Mouse Gastrointestinal Tract Research Articles

Regional Distribution and Relative Frequency of Gastrointestinal Endocrine Cells in the ddN Mice: An Immunohistochemical Study

The distributions and frequencies of some endocrine cells in the eight portions of the gastrointestinal (GI) tract - fundus, pylorus, duodenum, jejunum, ileum, cecum, colon and rectum of the ddN mouse, were studied with immunohistochemical method using seven types of antisera against chromogranin (Cg) A serotonin, somatostatin, glucagon, gastrin, cholecystokinin (CCK)-8 and human pancreatic polypeptide (hPP). In the GI tract of ddN mice, CgA, serotonin, somatostatin, glucagon, gastrin, CCK-8 immunoreactive (IR) cells were identified with various frequencies, but hPP-IR cells were not observed in this study. Most of IR cells in the intestinal portion were generally spherical or spindle in shape (open type cell) whereas cells showing round in shape (close type cell) were found in the intestinal gland and stomach regions occasionally. They showed the highest frequency in the pylorus or colon. CgA-IR cells were observed from the pylorus to ileum. Serotonin-IR cells were detected throughout the whole GI tract except for the fundus. Somatostatin-IR cells were demonstrated throughout the whole GI tract except for the cecum and colon. Gastrin and CCK-8-IR cells were restricted to the pylorus and duodenum. In addition, a few glucagon-IR cells were restricted to the fundus and rectum. In conclusion, the general distribution patterns and relative frequency of GI endocrine cells of the ddN mouse was similar to that of other strains of mice. However, some strain and/or species-dependent unique distributions and frequencies of endocrine cells were also observed in the present study.

Bacteria from drinking water supply and their fate in gastrointestinal tracts of germ-free mice: A phylogenetic comparison study

Microorganisms in drinking water sources may colonize in gastrointestinal (GI) tracts and this phenomenon may pose a potential health risk especially to immunocompromised population. The microbial community diversity of the drinking water was compared with the GI tracts of the mice using phylogenetic and statistical analyses of 16S rRNA gene sequences. A group of germ-free mice were fed with drinking water from public water supply that passed through an automated watering system with documented biofilm accumulation. From drinking water and GI tracts of the germ-free mice, 179 bacteria were isolated and 75 unique 16S rRNA gene phylotypes were sequenced as operational taxonomic unit (OTU, >97% similarity). Three major groups of the genus Acidovorax (21%), Variovorax (42%) and Sphingopyxis (15%) were found in drinking water. Three major groups of the genus Ralstonia (24%), Staphylococcus (20%) and Bosea (22%) were found in GI tracts. Ralstonia (6%, 24%), Sphingopyxis (15%, 2%), Bacillus (3%, 5%), Escherichia coli (3%, 2%) and Mesorhizobium (3%, 5%) were found in both sources – drinking water and GI tract. A lineage-per-time plot shows that the both bacterial communities have convex shape lines, suggesting an excess of closely related ecotypes. A significant F ST test (0.00000–0.00901) coupled with an insignificant P test (0.07–0.46) implies that the tree contained several clades of closely related bacteria. Both phylogenetic and statistical results suggest a correlation between the bacterial communities originating in the drinking water and those associated with the GI tracts. The GI tract showed a higher genetic diversity than the drinking water, but a similar lineage-per-time plot was obtained overall. It means a sudden evolutionary transformation and colonization occurred with high selective forces.

In Vitro Communities Derived from Oral and Gut Microbial Floras Inhibit the Growth of Bacteria of Foreign Origins

The gastrointestinal (GI) tract is home to trillions of microbes. Within the same GI tract, substantial differences in the bacterial species that inhabit the oral cavity and intestinal tract have been noted. While the influence of host environments and nutritional availability in shaping different microbial communities is widely accepted, we hypothesize that the existing microbial flora also plays a role in selecting the bacterial species that are being integrated into the community. In this study, we used cultivable microbial communities isolated from different parts of the GI tract of mice (oral cavity and intestines) as a model system to examine this hypothesis. Microbes from these two areas were harvested and cultured using the same nutritional conditions, which led to two distinct microbial communities, each with about 20 different species as revealed by PCR-based denaturing gradient gel electrophoresis analysis. In vitro community competition assays showed that the two microbial floras exhibited antagonistic interactions toward each other. More interestingly, all the original isolates tested and their closely related species displayed striking community preferences: They persisted when introduced into the bacterial community of the same origin, while their viable count declined more than three orders of magnitude after 4 days of coincubation with the microbial flora of foreign origin. These results suggest that an existing microbial community might impose a selective pressure on incoming foreign bacterial species independent of host selection. The observed inter-flora interactions could contribute to the protective effect of established microbial communities against the integration of foreign bacteria to maintain the stability of the existing communities.Electronic supplementary materialThe online version of this article (doi:10.1007/s00248-010-9711-9) contains supplementary material, which is available to authorized users.

