In Vivo Gastric and Intestinal Slow Waves in W/W<Superscript>v</Superscript> Mice

The aim was to investigate whether there are regular gastric and intestinal slow waves in conscious W/W^v mice. Eleven W/W^v mice and 11 wild-type mice were implanted with two pairs of electrodes in the stomach and small intestine. Gastrointestinal slow waves were recorded both under anesthesia and in the conscious state. Atropine and verapamil were given separately to an additional 10 W/W^v mice. Results were as follows. (1) The conscious W/W^v mice showed lower rhythmic slow waves in the small intestine (77.1 vs 93.5%; P < 0.001). However, the frequency (10.7 vs 18.8 cpm; P < 0.0001) and the antregrade propagation of intestinal slow waves in W/W^v mice were significantly lower than in the controls. In the stomach, regular slow waves were recorded in both groups, with no difference between the two groups. (2) Anesthesia significantly impaired both gastric and intestinal slow waves in both groups. (3) Atropine and verapamil had no effects on the rhythmicity of intestinal slow waves. We conclude that ICC-MY may not be the sole pacemaker cells for slow waves in the small intestine.There may be some abnormality of smooth muscle cells in W/W^v mice that causes a reduction in the frequency, rhythmicity, and antegrade propagation of slow waves.Xiaohua Hou is a Visiting Scientist from the Division of Gastroenterology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.     

Xiangbinfang granules enhance gastric antrum motility via intramuscular interstitial cells of Cajal in mice

BACKGROUND Interdigestive migrating motor complexes(MMC)produce periodic contractions in the gastrointestinal tract,but the exact mechanism of action still remains unclear.Intramuscular interstitial cells of Cajal(ICC-IM)participate in gastrointestinal hormone and neuromodulation,but the correlation between ICCIM and MMC is also unclear.We found that xiangbinfang granules(XBF)mediated the phase III contraction of MMC.Here,the effects of XBF on gastric antrum motility in W/Wv mice and the effects of ICC-IM on gastric antrum MMC are reported.AIM To observe the effects of ICC-IM on gastric antrum motility and to establish the mechanism of XBF in promoting gastric antrum motility.METHODS The density of c-kit-positive ICC myenteric plexus(ICC-MP)and ICC-IM in the antral muscularis of W/Wv and wild-type(WT)mice was examined by confocal microscopy.The effects of XBF on gastric antrum slow waves in W/Wv and WT mice were recorded by intracellular amplification recording.Micro-strain-gauge force transducers were implanted into the gastric antrum to monitor the MMC and the effect of XBF on gastric antrum motility in conscious W/Wv and WT mice.RESULTS In the gastric antrum of W/Wv mice,c-kit immunoreactivity was significantly reduced,and no ICC-IM network was observed.Spontaneous rhythmic slow waves also appeared in the antrum of W/Wv mice,but the amplitude of the antrum slow wave decreased significantly in W/Wv mice(22.62±2.23 mV vs 2.92±0.52 mV,P<0.0001).MMCs were found in 7 of the 8 WT mice but no complete MMC cycle was found in W/Wv mice.The contractile frequency and amplitude index of the gastric antrum were significantly increased in conscious WT compared to W/Wv mice(frequency,3.53±0.18 cpm vs 1.28±0.12 cpm;amplitude index,23014.26±1798.65 mV·20 min vs 3782.16±407.13 mV·20 min;P<0.0001).XBF depolarized smooth muscle cells of the gastric antrum in WT and W/Wv mice in a dose-dependent manner.Similarly,the gastric antrum motility in WT mice was significantly increased after treatment with XBF 5 mg(P<0.05).Atropine(0.1 mg/kg)blocked the enhancement of XBF in WT and W/Wv mice completely,while tetrodotoxin(0.05 mg/kg)partially inhibited the enhancement by XBF.CONCLUSION ICC-IM participates in the regulation of gastric antrum MMC in mice.XBF induces MMC III-like contractions that enhance gastric antrum motility via ICCIM in mice.     

Differential gene expression in the murine gastric fundus lacking interstitial cells of Cajal

Background  

The muscle layers of murine gastric fundus have no interstitial cells of Cajal at the level of the myenteric plexus and only possess intramuscular interstitial cells and this tissue does not generate electric slow waves. The absence of intramuscular interstitial cells in W/W ^V mutants provides a unique opportunity to study the molecular changes that are associated with the loss of these intercalating cells.     

