Inhibition of Bacterial Translocation from the Gastrointestinal Tract of Mice Injected with Cyclophosphamide

Effects of intraperitoneal injection of cyclophosphamide, an immunosuppressant, on the degree of bacterial translocation and morphological changes of Peyer's patches (PP) in the intestine were investigated with antibiotic-decontaminated SPF mice and germfree mice monoassociated with Escherichia coli C25. It has been reported that treatment with cyclophosphamide induces bacterial translocation. Cyclophosphamide treatment in this study, however, significantly decreased E. coli C25 translocation from the gastrointestinal tract to the mesenteric lymph nodes (MLN), although the numbers of lymphoid cells, especially B cells, in the PP, MLN, and spleen were remarkably reduced. Four injections of cyclophosphamide at a dose of 100 mg/kg inhibited bacterial translocation more than one injection at a dose of 200 mg/kg in SPF mice. Germfree mice, however, treated with one dose of 200 mg/kg showed the same inhibition of bacterial translocation as those given 100 mg/kg four times. In cyclophosphamide-treated mice, lymph follicles in the PP were obviously smaller than those in control mice, M-cells were similar in appearance to absorption epithelial cells except for short microvilli, and immune cells among the M-cells had disappeared. These data suggested that inhibition of bacterial translocation in mice treated with cyclophosphamide may be the result of morphological and physiological changes of epithelial cells in the gastrointestinal tract, especially M-cells, as a point of entry of invading bacteria, independent of the changes in immunological function. Received: 16 November 1995 / Accepted: 12 December 1995     

Bacterial translocation from the gastrointestinal tracts of thymectomized mice

The incidence of translocation of viable indigenous bacteria from the gastrointestinal tract to the mesenteric lymph node, spleen, liver, and kidney was compared in neonatally thymectomized mice and sham-thymectomized specific pathogen-free mice. The immunologic responses of the thymectomized mice to sheep erythrocytes were decreased compared to the responses of sham-thymectomized mice. Strictly anaerobic bacteria were isolated from only 1.8% of the organs from thymectomized mice and from none of the organs of shamthymectomized mice. Aerobic or facultatively anaerobic bacteria were cultured from 27.4% of the organs of thymectomized mice. Of the thymectomized mice, 70.7% contained viable aerobic or facultatively anaerobic bacteria in one or more of their organs tested, compared with only 10% of the sham-thymectomized mice.Escherichia coli was the predominant bacterial species isolated from these organs, althoughStaphylococcus aureus, Streptococcus, andCorynebacterium also were present.Bacteroides were the only strictly anaerobic bacteria cultured. Neonatal thymectomy promotes the translocation of certain indigenous bacteria from the gastrointestinal tract to the mesenteric lymph node, spleen, liver, and kidney.     

Bacterial translocation from the gastrointestinal tract in various mouse models for human diabetes.

Bacterial translocation from the gastrointestinal (GI) tract to other internal organs was examined in multiple low-dose streptozotocin-injected (M-STZ), single large-dose streptozotocin-injected (S-STZ), alloxan-injected (Alloxan), and non-obese diabetic (NOD) mice. The incidence of bacterial translocation from the GI tract to the tested organs among diabetic mice was in the order of M-STZ mice greater than S-STZ mice greater than NOD, Alloxan, and control mice. The injections of insulin to M-STZ mice did not decrease the incidence of translocation. These results suggest that bacterial translocation from the GI tract in diabetic mice is not induced by diabetes.     

The chromosome-damaging action of 2 chemical vaccine contaminants on mice

The cytogenetic effects of two chemical agents, hydroxylamine used for the destruction of bacterial cells and thimerosal added to many immunobiological preparations as preservative, were studied in vivo by their action on the marrow cells of C57BL/6J mice. The preparations under study, when injected intraperitoneally in a wide range of doses, including subtoxic ones, induced no chromosomal aberrations. At the same time cyclophosphamide, an antitumor cytostatic agent used for positive control, produced a pronounced damaging effect on chromosomes.     

Effect of age on bacterial translocation from the gastrointestinal tract in mice.

