present at intraepithelial sites in the intestine, the proportion of this subset was rather low. The ratio of CD4 to CD8 tended to decrease among natural killer T (NKT) cells and T cells at all intraepithelial sites in the intestine with age. A unique population of double-positive CD4+ CD8+ cells in the small intestine increased in old mice. B220+ T cells were found mainly in the appendix and colon, and the proportion of these T cells decreased in old mice. Conventional NKT cells were very few in Jalpha281-/- and CD1d-/- mice in the liver, while NKT cells which existed in the appendix remained unchanged even in these mice. This was because unconventional CD8+ NKT cells were present in the intestine. The present results suggest that despite the fact that both the liver and intraepithelial sites in the intestine carry many extrathymic T cells, the distribution of lymphocyte subsets and their age-associated variation are site-specific.

Aging↗

Pathogenesis of Campylobacter spp. in athymic and euthymic germfree mice.

Adult athymic (nu/nu) and euthymic (+/nu) germfree BALB/c mice were orally challenged with pure cultures of Campylobacter jejuni (human clinical fecal strains) and a human blood isolate of Campylobacter fetus subsp. fetus. After a period of adaptation to the mouse intestinal tract, all three C. jejuni strains caused disease in gnotobiotic mice. Mouse-adapted, weakly cytotoxic C. jejuni 45100 consistently induced disease symptoms (transient diarrhea, cecal shrinkage, and acute inflammatory changes with eosinophilia in the lower intestinal mucosa) in nu/nu mice 7 to 9 days after oral challenges. Conversely, no overt disease or histopathology was evident in +/nu mice challenged with the same strain (45100). After periods of adaptation in the murine alimentary tract, the two C. jejuni strains, 24 and INN 73-83, with greater cytotoxin-producing capacities, decreased cecal size and caused minor mucosal inflammatory changes in both nu/nu and +/nu BALB/c mice 1 to 2 weeks after intestinal colonization. A transient splenomegaly was also evident at 1 to 2 weeks after germfree nu/nu mice were colonized with each of the three C. jejuni strains used in this study. Occult blood was observed in a small percentage (approximately 11%) of nu/nu and +/nu BALB/c mice that were colonized with C. jejuni strains 45100 and INN 73-83. C. fetus subsp. fetus 255 colonized the alimentary tract of gnotobiotic mice, but neither morbidity nor mortality was evident. The disease we observed in the gnotobiotic mice, along with the histological changes in the intestinal tract after oral challenges, resembles symptoms of campylobacteriosis in humans. The gnotobiotic BALB/c mouse model of Campylobacter disease provides a unique opportunity to detail basic aspects of the acute and chronic pathogenesis of and immunity to this recently recognized disease.

Animals↗

Hyperosmotic saline dextran for resuscitation from traumatic-hemorrhagic hypotension: effect on regional blood flow.

The macro- and microcirculatory effect of small-volume resuscitation with hyperosmotic-hyperoncotic solutions was analyzed in 21 anesthetized beagles subjected to standardized traumatic-hemorrhagic hypotension (laparotomy and exteriorization of the intestine; MAP 40 mmHg for 75 min). Primary resuscitation consisted of bolus infusion of 10% of the blood loss (approx. 4 ml/kg) of either hyperosmotic (7.2%) saline -HSS-, hyperoncotic (10%) dextran 60 -HDS-, or hyperosomotic-hyperoncotic saline dextran (10% dextran 60 in 7.2% saline; HHS). Within 5 min CO was restored and systemic pressure significantly increased. In the HHS-group nutritional blood flow (RBF, measured by radiolabeled microspheres phi 15 microns) in kidneys, gastric mucosa, small intestine, colon, and pancreas was completely restored, while RBF to the myocardium, brain, and skeletal muscles exceeded baseline values. Despite the identical response in central hemodynamics, RBF to gastric mucosa, intestine, pancreas, and kidneys was significantly lower in HSS-animals (P less than 0.05 vs. HHS). In contrast, in the HDS-group CO, splanchnic, myocardial, and renal blood flow remained significantly reduced (P less than 0.05 vs. HHS). Despite the normalization of cardiac output by small volumes of hypertonic solutions, 7.2% saline alone failed to fully restore RBF after protracted traumatic hemorrhage. For the concept of small-volume resuscitation, the hyperosomotic-hyperoncotic solution of 10% dextran 60 in 7.2% saline appears to be most effective to improve organ perfusion during the prehospital period of trauma patients.

