Increase in rat intestinal permeability to endotoxin during hyperthermia

Victims of heat stroke exhibit several clinical features which are also encountered in endotoxaemia. In order to investigate these similarities hyperthermic rats were used to explore the possibility that high body temperature results in increased permeability of intestinal wall to endotoxin. 125I endotoxin was introduced into intestinal segments taken from non-heat exposed rats. The segments were then incubated at 37 degrees C or 45 degrees C. Intestinal segments from heat stressed rats were similarly prepared and incubated at 37 degrees C. Leakage of endotoxin from segments taken from heat stressed rats was three times greater than from those from non-heat stressed rats, as were the segments from non-heat stressed rats which were incubated at 45 degrees C. These results indicate that the intestinal membrane is damaged by heat and that an increase in outward leakage of microbial endotoxins from the gut then occurs. This might contribute to the pathophysiological picture of heatstroke.     

非酒精性脂肪性肝炎患者肠道菌群失调与肠道通透性及血清内毒素的相关性研究

目的 探讨非酒精性脂肪性肝炎患者肠道菌群失调与肠道通透性及血清内毒素的相关性.方法 选择肠道菌群中具有代表性的细菌共8种进行培养.研究对象为健康成人(A组)和非酒精性脂肪性肝炎(B组)各30例,计数两组肠道菌群中8种细菌的数量,检测所有被研究者的血清内毒素、二胺氧化酶(DAO)、D-乳酸及TNF-α的浓度,比较两组细菌数量和血清指标的变化,并进行相关分析.结果 与A组比较,B组双歧杆菌、乳杆菌和拟杆菌的菌落数显著减少(P＜0.01或P＜0.05),而肠球菌、肠杆菌的菌落数则有显著增加(P＜0.01或P＜0.05);酵母真菌、葡萄球菌、梭菌菌落数没有发生显著改变(P＞0.05);血清内毒素、DAO、D-乳酸、TNF-α水平显著增高(P＜0.01);相关分析显示肠杆菌与内毒素、DAO、D-乳酸相关(r=0.644,P＜0.001;r=0.415,P=0.023;r =0.383,P=0.037);血清内毒素和DAO、D-乳酸、TNF-α显著相关(r=0.485,P=0.007;r=0.477,P=0.008;r=0.490,P=0.006);TNF-α则与DAO、D-乳酸相关(r=0.426,P =0.019;r =0.440,P=0.015).结论 非酒精性脂肪性肝炎患者存在肠道菌群失调、肠道通透性增高及肠源性内毒素血症,肠杆菌的过度生长与肠源性内毒素血症及肠道通透性密切相关.     

Increased intestinal permeability correlates with sigmoid mucosa alpha-synuclein staining and endotoxin exposure markers in early Parkinson's disease

Parkinson's disease (PD) is the second most common neurodegenerative disorder of aging. The pathological hallmark of PD is neuronal inclusions termed Lewy bodies whose main component is alpha-synuclein protein. The finding of these Lewy bodies in the intestinal enteric nerves led to the hypothesis that the intestine might be an early site of PD disease in response to an environmental toxin or pathogen. One potential mechanism for environmental toxin(s) and proinflammatory luminal products to gain access to mucosal neuronal tissue and promote oxidative stress is compromised intestinal barrier integrity. However, the role of intestinal permeability in PD has never been tested. We hypothesized that PD subjects might exhibit increased intestinal permeability to proinflammatory bacterial products in the intestine. To test our hypothesis we evaluated intestinal permeability in subjects newly diagnosed with PD and compared their values to healthy subjects. In addition, we obtained intestinal biopsies from both groups and used immunohistochemistry to assess bacterial translocation, nitrotyrosine (oxidative stress), and alpha-synuclein. We also evaluated serum markers of endotoxin exposure including LPS binding protein (LBP). Our data show that our PD subjects exhibit significantly greater intestinal permeability (gut leakiness) than controls. In addition, this intestinal hyperpermeability significantly correlated with increased intestinal mucosa staining for E. coli bacteria, nitrotyrosine, and alpha-synuclein as well as serum LBP levels in PD subjects. These data represent not only the first demonstration of abnormal intestinal permeability in PD subjects but also the first correlation of increased intestinal permeability in PD with intestinal alpha-synuclein (the hallmark of PD), as well as staining for gram negative bacteria and tissue oxidative stress. Our study may thus shed new light on PD pathogenesis as well as provide a new method for earlier diagnosis of PD and suggests potential therapeutic targets in PD subjects. TRIAL REGISTRATION: Clinicaltrials.gov NCT01155492.     