Orally Administered Bifidobacteria as Vehicles for Delivery of Agents to Systemic Tumors

Certain bacteria have emerged as biological gene vectors with natural tumor specificity, capable of specifically delivering genes or gene products to the tumor environment when intravenously (i.v.) administered to rodent models. We show for the first time that oral administration of bacteria to mice resulted in their translocation from the gastrointestinal tract (GIT) with subsequent homing to and replication specifically in tumors. The commensal, nonpathogenic Bifidobacterium breve UCC2003 harboring a plasmid expressing lux fed to mice bearing subcutaneous (s.c.) tumors were readily detected specifically in tumors, by live whole-body imaging, at levels similar to i.v. administration. Reporter gene expression was visible for >2 weeks in tumors. Mice remained healthy throughout experiments. Cytokine analyses indicated a significant upregulation of interferon-gamma (IFN-gamma) in the GIT of bifidobacteria-fed mice, which is associated with increases in epithelial permeability. However, B. breve feeding did not increase systemic levels of other commensal bacteria. The presence of tumor was not necessary for translocation to systemic organs to occur. These findings indicate potential for safe and efficient gene-based treatment and/or detection of tumors via ingestion of nonpathogenic bacteria expressing therapeutic or reporter genes.

Cadmium chloride exhibits a profound toxic effect on bacterial microflora of the mice gastrointestinal tract

Cadmium (Cd²(+)), a naturally occurring heavy metal, is an important environmental pollutant and a potent toxicant to bacteria. The gastrointestinal (GI) tract microflora has a marked capacity to cope with the increased load of ingested metals. However, heavy metals may have harmful effects on GIT microflora. Under the conditions of experimental exposure to cadmium, changes in the population of intestinal microflora in healthy mice were examined. Five experimental groups received 23 to 50 mg kg⁻¹ cadmium in drinking water and control group was given water free from cadmium for 45 days. Intestinal contents and biopsy samples were aseptically collected and bacterial counts were performed. The microflora of the intestine in control group was represented by bacteria of the genera Bacillus cereus, Lactobacillus spp., Clostridium spp., Escherichia coli, Klebsiella spp., Pseudomonas spp., Enterococcus spp. and Proteus spp. As the result of dysbiosis induced by the introduction of cadmium, a sharp decrease in the population of all microbial species in the intestine was observed. The deleterious effect of cadmium appeared to be less in the large intestine and rectum than that of small intestine, suggesting a site-specific influence of cadmium. The gram-negative bacteria tested were less sensitive to cadmium compared to the gram-positive bacteria because of their possible different ability to uptake the metal ions.

W1167 Fluorescence Molecular Tomographic (FMT) Imaging of Protease Activities and Integrins in the Inflamed Gastrointestinal Tract of Mice

W1167 Fluorescence Molecular Tomographic (FMT) Imaging of Protease Activities and Integrins in the Inflamed Gastrointestinal Tract of Mice

Genetic diversity in Campylobacter jejuni is associated with differential colonization of broiler chickens and C57BL/6J IL10-deficient mice