Reconstitution by bone marrow grafting of the defective protective capacity at the migratory phase but not at the intestinal phase of Nippostrongylus brasiliensis infection in W/Wv mice

After a primary infection by subcutaneous inoculation with the infective larvae (L₃) of Nippostrongylus brasiliensis, the intestinal worm burden was higher and expulsion was slower in W/W^v mice than in ^+/+ mice. When the course of infection was segregated into the migratory and intestinal phases, protection during the migratory phase examined by the larval recovery from the lungs and that during the intestinal phase measured by worm burden after intraduodenal implantation with adult worms were both defective in W/W^v mice. The higher susceptibility of W/W^v mice during the migratory phase was normalized by bone marrow re constitution. On the other hand, higher susceptibility of W/W^v mice during the intestinal phase, which was measured by worm burden 24 h after intraduodenal implantation of the larvae recovered from the lungs of rats, was not normalized by bone marrow grafting. Furthermore, slower expulsion seen in W/W^v mice after intraduodenal implantation with adult worms was not hastened by bone marrow reconstitution. These results indicate that the protective mechanisms against N. brasiliensis operating during the migratory phase and those during the intestinal phase were different in terms of bone marrow dependency and that nonmyeloid cells utilizing c-kit ligand/receptor system to express their functions are involved in the rnucosal defence against N. brasiliensis.     

Evaluation of the Role of Mast Cells in the Progression of Acetic Acid-Induced Colitis in Mice

Background: Mast cells are widely distributed in the gastrointestinal mucosa. However, their role in the pathogenesis of inflammatory bowel disease remains unsettled. The aim of the present study is to clarify the relative importance of mast cells in the progression of acetic acid-induced colitis in mice. Methods: Mast cell-deficient W/W^v and their normal littermate +/+ mice were given intrarectal administration of 5% acetic acid. The severity of colonic damage, the number of mast cells, and myeloperoxidase (MPO) activities in the colonic tissues were examined. Results: The severity of colonic damage was comparable between W/W^v and +/+ mice. In both groups of animals kinetic changes of the severity of the mucosal damage agreed well with that of MPO activities in the colonic mucosa. Pretreatment with a mast cell stabilizer, ketotifen, did not affect the severity of colitis in +/+ mice. Conclusions: These results discount, but do not disprove, the role of mast cells in the progression of acetic acid-induced colitis in mice.     

Roles of Interstitial Cells of Cajal in Intestinal Transit and Exogenous Electrical Pacing

The aims of this study were to investigate the role of interstitial cells of Cajal (ICCs) on small intestinal transit and its responses to exogenous pacing in W/W^v mice. Eleven W/W^v mice and their controls implanted with four pairs of gastrointestinal electrodes were used for testing the entrainment of slow waves. Another 20 W/W^v mice and their controls equipped with a duodenal catheter and one pair of intestinal electrodes were used to test small intestinal transit represented by the geometric center (GC). Results were as follows. (1) The effect of pacing on slow wave frequency was sustained only in controls, and not in W/W^v mice. (2) Both gastric and intestinal slow waves were completely entrained in controls and W/W^v mice. Higher energy was required for pacing the stomach than the small intestine. (3) There was no significant difference in small intestinal transit between the controls and the W/W^v mice (GC: 5.4 vs. 5.5). (4) Pacing showed no effects on small intestinal transit in either wild-type (GC: 5.4 vs. 5.6) or W/W^v mice (GC: 5.5 vs. 5.7). We conclude that myenteric ICCs may not play an important role in the regulation of small intestinal transit in conscious mice. Gastric and intestinal pacing can be achieved without ICCs.     