Mouse intestinal sensitivity to cycloheximide

Summary Cycloheximide 40 mg/kg was injected into adult male mice. Biochemical, histochemical and morphological modifications of the mucosal cells of the duodenum have been recorded after one or several injections. Biochemical experiments have shown a rapid decrease of alkaline phosphatase activity in the duodenum and a significant increase in the liver. Histochemical studies have shown a total disappearance of the alkaline phosphatase activity in the Golgi zone of the absorbing cells, two hours after one injection. Acid phosphatase, thiamine-pyrophosphatase and glucose-6-phosphatase did not exhibit the same pattern. Alterations of the ultrastructure of the villus cells have been observed after several injections. Atrophy of the Golgi cisternae, appearance of autophagic vacuoles and decrease of the goblet cells secretory granules have been the most frequent modifications.This work was supported by grants from the Medical Research Council of Canada and from the National Defense Board of Canada.The authors are greatly indebted to Mr. M. Couture for his skillful technical help.Mr. Charuel is a recipient of a studentship from the France-Quebec agreement.     

Mesenchymal stem cells stimulate intestinal stem cells to repair radiation-induced intestinal injury

The loss of stem cells residing in the base of the intestinal crypt has a key role in radiation-induced intestinal injury. In particular, Lgr5⁺ intestinal stem cells (ISCs) are indispensable for intestinal regeneration following exposure to radiation. Mesenchymal stem cells (MSCs) have previously been shown to improve intestinal epithelial repair in a mouse model of radiation injury, and, therefore, it was hypothesized that this protective effect is related to Lgr5⁺ ISCs. In this study, it was found that, following exposure to radiation, transplantation of MSCs improved the survival of the mice, ameliorated intestinal injury and increased the number of regenerating crypts. Furthermore, there was a significant increase in Lgr5⁺ ISCs and their daughter cells, including Ki67⁺ transient amplifying cells, Vil1⁺ enterocytes and lysozyme⁺ Paneth cells, in response to treatment with MSCs. Crypts isolated from mice treated with MSCs formed a higher number of and larger enteroids than those from the PBS group. MSC transplantation also reduced the number of apoptotic cells within the small intestine at 6 h post-radiation. Interestingly, Wnt3a and active β-catenin protein levels were increased in the small intestines of MSC-treated mice. In addition, intravenous delivery of recombinant mouse Wnt3a after radiation reduced damage in the small intestine and was radioprotective, although not to the same degree as MSC treatment. Our results show that MSCs support the growth of endogenous Lgr5⁺ ISCs, thus promoting repair of the small intestine following exposure to radiation. The molecular mechanism of action mediating this was found to be related to increased activation of the Wnt/β-catenin signaling pathway.The epithelium of the small intestine contains crypts and villi. Intestinal stem cells (ISCs) reside in the base of the crypts and are responsible for maintaining intestinal epithelial homeostasis and regeneration following injury.^{1, 2} Recent studies have identified two populations of stem cells in the small intestine of mice called Lgr5⁺ and Bmi1⁺ ISCs.^{3, 4, 5, 6, 7, 8, 9, 10, 11} Lgr5⁺ ISCs, also known as crypt base columnar cells (CBCs), are interspersed among the Paneth cells and are active rapidly cycling stem cells.¹² A single Lgr5⁺ ISC can grow to form ‘enteroids'' in vitro that develop into all the differentiated cell types found in the intestinal crypt.¹³ Conversely, Bmi1⁺ cells are a population of ISCs located at position +4 relative to the base of the crypt, and are quiescent, slowly cycling stem cells.¹⁴ The loss of ISCs has a critical role in radiation-induced intestinal injury (RIII).^{15, 16, 17, 18} Apoptosis of stem cells because of exposure to radiation prevents normal re-epithelialization of the intestines. Therefore, enhancing the survival of ISCs following radiation is a potential effective treatment for RIII.Mesenchymal stem cells (MSCs) possess significant potential as a therapeutic for tissue damage because of their ability to regulate inflammation, inhibit apoptosis, promote angiogenesis, and support the growth and differentiation of local stem and progenitor cells.^{19, 20} However, the mechanisms by which MSCs mediate these beneficial effects remain unclear, although it has been suggested that MSCs may actively secrete a broad range of bioactive molecules with immunomodulatory (PGE2, IDO, NO, HLA-G5, TSG-6, IL-6, IL-10 and IL-1RA), mitogenic (TGFα/β, HGF, IGF-1, bFGF and EGF), angiogenic (VEGF and TGF-β1) and/or anti-apoptotic (STC-1 and SFRP2) properties that function to modulate the regenerative environment at the site of injury.²¹ Upon re-establishment of the microenvironment following damage, the surviving endogenous stem and progenitor cells can then regenerate the injured tissue completely.Our previous study, as well as other published studies, has found that systemic administration of MSCs improves intestinal epithelial repair in an animal model of radiation injury.^{22, 23, 24, 25} Following MSC treatment, radiation-induced lesions in mice were significantly smaller than those in the control group. However, the mechanism behind this protective effect is not fully understood. Lgr5⁺ ISCs have been previously shown to be indispensable for radiation-induced intestinal regeneration.²⁶ Therefore, in this study, we tested whether the therapeutic effects of MSCs in response to RIII are related to the Lgr5⁺ population of resident ISCs.     