Previous studies have demonstrated that Campylobacter jejuni, the leading causative agent of bacterial food-borne disease in the USA, exhibits high-frequency genetic variation that is associated with changes in cell-surface antigens and ability to colonize chickens. To expand our understanding of the role of genetic diversity in the disease process, we analysed the ability of three C. jejuni human disease isolates (strains 11168, 33292 and 81-176) and genetically marked derivatives to colonize Ross 308 broilers and C57BL/6J IL10-deficient mice. C. jejuni colonized broilers at much higher efficiency (all three strains, 23 of 24 broilers) than mice (11168 only, 8 of 24 mice). C. jejuni 11168 genetically marked strains colonized mice at very low efficiency (2 of 42 mice); however, C. jejuni reisolated from mice colonized both mice and broilers at high efficiency, suggesting that this pathogen can adapt genetically in the mouse. We compared the genome composition in the three wild-type C. jejuni strains and derivatives by microarray DNA/DNA hybridization analysis; the data demonstrated a high degree of genetic diversity in three gene clusters associated with synthesis and modification of the cell-surface structures capsule, flagella and lipo-oligosaccharide. Finally, we analysed the frequency of mutation in homopolymeric tracts associated with the contingency genes wlaN (GC tract) and flgR (AT tracts) in culture and after passage through broilers and mice. C. jejuni adapted genetically in culture at high frequency and the degree of genetic diversity was increased by passage through broilers but was nearly eliminated in the gastrointestinal tract of mice. The data suggest that the broiler gastrointestinal tract provides an environment which promotes outgrowth and genetic variation in C. jejuni; the enhancement of genetic diversity at this location may contribute to its importance as a human disease reservoir.

Adherence, colonization and dissemination ofCandida dubliniensisand otherCandidaspecies

Adherence to host cells is essential for yeasts to develop their full pathogenic potential since it triggers the process that leads to colonization and enables their persistence in the host. The aim of this work was to study the in vitro adherence of Candida dubliniensis and other Candida species, as well as the relation of adherence with the colonization and dissemination of these yeasts in an experimental mice model. Clinical isolates of Candida dubliniensis, Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis and Candida tropicalis were tested for their in vitro ability to adhere to buccal epithelial cells and in vivo to colonize and disseminate in an experimental infant mice model. Although C. dubliniensis isolates showed variable adherence values, their ability to colonize and disseminate in mice tissue was almost null. All C. albicans strains showed high levels of adherence and a prolonged gastrointestinal (GI) tract colonization. Both C. glabrata and C. krusei, showed a minor in vitro adherence and limited colonization time in infant mice GI tract. C. albicans and C. parapsilosis demonstrated a higher ability to disseminate, but the other non-C. albicans Candida strains showed a lower ability to disseminate. This study demonstrates that C. dubliniensis has a low GI tract colonization ability, as well as low dissemination ability in relation to C. albicans.

Inulin-Type Fructans Stimulated the Growth of Exogenously AdministeredLactobacillus plantarumNo. 14 in the Mouse Gastrointestinal Tract

This study examined the effects of the inulin-type fructans known as fructo-oligosaccharide (FOS) and inulin on the growth and persistence of Lactobacillus plantarum No. 14 (LP14) in the gut. Cultivation in medium that contained either FOS or inulin revealed that both fructans supported the growth of LP14. Fecal recovery of viable L. plantarum at 24 and 30 h following administration of LP14 were highest in mice fed inulin, followed by mice fed FOS and the control diet. However, neither of the fructans prolonged the appearance of LP14 in the gut. Cultivation in fecal extract medium prepared from mice fed each of the test diets showed higher growth of LP14 in the mice fed fructans. Histological analysis following fluorescein-stained LP14 administration showed that LP14 was largely localized to the luminal contents. These results suggest that inulin-type fructans support the growth of LP14 in the luminal contents only during their passage through the gut.

A Mineral-Rich Red Algae Extract Inhibits Polyp Formation and Inflammation in the Gastrointestinal Tract of Mice on a High-Fat Diet

The purpose of this study was to determine whether a mineral-rich extract derived from the red marine algae Lithothamnion calcareum could be used as a dietary supplement for chemoprevention against colon polyp formation. A total of 60 C57bl/6 mice were divided into 3 groups based on diet. One group received a low-fat, rodent chow diet (AIN76A). The second group received a high-fat "Western-style" diet (HFWD). The third group was fed the same HFWD with the mineral-rich extract included as a dietary supplement. Mice were maintained on the respective diets for 15 months. Autopsies were performed at the time of death or at the completion of the study. To summarize, the cumulative mortality rate was higher in mice on the HFWD during the 15-month period (55%) than in mice from the low-fat diet or the extract-supplemented high-fat diet groups (20% and 30%, respectively; P < .05 with respect to both). Autopsies revealed colon polyps in 20% of the animals on the HFWD and none in animals of the other 2 groups (P < .05). In addition to the grossly visible polyps, areas of hyperplasia in the colonic mucosa and inflammatory foci throughout the gastrointestinal tract were observed histologically in animals on the high-fat diet. Both were significantly reduced in animals on the low-fat diet and animals on the extract-supplemented HFWD.These data suggest that the mineral-rich algae extract may provide a novel approach to chemoprevention in the colon.