Gastric Inflammation During Systemic Anaphylaxis (Neutrophil Recruitment in Stomach Wall of Mice Does Not Require Mast Cell Participation)

We determined whether neutrophil infiltrationinto the stomach wall occurred during systemicanaphylaxis in mice and assessed the participation ofmast cells in the response. Normal mice sensitized and challenged with antigen exhibited significantneutrophil infiltration in the gastric mucosa andsubmucosa compared with saline-challenged mice. Thedevelopment of clinical signs of anaphylaxis and extent of gastric neutrophil infiltration was similarin mast cell-deficient Kit^W/Kit^W-vor Mgf^Sl/Mgf^Sl-d mice and therespective normal congenic mice. Pretreatment withsodium cromoglycate prevented the clinical signs of anaphylaxis and significantlydiminished the infiltration of neutrophils in +/+ orKit^W/Kit^W-v mice. Systemicanaphylaxis is associated with neutrophil infiltrationinto the stomach wall in mice, and mast cells are not required for thedevelopment of either the clinical manifestations orgastric neutrophil infiltration observed in theresponse.     

Haematopoietic Defects of W/Wv Mice Studied with the Spleen Colony,Agar Colony,and Diffusion Chamber Techniques

Bone marrow progenitor cells from anaemic W/W^v mice were compared with normal +/+ cells utilizing the spleen colony, the agar colony and the diffusion chamber techniques. Spleen colony formation from W/W^v cells was markedly defective, and more so for erythroid than for granuloid colonies. The progenitor cell concentration was apparently normal as measured by the two other techniques. The concentration of circulating progenitor cells also seemed to be normal. On the other hand, the cell formation per progenitor cell was subnormal in all three assay systems. The initial proliferative response of W/W^v spleen colony-formers and agar colony-formers to short-term diffusion chamber culturing was apparently normal. The incorporation of ³H-thymidine, related to the number of proliferative granulocytes present in the chambers, also seemed to be normal. The results indicate that the W/W^v defect is not limited to the multipotent stem cells. A possible interpretation is that it is the capacity for continued self-renewal of immature cells that is defective.     

Enhanced mucosal permeability and nitric oxide synthase activity in jejunum of mast cell deficient mice

Background—Recent reports have described amodulating influence of nitric oxide (NO) on intestinal mucosalpermeability and have implicated a role for mast cells in this NOmediated process.
Aims—To assess further the contribution of mastcells to the mucosal permeability changes elicited by the NO synthase(NOS) inhibitor N^G-nitro-L-arginine methylester(L-NAME), using mast cell deficient (W/W^V) andmast cell replete mice (+/+).
Methods—Chromium-51 EDTA clearance (from blood tojejunal lumen), jejunal NOS and myeloperoxidase (MPO) activities, andplasma nitrate/nitrite levels were monitored.
Results—The increased EDTA clearance elicited byintraluminal L-NAME in W/W^V mice (4.4-fold) wassignificantly greater than the response observed in control (+/+) mice(1.8-fold). The exacerbated response in W/W^v mice wasgreatly attenuated by pretreatment with either dexamethasone (1.3-fold)or the selective inducible NOS inhibitor, aminoguanidine (1.4-fold),and partially attenuated by the mast cell stabiliser, lodoxamide(2.9-fold). Jejunal inducible NOS activity was significantly higher inW/W^V than in +/+ mice, while jejunal MPO was lower inW/W^V mice than in +/+ mice, suggesting that the higherinducible NOS in W/W^V does not result from the recruitmentof inflammatory cells into the gut. The higher inducible NOS activityin the jejunum of W/W^V was significantly reduced bydexamethasone treatment.
Conclusions—Our results suggest that mast cellsnormally serve to inhibit inducible NOS activity tonically in the gutand that inhibitors of NOS elicit a larger permeability response when this tonic inhibitory influence is released by mast cell depletion.

Keywords:aminoguanidine; c-kit; dexamethasone; epithelium; neutrophils

     

Synergistic protecting effect of cord blood CD34+ cells over-expressing both interleukin-3 and Flt3 ligand on lethally irradiated mice