Nuclear scintigraphic assessment of radiation-induced intestinal dysfunction

Radiation-induced damage to the intestine can be measured by abnormalities in the absorption of various nutrients. Changes in intestinal absorption occur after irradiation because of loss of the intestinal absorptive surface and a consequent decrease in active transport. In our study, the jejunal absorption of (99m)Tc-pertechnetate, an actively transported gamma-ray emitter, was assessed in C3H/Kam mice given total-body irradiation with doses of 4, 6, 8 and 12.5 Gy and correlated with morphological changes in the intestinal epithelium. The absorption of (99m)Tc-pertechnetate from the intestinal lumen into the circulation was studied with a dynamic gamma-ray-scintigraphy assay combined with a multichannel analyzer to record the radiometry data automatically in a time-dependent regimen. The resulting radioactivity-time curves obtained for irradiated animals were compared to those for control animals. A dose-dependent decrease in absorptive function was observed 3.5 days after irradiation. The mean absorption rate was reduced to 78.8 +/- 9.3% of control levels in response to 4 Gy total-body irradiation (mean +/- SEM tracer absorption lifetime was 237 +/- 23 s compared to 187 +/- 12 s in nonirradiated controls) and to 28.3 +/- 3.7% in response to 12.5 Gy (660 +/- 76 s). The decrease in absorption of (99m)Tc-pertechnetate at 3.5 days after irradiation correlated strongly (P < 0.001) with TBI dose, with the number of cells per villus, and with the percentage of cells in the crypt compartment that were apoptotic or mitotic. A jejunal microcolony assay showed no loss of crypts and hence no measured dose-response effects after 4, 6 or 8 Gy TBI. These results show that dynamic enteroscintigraphy with sodium (99m)Tc-pertechnetate is a sensitive functional assay for rapid evaluation of radiation-induced intestinal damage in the clinically relevant dose range and has a cellular basis.     

Influence of physical loading on the content of intestinal microflora endotoxin in blood plasma and immunity indices to endotoxin

The content of endotoxin of Gram-negative bacteria and humoral and granulocytic immunity indices to endotoxin in sportsmen and untrained persons before and after undergoing physical load were studied. The study revealed that prior to physical load the content of intestinal microflora endotoxin in the blood stream of volunteer students increased and the humoral immunity indices to endotoxin decreased. Judging by the content of cortisol in the blood, the state of stress increased after a physical load. In the groups with good adaptation a decreased endotoxin content in the blood stream was noted, especially 24 hours after load. On the contrary, in the groups with dysadaptation the content of endotoxin in blood plasma increased after a load and was accompanied by a decrease in antibody titers and in the reserve of leukocytes capable of binding endotoxin.     