Effect of Recql5 deficiency on the intestinal tumor susceptibility ofApcminmice

To investigate whether Recql5, a DNA helicase that plays an important role in the maintenance of genome integrity, is a tumor suppressor in the gastrointestinal tract in mice. We generated cohorts of both Recql5-proficient and Recql5-deficient Apc(min/+) mice and compared the tumor susceptibility in their gastrointestinal tracts. Recql5 deficiency in Apc(min/+) mice resulted in a significant increase in the tumor incidence in both the colon (P = 0.0162) and the small intestine (P < 0.01). These findings have provided the first genetic evidence for a tumor suppression role of Recql5 in the gastrointestinal tract of mice. Importantly, since mouse Recql5 and human RECQL5 are highly conserved, these findings also suggest that RECQL5 may be a tumor suppressor for human colon cancer. Recql5 has a tumor suppression role in the mouse gastrointestinal tract.

Dietary ratio of protein to carbohydrate induces plastic responses in the gastrointestinal tract of mice

Some vertebrates change the size of their digestive system in response to quantity and fibre content of ingested food, but the effects of dietary nutrients on gut structure remain poorly understood. Here we investigate how the protein to carbohydrate ratio of diets affects the mass of the gastrointestinal tract in mice. We fed 6-week-old male mice one of five isocaloric diets differing only in protein to carbohydrate ratio (the "no-choice" treatments), while a further four treatment groups received nutritionally complementary food pairings from which they could self-select a diet (the "choice" treatments). After 32 days, we measured the resulting dry mass of stomachs, intestines, caeca and colons. In the no-choice treatments, the stomachs were heavier in the mice fed diets containing more protein and less carbohydrate, indicating that larger stomachs may be needed for efficient digestion of the protein-rich food. In contrast, intestines, caeca and colons were heavier when diets contained more carbohydrates and less protein. This response may function to increase the digestive rate of carbohydrates when the dietary content of this macronutrient increases, but it may also indicate a compensatory response to increase amino acid uptake from a protein-deficient food. Mice in the choice treatments self-selected a diet with a protein to carbohydrate ratio of 0.46, and had gut dimensions similar to the expectation derived from no-choice treatments for this diet composition. Our results provide an example of plasticity in the differential allocation of resources to organ function, which is triggered by variation in resource quality.

Role of PSD95 in membrane association and catalytic activity of nNOSα in nitrergic varicosities in mice gut

We have recently shown that membrane association of neuronal nitric oxide synthase-alpha (nNOSalpha) is critical in the regulation of synthesis of NO during nitrergic neurotransmission. The purpose of this study was to examine the role of the synapse-associated proteins (SAPs) in membrane association of nNOSalpha. Varicosities (swellings on terminal axons) were isolated from mice gastrointestinal tract and examined for nNOSalpha, postsynaptic density protein 95 (PSD95), and membrane interactions by coimmunoprecipitation and SDS-PAGE. Our results show that PSD95 protein was present in the membrane fraction of the nerve varicosity, whereas both PSD95 and SAP97 were present in the cytosol. nNOSalpha was associated with PSD95 but not SAP97. nNOSalpha-PSD95 complex was bound to the membrane via palmitoylation of PSD95. Depalmitoylation of PSD95 with 2-bromopalmitate dislocates nNOSalpha and PSD95 from the varicosity membrane and abolishes NO production. These studies show that palmitoylation of PSD95 anchors nNOSalpha to the varicosity membrane and that it is obligatory for NO production by the enzyme. Palmitoylation of PSD95 may provide a novel target for regulation of nitrergic neurotransmission.