Animals↗

The origin of sensory innervation of the peritoneum in the rat.

The distribution of sensory neurons innervating the peritoneum was studied using axonal transport of fluoro-gold. The tracer was injected into parietal peritoneum, diaphragm, mesentery, mesocolon, visceral peritoneum covering the stomach, small intestine, colon, liver, spleen, kidney, urinary bladder or uterus. After ten days of survival bilateral dorsal root ganglia from C2 to S6, and the nodose ganglia were dissected. The cryostat sections of these ganglia were mounted on glass slides and observed with a fluorescence microscope. In cases where the tracer was placed on the peritoneum covering the abdominal wall, labeled neurons were observed only in the ipsilateral dorsal root ganglia. A small number of neurons in nodose and cervical dorsal root ganglia of both sides were labeled after placing the tracer on the central part of the diaphragm. When fluoro-gold was applied to the peripheral part of the diaphragm, nodose ganglion was negative, and dorsal root ganglia from T6 to T12 were positive. Many neurons in the nodose ganglia in addition to somata in the dorsal root ganglia from T4 to T13 were labeled when the tracer was placed on the peritoneum lining the stomach, small intestine or caecum. After applying the tracer onto the colon, labeled neurons were observed in the dorsal root ganglia from T13 to L2 and L5 to S1. Ganglion cells in the nodose and dorsal root ganglia from T5 to T13 were positive when fluoro-gold was placed on the mesentery. No labeled neurons were observed in any ganglia when the tracer was applied to the peritoneum covering the spleen, kidney, uterus, urinary bladder and liver. These results suggest that most of the parietal peritoneum receives sensory nerves from dorsal root ganglia and the visceral peritoneum from both spinal nerves and the vagus nerve.

Animals↗

Expression and release of IL-18 binding protein in response to IFN-gamma.

IL-18 and IL-18 binding protein (IL-18BP) are two newly described opponents in the cytokine network. Local concentrations of these two players may determine biological functions of IL-18 in the context of inflammation, infection, and cancer. As IL-18 appears to be involved in the pathogenesis of Crohn's disease and may modulate tumor growth, we investigated the IL-18/IL-18BPa system in the human colon carcinoma/epithelial cell line DLD-1. In this study, we report that IFN-gamma induces expression and release of IL-18BPa from DLD-1 cells. mRNA induction and secretion of IL-18BPa immunoreactivity were associated with an activity that significantly impaired release of IFN-gamma by IL-12/IL-18-stimulated PBMC. Inducibility of IL-18BPa by IFN-gamma was also observed in LoVo, Caco-2, and HCT116 human colon carcinoma cell lines and in the human keratinocyte cell line HaCaT. Induction of IL-18BPa in colon carcinoma/epithelial cell lines was suppressed by coincubation with sodium butyrate. IFN-gamma-mediated IL-18BPa and its suppression by sodium butyrate were confirmed in organ cultures of intestinal colonic biopsy specimens. In contrast, sodium butyrate did not modulate expression of IL-18. The present data suggest that IFN-gamma may limit biological functions of IL-18 at sites of colonic immune activation by inducing IL-18BPa production. Down-regulation of IL-18BPa by sodium butyrate suggests that reinforcement of local IL-18 activity may contribute to actions of this short-chain fatty acid in the colonic microenvironment.

Butyrates↗

The influence of a gastrointestinal microflora on natural killer cell activity.