The intestinal radiation syndrome: sepsis and endotoxin

Rats were whole-body irradiated with 8-MeV cyclotron-produced neutrons and 137Cs gamma rays to study the role of enteric bacteria and endotoxin in the intestinal radiation syndrome. Decrease in intestinal weight was used as an index of radiation-induced breakdown of the mucosa. Neutron and gamma-ray doses that were sublethal for intestinal death resulted in a dose-dependent decrease in intestinal weight, reaching minimal values 2 to 3 days after exposure, followed by recovery within 5 days after irradiation. Neutron and photon doses that caused intestinal death resulted in greater mucosal breakdown with little or no evidence of mucosal recovery. The presence of fluid in the intestine and diarrhea, but not bacteremia or endotoxemia, were related to mucosal breakdown and recovery. Neither sepsis nor endotoxin could be detected in liver samples taken at autopsy from animals which died a short time earlier from intestinal injury. These results suggest that overt sepsis and endotoxemia do not play a significant role in the intestinal radiation syndrome.     

Phlorizin increases the permeability of intestinal mucosal membrane to sodium

We reported previously that when jejunal transmural glucose transport was inhibited by phlorizin the ratio of Na:glucose transport increased from 2.0:1 (in controls) to 3.3:1. To elucidate the mechanism of this increased ratio of Na:glucose transport, in the present study we have investigated the effect of phlorizin on Na uptake by brush border membrane vesicles and by everted sacs of hamster jejunum. In experiments on membrane vesicles the following observations were made. The time course of Na uptake showed that the control vesicles were in complete equilibrium with a Na-containing (100 mM) medium between 30 and 90 min incubation. In these periods of incubation, the vesicles incubated with phlorizin presumably also equilibrated with the medium, but lost their intravesicular Na during Millipore filtration and washing, and consequently the residual Na content was lower than that of controls. This effect of phlorizin was concentration dependent, and appeared to be unrelated to Na-coupled glucose transport, because it was also observed in the absence of glucose. This loss of Na during Millipore filtration and washing was also observed (i) when vesicles were equilibrated in a Na-containing solution in the absence of phlorizin and then exposed to a similar solution containing phlorizin, or (ii) when vesicles were equilibrated in a Na-containing solution in the presence of phlorizin and then washed repeatedly following Millipore filtration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Early detection of radiation-induced glomerular injury by albumin permeability assay

Renal irradiation leads predictably to glomerular vascular injury, cell lysis, matrix accumulation, sclerosis and loss of renal function. The immediate effects of renal irradiation that may be associated with glomerular pathology and proteinuria are not clear in the human disease or its rat model. We hypothesized that radiation-induced injury causes immediate and subtle alterations in glomerular physiology independent of the neurohumoral and hemodynamic regulatory mechanisms. We employed a sensitive in vitro functional assay of glomerular albumin permeability (P(alb)) to demonstrate radiation-induced damage to the glomerular filtration barrier immediately after total-body irradiation of rats. In blinded experiments, control rats were sham-treated, and experimental rats received 9.5 Gy X rays. Rats were killed humanely at 1 h to 9 weeks after irradiation and glomeruli were isolated. In parallel experiments, glomeruli were isolated from normal rats and irradiated in vitro. The change in glomerular capillary permeability due to an experimental oncotic gradient was determined using videomicroscopy and P(alb) was calculated. Results show that in vivo or in vitro irradiation of glomeruli caused an increased P(alb) at 1 h. Increased P(alb) was observed up to 3 weeks after irradiation. Glomeruli from mice irradiated with 9.5 or 19.0 Gy X rays did not show increased P(alb) at 1 h postirradiation. We conclude that glomerular protein permeability of irradiated rats increases in a dose-dependent manner immediately after irradiation and that it appears to be independent of hemodynamic or systemic influences.     

Association of Treponema hyodysenteriae with porcine intestinal mucosa

The association of Treponema hyodysenteriae with porcine caecal and colonic mucosal surfaces was studied by electron microscopy after orogastric inoculation of pigs with pure cultures. Examination of caecal and colonic mucosa from infected and control animals revealed that large numbers of the spirochaete were associated only with intestinal mucosal surfaces of infected animals. Further examination of the intestinal mucosa from infected pigs showed that T. hyodysenteriae colonized two sites preferentially: the mucus-filled crypts of Lieberkühn and the mucus gel covering the epithelium. Furthermore, no evidence of either specific or nonspecific adhesion to the epithelium proper was found, suggesting that penetration of, or trapping in the mucus gel may be the predominant mechanism of mucosal association by T. hyodysenteriae. Moreover, T. hyodysenteriae was also observed to be highly motile in intestinal mucus, moving faster than any other organism present, and this 'high speed' motility appeared to facilitate penetration into the mucosa. The pattern of motility observed was also highly suggestive of chemotaxis, and this was subsequently confirmed using an in vitro assay to porcine mucus material. It is suggested, therefore, that motility and chemotaxis are important factors/mechanisms in the association and colonization of porcine intestinal mucosa by T. hyodysenteriae.     