The objective of the present work was to study the effect of interleukin-3 (IL-3) and Flt3 ligand (FL) over-expression in human cord blood CD34⁺ cells on rescuing lethally irradiated mice by transplantation. CD34⁺ cells were isolated from human umbilical cord blood (UCB) and infected with recombinant retrovirus expressing FL, IL-3 or FL/IL-3 genes, respectively. Stably transduced CD34⁺ cells were transplanted i.v. into NOD/SCID/IL2rγ^null mice that had been conditioned with 8.0 Gy of irradiation. At 6 weeks post-irradiation, chimerism in the animal bone marrow (BM) and the spleen were investigated. Recovery of peripheral blood cell and animal survival were recorded 2, 4, and 6 weeks post-irradiation. The chimerism was further investigated by serial transplantation assay. At 6 weeks post-transplantation, each of the three transduced human UCB CD34⁺ cell types could be observed in all examined BM of chimeric mice; however, more human cells could be found in BM, spleen and peripheral blood of those mice transplanted with CD34⁺ cells over-expressing IL-3/FL cells. Over-expression of IL-3 and FL at mRNA and protein levels could be detected in the BM cells of chimeric mice. Accordingly, hIL-3/FL co-overexpressing mice showed greater recovery of peripheral blood counts as early as 2 weeks after irradiation, and a much higher survival rate (11/12) than other groups at 6 weeks post-irradiation. Serial transplantation assay indicated that human cord blood CD34⁺ cells could recover potentials of proliferation and self-renewal after being transplanted into mice. Our results suggested that transplant of CD34⁺ cord blood cells over-expressing IL-3/FL genes improved rescue of radiation-injured mice, which probably results from the synergistic effect between hIL-3 and hFL causing the rapid reconstitution of hematopoiesis.     

Bone marrow characterization in sickle cell disease: inflammation and stress erythropoiesis lead to suboptimal CD34 recovery

Stress erythropoiesis and chronic inflammation in subjects with sickle cell disease (SCD) may have an impact on the bone marrow (BM) haematopoietic stem and progenitor cell (HSPC) quality and yield necessary for effective autologous, ex vivo HSPC gene therapy. BM from 19 subjects with SCD and five volunteers without SCD (non-SCD) was collected in different anticoagulants and processed immediately (day 0) or the following day (day 1). Inflammatory, contamination and aggregation markers within the mononuclear layer, and CD34, CD45 and Glycophorin-A (GPA) expression on HSPCs after CD34⁺ selection were analysed by conventional and imaging flow cytometry. Compared to non-SCD BM, multiple markers of inflammation, contamination (red cells, P < 0·01; platelets, P < 0·01) and aggregates (platelet/granulocytes, P < 0·01; mononuclear/red cells, P < 0·01) were higher in SCD BM. Total CD34⁺ cell count was lower in SCD BM (P < 0·05), however CD34⁺ count was higher in SCD BM when collected in acid citrate dextrose-A (ACDA) versus heparin (P < 0·05). Greater than 50% of CD34⁺ HSPCs from SCD BM are CD34^dim due to higher erythroid lineage expression (P < 0·01) as single cell CD34⁺CD45⁺GPA⁺ (P < 0·01) and CD34⁺CD45⁻GPA⁺ (P < 0·01) HSPCs. SCD BM is characterized by increased inflammation, aggregation and contamination contributing to significant differences in HSPC quality and yield compared to non-SCD BM.     

Exogenous Interleukin-6 Facilitated the Contraction of the Colon in a Depression Rat Model

Background

Gut dysmotility is closely associated with proinflammatory cytokines both in irritable bowel syndrome and inflammatory bowel disease. There is a dose–response relationship between depression and these inflammatory cytokines.

Aims

In the present study, we aimed to investigate the effect of Interleukin-6 (IL-6) on colon motility in a rat model of depression induced by chronic unpredictable mild stress (CUMS).

Methods

The contraction of the circular muscle strips of proximal colon was monitored by a polygraph. IL-6 and IL-6 receptor (IL-6R) mRNA was assayed by real-time quantitative PCR. Immunohistochemistry staining was used to locate the IL-6 and IL-6R in the rat colon.

Results

IL-6 and IL-6R were expressed in the mucosal layer, smooth muscle cells, and myenteric plexus of the colon. Exogenous IL-6 (20 ng/ml) increased the contraction of the circular muscle strip. Pretreatment of tetrodotoxin (blocker of voltage-dependent Na⁺ channel on nerve fiber) blocks the excitatory effect of IL-6 on the contraction of the colon in non-stressed rats, but partially inhibited IL-6-induced excitatory effect on the muscle strips in CUMS-treated rats.