Persistence of colicinogenic Escherichia coli in the mouse gastrointestinal tract

BackgroundThe ability of a bacterial strain to competitively exclude or displace other strains can be attributed to the production of narrow spectrum antimicrobials, the bacteriocins. In an attempt to evaluate the importance of bacteriocin production for Escherichia coli strain residence in the gastrointestinal tract, a murine model experimental evolution study was undertaken.ResultsSix colicin-producing, yet otherwise isogenic, E. coli strains were administered and established in the large intestine of streptomycin-treated mice. The strains' persistence, population density, and doubling time were monitored over a period of 112 days. Early in the experiment only minor differences in population density between the various colicin-producing and the non-producing control strains were detected. However, over time, the density of the control strains plummeted, while that of the colicin-producing strains remained significantly higher (F(7,66) = 2.317; P < 0.0008).ConclusionThe data presented here support prior claims that bacteriocin production may play a significant role in the colonization of E. coli in the gastrointestinal tract. Further, this study suggests that the ability to produce bacteriocins may prove to be a critical factor in determining the success of establishing probiotic E. coli in the gastrointestinal tract of humans and animals.

Telomerase modulates Wnt signalling by association with target gene chromatin

Stem cells are controlled, in part, by genetic pathways frequently dysregulated during human tumorigenesis. Either stimulation of Wnt/beta-catenin signalling or overexpression of telomerase is sufficient to activate quiescent epidermal stem cells in vivo, although the mechanisms by which telomerase exerts these effects are not understood. Here we show that telomerase directly modulates Wnt/beta-catenin signalling by serving as a cofactor in a beta-catenin transcriptional complex. The telomerase protein component TERT (telomerase reverse transcriptase) interacts with BRG1 (also called SMARCA4), a SWI/SNF-related chromatin remodelling protein, and activates Wnt-dependent reporters in cultured cells and in vivo. TERT serves an essential role in formation of the anterior-posterior axis in Xenopus laevis embryos, and this defect in Wnt signalling manifests as homeotic transformations in the vertebrae of Tert(-/-) mice. Chromatin immunoprecipitation of the endogenous TERT protein from mouse gastrointestinal tract shows that TERT physically occupies gene promoters of Wnt-dependent genes. These data reveal an unanticipated role for telomerase as a transcriptional modulator of the Wnt/beta-catenin signalling pathway.

Hydrogen sulphide synthesis in the rat and mouse gastrointestinal tract

Hydrogen sulphide (H2S) exerts several anti-inflammatory effects, accelerates the healing of experimental gastric ulcers, and can stimulate intestinal secretion. Little is known about H2S synthesis in the gastrointestinal tract. The aim of this study was to characterize H2S synthesis throughout the gastrointestinal tract. H2S synthesis in various gastrointestinal tissues of rats and mice was determined. The effects and selectivity of inhibitors of two key enzymes for H2S synthesis, cystathionine-gamma-lyase and cystathionine-beta-synthase, were examined. Cystathionine-gamma-lyase and cystathionine-beta-synthase expression was evaluated by Western blotting and immunohistochemistry. Cystathionine-gamma-lyase and cystathionine-beta-synthase expression in biopsies of human colon was also examined. H2S synthesis was variable throughout the gastrointestinal tract in parallel with variations in cystathionine-gamma-lyase and cystathionine-beta-synthase expression. The efficacy of cystathionine-beta-synthase and cystathionine-gamma-lyase inhibitors to reduce H2S synthesis in these tissues was also variable. Cystathionine-beta-synthase is the predominant source of H2S synthesis in the colon of rodents. Cystathionine-gamma-lyase and cystathionine-beta-synthase were also expressed in healthy human colon biopsies. The capacity for H2S synthesis varies throughout the rodent gastrointestinal tract, as does the distribution and contribution of the two key enzymes. Investigation of additional enzymatic sources of H2S and the development of more selective inhibitors are suggested.

Behavior of the meat-borne bacterium Lactobacillus sakei during its transit through the gastrointestinal tracts of axenic and conventional mice.