These studies demonstrate that the natural cytotoxicity of BALB/c mouse spleen cells for 51Cr-labeled YAC-1 cells can be significantly enhanced by microorganisms in the alimentary tract. Spleen cells from germfree BALB/c mice, euthymic, athymic, or non-nude background (+/+), had natural cell-mediated cytotoxicity for YAC-1 cells. Intestinal colonization with a few (flora-defined) or many (complex flora-conventionalized) microorganisms significantly enhanced natural cell-mediated cytotoxicity of athymic and euthymic mice over their germfree counterparts. Conversely, colonization of the alimentary tract of athymic and euthymic germfree mice with a pure culture of Candida albicans or colonization with Candida and a Bacillus sp. did not enhance natural cell-mediated cytotoxic activity over germfree levels. Spleen cells from germfree athymic mice were significantly more cytotoxic than spleen cells from germfree BALB/c mice that did not carry the nude gene (ie, +/+). In the germfree or gnotobiotic state, no difference in natural killer cell activity was evident between athymic (nu/nu) and heterozygous (+/nu) littermate mice; however, athymic (nu/nu) flora-defined or conventionalized mouse spleen cells were significantly more cytotoxic for YAC-1 cells than splenocytes from flora-defined or conventionalized heterozygous (+/nu) littermates. Spleen cells from BALB/c mice that were athymic (nu/nu) and colonized with a complex microbial flora (ie, conventionalized) had the highest percentage of cytotoxicity, at three different effector to target ratios, for YAC-1 cells. These studies indicate that the intestinal microflora can alter murine natural cell-mediated cytotoxicity.

Animals↗

Comparative immunocytochemical localization of lysyl oxidase (LOX) and the lysyl oxidase-like (LOXL) proteins: changes in the expression of LOXL during development and growth of mouse tissues.

Lysyl oxidase (LOX) and lysyl oxidase-like (LOXL) are extracellular enzymes that deaminate peptidyl lysyl residues involved in the cross-linking of fibrillar collagens and elastin. While LOX is required for the survival of newborn mice, the role of LOXL during development remains unclear. Studies have shown that the same cell types express LOX and LOXL in the same tissues, but no functional differences have been established. We have compared the immunohistochemical localization of LOX and LOXL in various tissues from normal, young adult mice. LOX and LOXL were co-localized in the skin, aorta, heart, lung, liver and cartilage, but were localized to different areas in the kidney, stomach, small intestine, colon, retina, ovary, testis and brain. LOXL expression was further examined in tissues from different developmental stages. In embryonic mice (10.5-14.5 dpc), LOXL immunostaining was abundant in the heart, liver, intestine, and neural tube. LOXL was present in most major organs in late fetal (16.5 dpc) and newborn mice, but generally diminished as animals aged. Immunoreactivity was significantly reduced in the heart, lung, kidney and liver of 2 year-old mice, but remained prevalent in the skin and tongue. LOX and LOXL were also found in the nuclei of cells in a number of tissues. These results indicate that LOXL has a role during mouse development and in the maintenance of adult tissues.

Aging↗

Effect of oral Bacillus coagulans administration on the density of vancomycin-resistant enterococci in the stool of colonized mice.

AIMS: A mouse model of vancomycin-resistant enterococcus (VRE) stool colonization was used to study the effect of Bacillus coagulans, a biotherapeutic agent, on the density of colonization. METHODS AND RESULTS: VRE-colonized mice received orally administered B. coagulans (107 cfu) or saline daily for four days. For one VRE strain, the density of VRE at one and four days after treatment was 1.4 log10cfu x g(-1) lower in experimental vs. control mice (P=0.03), and 35% of experimental vs. 0% of control mice had no detectable VRE four days after treatment (P=0.03). For two additional strains, there was no statistically significant reduction of VRE density in the B. coagulans groups. CONCLUSION: B. coagulans therapy reduced the density of colonization for one of three VRE strains tested. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests a potential role for biotherapeutic agents as a means to reduce the density of VRE intestinal colonization.

Animals↗

Cloning and characterization of a ninth member of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family, ppGaNTase-T9.