Conclusions

These results suggest that IL-6-induced the contraction of the colonic strip by acting on the gut’s nervous system and acting directly on the smooth muscle in rats with depression.     

Interstitial cells of Cajal mediate inhibitory neurotransmission in the stomach.

The structural relationships between interstitial cells of Cajal (ICC), varicose nerve fibers, and smooth muscle cells in the gastrointestinal tract have led to the suggestion that ICC may be involved in or mediate enteric neurotransmission. We characterized the distribution of ICC in the murine stomach and found two distinct classes on the basis of morphology and immunoreactivity to antibodies against c-Kit receptors. ICC with multiple processes formed a network in the myenteric plexus region from corpus to pylorus. Spindle-shaped ICC were found within the circular and longitudinal muscle layers (IC-IM) throughout the stomach. The density of these cells was greatest in the proximal stomach. IC-IM ran along nerve fibers and were closely associated with nerve terminals and adjacent smooth muscle cells. IC-IM failed to develop in mice with mutations in c-kit. Therefore, we used W/W(V) mutants to test whether IC-IM mediate neural inputs in muscles of the gastric fundus. The distribution of inhibitory nerves in the stomachs of c-kit mutants was normal, but NO-dependent inhibitory neuro-regulation was greatly reduced. Smooth muscle tissues of W/W(V) mutants relaxed in response to exogenous sodium nitroprusside, but the membrane potential effects of sodium nitroprusside were attenuated. These data suggest that IC-IM play a critical serial role in NO-dependent neurotransmission: the cellular mechanism(s) responsible for transducing NO into electrical responses may be expressed in IC-IM. Loss of these cells causes loss of electrical responsiveness and greatly reduces responses to nitrergic nerve stimulation.     

Effect of aged bone marrow microenvironment on mesenchymal stem cell migration

Mesenchymal stem cells (MSCs) are known to have many notable features, especially their multiple differentiation ability and immunoregulatory capacity. MSCs are important stem cells in the bone marrow (BM), and their characteristics are affected by the BM microenvironment. However, effects of the BM microenvironment on the properties of MSCs are not well understood. In this study, we found that BM from aged mice decreased MSC colony formation. Flow cytometry data showed that the proportion of B220⁺ cells in BM from aged mice was significantly lower than that in BM from young mice, while the proportion of CD11b⁺, CD3⁺, Gr-1⁺, or F4/80⁺ cells are on the contrary. CD11b⁺, B220⁺, and Ter119⁺ cells from aged mice were not the subsets that decreased MSC colony formation. We further demonstrated that both BM from aged mice and young mice exhibited similar effects on the proliferation of murine MSC cell line C3H10T1/2. However, when cocultured with BM from aged mice, C3H10T1/2 showed slower migration ability. In addition, we found that phosphorylation of JNK (c-Jun N-terminal kinases) in C3H10T1/2 cocultured with BM from aged mice was lower than that in C3H10T1/2 cocultured with BM from young mice. Collectively, our data revealed that BM from aged mice could decrease the migration of MSCs from their niche through regulating the JNK pathway.     

Differential gene expression profile in the small intestines of mice lacking pacemaker interstitial cells of Cajal

Background  

We previously identified eight known and novel genes differentially expressed in the small intestines of wild type and W/W ^V mice, which have greatly reduced populations of the interstitial cells of Cajal, that are responsible for the generation of electrical slow waves, by using a differential gene display method.     

Severer lupus erythematosus-like skin lesions in MRL/lpr mice with homozygous Kitwsh/wsh mutation

Objective: To clarify the roles of mast cells (MCs) on the pathogenesis of lupus erythematosus (LE)-like skin lesions on MRL/lpr mice.

Methods: MRL/lpr mice were mated with C57BL/6-Kit^wsh/wsh mice and the heterozygous F1 mice were 10 times backcrossed with the parental MRL/lpr to generate MRL/lpr-Kit^wsh/wsh mice. MC-deficient MRL/lpr-Kit^wsh/wsh mice were compared with MRL/lpr-Kit^+/+ and MRL/lpr-Kit^wsh/+ mice with intact MCs.