A Lactobacillus sakei strain named FLEC01 was isolated from human feces and characterized genotypically. Comparison of the genetic features of this strain with those of both the meat-borne L. sakei strain 23K and another human isolate, LTH5590, showed that they belong to different but closely related clusters. The three L. sakei strains did not persist and only transited through the gastrointestinal tracts (GITs) of conventional C3H/HeN mice. In contrast, they all colonized the GITs of axenic mice and rapidly reached a population of 10(9) CFU/g of feces, which remained stable until day 51. Five days after mice were fed, a first subpopulation, characterized by small colonies, appeared and reached 50% of the total L. sakei population in mice. Fifteen to 21 days after feeding, a second subpopulation, characterized by rough colonies, appeared. It coexisted with the two other populations until day 51, and its cell shapes were also affected, suggesting a dysfunction of the cell division or cell wall. No clear difference between the behaviors of the meat-borne strain and the two human isolates in both conventional and axenic mice was observed, suggesting that L. sakei is a food-borne bacterium rather than a commensal one and that its presence in human feces originates from diet. Previous observations of Escherichia coli strains suggest that the mouse GIT environment could induce mutations to increase their survival and colonization capacities. Here, we observed similar mutations concerning a food-grade gram-positive bacterium for the first time.

Invitro and invivo safety evaluation of the bacteriocin producer Streptococcusmacedonicus ACA-DC 198

Streptococcus macedonicus ACA-DC 198, a bacteriocin producer isolated from Greek Kasseri cheese, was used in a series of in vitro and in vivo experiments in order to evaluate its pathogenic potential. The strain was examined in vitro for haemolytic activity, antibiotic resistance and presence of pathogenicity genes encountered in Streptococcus pyogenes. Subsequently, the strain was orally administered to mice (8.9 log cfu daily), continuously over a period of 12 weeks, in order to ascertain the effects of its long term consumption on animal health and gastric inflammation. S. macedonicus ACA-DC 198 was found to be non-haemolytic and sensitive to ampicillin, chloramphenicol, ciprofloxacin, erythromycin, streptomycin, tetracycline, and vancomycin, with the only resistance observed against kanamycin. PCR amplification and DNA–DNA hybridization did not reveal the presence of any of the S. pyogenes pathogenicity genes examined, namely emm, scpA, hasA, speB, smez2, speJ, sagAB, hylA, ska, speF, speG, slo, hylP2 and mga. In the mouse study, no detrimental effects were observed in the behaviour, general well being, weight gain and water consumption of the animals receiving S. macedonicus ACA-DC 198. Histologic analysis showed no evidence of inflammation in the stomach of the animals receiving S. macedonicus ACA-DC 198, while faecal microbiological analysis revealed that the strain retained its viability passing through the mouse gastrointestinal tract. Finally, no evidence of translocation to the liver, spleen and mesenteric lymph nodes was observed. In conclusion, none of the examined virulence determinants were detected in S. macedonicus ACA-DC 198 and its long term, high dosage oral administration did not appear to induce any pathogenic effect in mice.

S1611 Elevation of UCP-2 By High Glucose Impairs Glucose-Induced GLP-1 Secretion in Mouse Gastrointestinal Tract

S1611 Elevation of UCP-2 By High Glucose Impairs Glucose-Induced GLP-1 Secretion in Mouse Gastrointestinal Tract

Ano1 is a selective marker of interstitial cells of Cajal in the human and mouse gastrointestinal tract

Populations of interstitial cells of Cajal (ICC) are altered in several gastrointestinal neuromuscular disorders. ICC are identified typically by ultrastructure and expression of Kit (CD117), a protein that is also expressed on mast cells. No other molecular marker currently exists to independently identify ICC. The expression of ANO1 (DOG1, TMEM16A), a Ca(2+)-activated Cl(-) channel, in gastrointestinal stromal tumors suggests it may be useful as an ICC marker. The aims of this study were therefore to determine the distribution of Ano1 immunoreactivity compared with Kit and to establish whether Ano1 is a reliable marker for human and mouse ICC. Expression of Ano1 in human and mouse stomach, small intestine, and colon was investigated by immunofluorescence labeling using antibodies to Ano1 alone and in combination with antibodies to Kit. Colocalization of immunoreactivity was demonstrated by epifluorescence and confocal microscopy. In the muscularis propria, Ano1 immunoreactivity was restricted to cells with the morphology and distribution of ICC. All Ano1-positive cells in the muscularis propria were also Kit positive. Kit-expressing mast cells were not Ano1 positive. Some non-ICC in the mucosa and submucosa of human tissues were Ano1 positive but Kit negative. A few (3.2%) Ano1-positive cells in the human gastric muscularis propria were labeled weakly for Kit. Ano1 labels all classes of ICC and represents a highly specific marker for studying the distribution of ICC in mouse and human tissues with an advantage over Kit since it does not label mast cells.