We have cloned, expressed and characterized the gene encoding a ninth member of the mammalian UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGaNTase) family, termed ppGaNTase-T9. This type II membrane protein consists of a 9-amino acid N-terminal cytoplasmic region, a 20-amino acid hydrophobic/transmembrane region, a 94-amino acid stem region, and a 480-amino acid conserved region. Northern blot analysis revealed that the gene encoding this enzyme is expressed in a broadly distributed manner across many adult tissues. Significant levels of 5- and 4.2-kilobase transcripts were found in rat sublingual gland, testis, small intestine, colon, and ovary, with lesser amounts in heart, brain, spleen, lung, stomach, cervix, and uterus. In situ hybridization to mouse embryos (embryonic day 14.5) revealed significant hybridization in the developing mandible, maxilla, intestine, and mesencephalic ventricle. Constructs expressing this gene transiently in COS7 cells resulted in no detectable transferase activity in vitro against a panel of unmodified peptides, including MUC5AC (GTTPSPVPTTSTTSAP) and EA2 (PTTDSTTPAPTTK). However, when incubated with MUC5AC and EA2 glycopeptides (obtained by the prior action of ppGaNTase-T1), additional incorporation of GalNAc was achieved, resulting in new hydroxyamino acid modification. The activity of this glycopeptide transferase is distinguished from that of ppGaNTase-T7 in that it forms a tetra-glycopeptide species from the MUC5AC tri-glycopeptide substrate, whereas ppGaNTase-T7 forms a hexa-glycopeptide species. This isoform thus represents the second example of a glycopeptide transferase and is distinct from the previously identified form in enzymatic activity as well as expression in embryonic and adult tissues. These findings lend further support to the existence of a hierarchical network of differential enzymatic activity within the diversely regulated ppGaNTase family, which may play a role in the various processes governing development.

Amino Acid Sequence↗

Colonization of streptomycin-treated mice by Aeromonas species.

Streptomycin-treated adult mice were investigated as a possible model for studying the enteropathogenicity of Aeromonas species. C57BL mice pre-treated with streptomycin (5.0 g/L drinking water, 48 hours) received a single intragastric dose (10(10) bacteria /10.5 mL) of one of six well-characterized, toxin-producing, human diarrhoeal isolates of A. veronii biovar sobria (n = 3) or A. hydrophila (n = 3). Their faeces were examined for Aeromonas for 10 days post-challenge. All strains colonized the antibiotic-treated mice. Colonization did not occur in mice which did not receive streptomycin. Strains of A. hydrophila were recovered in greater numbers than strains of A. veronii biovar sobria, and colonized ( > or = 10(3) cfu/g of faeces) a greater proportion of mice at day 10. Strains of the latter species, however, were more adherent in cell line assays used as models of intestinal adhesion. A. hydrophila strains localized in the large intestine and appeared not to be cell associated. This study, therefore, points to species-related differences in intestinal colonization mechanisms. The streptomycin-treated adult mouse model may prove useful for further investigation of some of these mechanisms. Diarrhoeal symptoms were, however, not produced in this model.

Aeromonas↗

Migration patterns of thymus-derived gamma delta T cells during chicken development.

Cell transfer experiments in congenic chick strains, one of which expresses the ov antigen marker, indicate that intestinal gamma delta T cells are derived from gamma delta+ thymocytes in embryos and newly hatched birds, and this early intestinal colonization occurs in two discrete waves. Here, we extend these studies to show that splenic colonization by gamma delta T cells occurs in essentially the same way. Following the engraftment of ov+ thymic lobes in thymectomized ov- recipients, gamma delta T cells migrate both to the spleen and intestine. By 1 week after hatching, a third generation of thymus-derived gamma delta T cells begins to migrate to both peripheral lymphoid organs, and this thymus-dependent seeding process is sustained over the first weeks of life. The survival time for splenic gamma delta migrants is significantly less than for the intestinal migrants. Tissue section analysis indicates that gamma delta T cells enter the intestinal epithelium at all villus levels. A shift in the gamma delta intraepithelial lymphocyte distribution toward the villus tip in thymectomized birds suggests the comigration of enterocytes and gamma delta intraepithelial lymphocytes. However, survival kinetics of the donor gamma delta population and a relatively high division rate of intestinal gamma delta T cells indicate that founder thymic migrants produce relatively long-lived clones of intestinal gamma delta T cells.

Animals↗