Results: MRL/lpr-Kit^wsh/wsh mice developed skin lesions without infiltrating MCs. As similar skin lesions on MRL/lpr-Kit^+/+ mice and MRL/lpr-Kit^wsh/+ mice contain comparable number of MCs, these mice were collectively analyzed as MRL/lpr mice with MCs. Compared with MRL/lpr mice with MCs, skin lesions developed earlier and showed consistently higher severity, with significantly higher mRNA expressions of many inflammatory cytokines in the dorsal skin on MRL/lpr mice without MCs. Furthermore, survival rate was significantly lower in MRL/lpr mice without MCs. The number of infiltrating MCs significantly increased in association with the severity of skin lesions in MRL/lpr mice with MCs.

Conclusions: These results demonstrated that MCs are infiltrated to suppress the progression of LE-like skin lesions in MRL/lpr mice.     

Acute Alcohol Intoxication Impairs the Hematopoietic Precursor Cell Response to Pneumococcal Pneumonia

Background: Alcohol abuse is associated with an increased incidence and severity of pneumonia. In both the general population and individuals consuming excess alcohol, Streptococcus pneumoniae is the most frequent lung infection pathogen. Alcoholic patients with pneumonia frequently present with granulocytopenia, which is predictive of increased mortality. The mechanisms underlying this impaired granulopoietic response to pneumococcal pneumonia have yet to be elucidated. Methods: Acute alcohol intoxication was induced in mice 30 minutes before intrapulmonary infection with S. pneumoniae. Bone marrow, lung, and blood samples were collected. Bone marrow cells were also isolated from naïve mice and treated in vitro with plasma from mice infected with S. pneumoniae. Results: Alcohol intoxication impaired the pneumococcal‐induced increase in granulocyte recruitment into the alveolar space, decreased bacterial clearance from the lung, and increased mortality. Pneumococcal pneumonia significantly increased bone marrow lineage^?c‐Kit⁺Sca‐1⁺ (LKS) cell number and colony‐forming unit—granulocytes and monocyte (CFU‐GM) activity of these cells. Both enhanced proliferation of LKS cells and re‐expression of Sca‐1 surface protein on downstream progenitor cells bearing lineage^?c‐Kit⁺Sca‐1^? surface markers accounted for the expansion of marrow LSK cells during pneumonia. Alcohol intoxication impaired these 2 mechanisms of LKS cell population expansion and was associated with a relative granulocytopenia during pneumococcal lung infection. Conclusions: Alcohol inhibits the hematopoietic precursor cell response to pneumonia, which may serve as a mechanism underlying the granulocytopenia and impaired host defense in alcohol abusers with bacterial pneumonia.     

Rapid retroviral infection of human haemopoietic cells of different lineages: efficient transfer in fresh T cells

In order to develop a clinically feasible gene marking approach, we have used the recently described PINCO retroviral expression system, composed of the enhanced green fluorescence protein (EGFP) cDNA driven by Moloney MLV LTR and packaged in the Phoenix amphotropic cell line. Two T, five B, one erythromyeloid and three myeloid cell lines were successfully infected with % GFP⁺ cells ranging from 4% to 79%, showing a lineage-dependent difference in infection susceptibility, with the myeloid cells being the least efficiently infected. We also infected normal mononuclear peripheral cells cultured in PHA and rhIL-2 for 2 d, and obtained an average of 30% GFP⁺ cells, all present within the CD3⁺ population, with CD4⁺ and CD8⁺ cells being equally infected. Finally, the tonsillar purified B population showed lower levels of infectivity (6%) whereas high susceptibility was shown by normal human umbilical vein endothelial cells (57%). Highly purified CD34⁺ cells were also susceptible, varying from 6% to 10% GFP⁺ cells. Immature myeloid/erythroid progenitors have been infected which stably expressed the GFP protein during further differentiation in culture. The GFP⁺ T cells were FACS-sorted rapidly upon infection, subsequently cultured and the fluorescence intensity monitored. In all cases the difference in percentage of GFP⁺ cells did not correlate with the percentage of S/G₂/M cycling cells as determined at the moment of infection or with the expression levels of Ram-1 amphotrophic receptor. The improved safety of this retroviral system, the rapidity of the technique, the high efficiency of infection with respect to normal T lymphocytes (in this last case higher than previously reported) and the lack of need for in vitro selection make this system favourable for clinical development.