Association of endogenous retroviruses with radiation-induced leukemias of BALB/c mice.

X-irradiation of BALB/c mice in the second month of life induced a high incidence of generalized lymphatic leukemia of T-cell origin, beginning at 7 months of age. Infectious ecotropic murine leukemia virus (B-tropic predominant over N-tropic) was isolable from all tumor extracts but exhibited a wide titer range among individual leukemias. Detection of infectious xenotropic virus usually required extensive amplification on indicator cells. Dual-tropic (mink cell focus-forming) virus has not been found in the leukemias. Expression of ecotropic virus in tail extracts prepared at 6.5 months of age, although greatly enhanced compared with unirradiated controls, was not found to be prognostic of tumor development in individual mice. We conclude that leukemogenesis does not show a simple dependence on infectious murine leukemia virus expression in these mice.     

Folic acid deficiency increases chromosomal instability, chromosome 21 aneuploidy and sensitivity to radiation-induced micronuclei

Folic acid deficiency can lead to uracil incorporation into DNA, hypomethylation of DNA, inefficient DNA repair and increase chromosome malsegregation and breakage. Because ionising radiation increases demand for efficient DNA repair and also causes chromosome breaks we hypothesised that folic acid deficiency may increase sensitivity to radiation-induced chromosome breakage. We tested this hypothesis by using the cytokinesis-block micronucleus assay in 10 day WIL2-NS cell cultures at four different folic acid concentrations (0.2, 2, 20, and 200 nM) that span the "normal" physiological range in humans. The study showed a significant dose-dependent increase in frequency of binucleated cells with micronuclei and/or nucleoplasmic bridges with decreasing folic acid concentration (P<0.0001, P=0.028, respectively). These biomarkers of chromosomal instability were also increased in cells irradiated (1.5 Gy gamma-rays) on day 9 relative to un-irradiated controls (P<0.05). Folic acid deficiency and gamma-irradiation were shown to have a significant interactive effect on frequency of cells containing micronuclei (two-way ANOVA, interaction P=0.0039) such that the frequency of radiation-induced micronucleated cells (i.e. after subtracting base-line frequency of un-irradiated controls) increased with decreasing folic acid concentration (P-trend<0.0001). Aneuploidy of chromosome 21, apoptosis and necrosis were increased by folic acid deficiency but not by ionising radiation. The results of this study show that folate status has an important impact on chromosomal stability and is an important modifying factor of cellular sensitivity to radiation-induced genome damage.     

Association of ionizing radiation-induced foci of NBS1 with chromosomal instability and breast cancer susceptibility

NBS1, a protein essential for DNA double-strand break repair, relocalizes into subnuclear structures upon induction of DNA damage by ionizing radiation, forming ionizing radiation-induced foci. We compared radiation-induced NBS1 foci in peripheral blood lymphocytes (PBLs) from 46 sporadic breast cancer patients and 30 healthy cancer-free volunteers. The number of persistent radiation-induced NBS1 foci per nucleus at 24 h after irradiation for patients with invasive cancer was significantly higher than for normal healthy volunteers. The frequency of spontaneous chromosome aberration increased as the number of persistent radiation-induced NBS1 foci increased, indicating that the number of persistent radiation-induced NBS1 foci might be associated with chromosome instability. There was also an inverse correlation between the number of radiation-induced NBS1 foci and the activity of DNA-dependent protein kinase (DNA-PK), which plays an important role in the nonhomologous end-joining (NHEJ) pathway, another mechanism of DNA DSB repair, indicating a close interrelationship between homologous recombination (HR) and NHEJ in DNA DSB repair. In conclusion, the number of persistent radiation-induced NBS1 foci is associated with chromosomal instability and risk of sporadic breast cancer and hence might be used to select individuals for whom a detailed examination is necessary because of their increased susceptibility to breast cancer, although refinement of the techniques for technical simplicity and accuracy will be required for clinical use.