To clarify the effects of age on bacterial translocation from the gastrointestinal tract, mice at the age of 1, 2, 4, 6, 12, and 15 months were antibiotic-decontaminated for 4 days and then inoculated orally with streptomycin-resistant Escherichia coli C25. Mice treated with cyclophosphamide and untreated controls were tested for bacterial translocation to the mesenteric lymph nodes (MLN) 2 days later. The population levels of E. coli C25 in cyclophosphamide-treated and untreated mice were approximately 10(9.3) and 10(9.5) per gram of cecum, respectively, at each tested age. There were no significant differences in the incidence of translocation of E. coli C25 to MLN at any of the tested ages, whereas the number of E. coli C25 detected in MLN was higher in young mice than in aged mice in both the cyclophosphamide-treated and untreated groups. These findings suggest that bacterial translocation from the GI tract may be a more important problem in young animals than in aged animals.     

Bacterial translocation from the intestines

Bacterial translocation is defined as the passage of viable bacteria from the gastrointestinal (GI) tract through the mucosal epithelium to other sites, such as the mesenteric lymph nodes, spleen, liver and blood. This paper reviews results from animal models utilized to obtain information concerning the defense mechanisms operating in the healthy host to confine bacteria to the GI tract. Gnotobiotic and antibiotic-decontaminated mice colonized with particular bacteria demonstrated that the indigenous GI flora maintains an ecologic equilibrium to prevent intestinal bacterial overgrowth and translocation from the GI tract. Studies with athymic (nu/nu) mice, thymus-grafted (nu/nu) mice, neonatally thymectomized mice, and mice injected with immunosuppressive agents demonstrated that the host immune system is another defense mechanism inhibiting bacterial translocation from the GI tract. Ricinoleic acid given orally to mice disrupted the intestinal epithelial barrier allowing indigenous bacteria to translocate from the GI tract. Thus, bacterial translocation from the GI tract of healthy adult mice is inhibited by: (a) an intact intestinal epithelial barrier, (b) the host immune defense system, and (c) an indigenous GI flora maintaining ecological equilibrium to prevent bacterial overgrowth. Deficiencies in host defense mechanisms act synergistically to promote bacterial translocation from the GI tract as demonstrated by animal models with multiple alterations in host defenses. Bacterial translocation occurred to a greater degree in mice with streptozotocin-induced diabetes, mice receiving nonlethal thermal injury, and mice receiving the combination of an immunosuppressive agent plus an oral antibiotic than in mice with only a primary alteration in host defenses. The study of bacterial translocation in these complex models suggests that opportunistic infections from the GI tract occur in discrete stages. In the healthy adult animal, bacterial translocation from the GI tract either does not occur or occurs at a very low level and the host immune defenses eliminate the translocating bacteria. Bacterial translocation does take place if one of the host defense mechanisms is compromised, such as a deficiency in the immune response, bacterial overgrowth in the intestines, or an increase in the permeability of the intestinal barrier. In this first stage, the bacteria usually translocate in low numbers to the mesenteric lymph node, and sometimes spleen or liver, but do not multiply and spread systemically.(ABSTRACT TRUNCATED AT 400 WORDS)     

Translocation ofLactobacillus murinus from the gastrointestinal tract

Bacterial translocation is defined as the passage of viable bacteria from the gastrointestinal tract to the mesenteric lymph nodes and other extraintestinal sites. The translocation rate of a newly described species of indigenous bacteria,Lactobacillus murinus, was compared with the translocation rates of indigenousLactobacillus acidophilus and nonindigenousSalmonella enteritidis. Groups of germfree or antibiotic-decontaminated, specific pathogen-free mice were monoassociated with each of these bacterial strains and tested at various intervals for translocation to the mesenteric lymph nodes. The translocation rates of the various bacteria expressed in decreasing order as the numbers of translocating bacteria per gram mesenteric lymph node wereS. enteritidis, L. murinus, andL. acidophilus. The degree of histologic damage to the gastrointestinal mucosa after monoassociation with these strains followed the same pattern. Thus,L. murinus translocates from the GI tract at a surprisingly high rate for an indigenous bacterial strain, and its translocation appears to be associated with mucosal alterations.     

Low translocation of Bacillus thuringiensis israelensis to inner organs in mice after pulmonary exposure to commercial biopesticide

Translocation of viable cells from a Bacillus thuringiensis israelensis-based biopesticide to inner organs in a mouse model was studied. Mice were exposed to the originally formulated product through the lungs and gastrointestinal tract by intratracheal instillation. Colony forming units (CFU) were grown from lungs, caecum, spleen and liver on Bacillus cereus-specific agar (BCSA) after 24 h and finally determined to be biopesticide strain B. t. israelensis by large plasmid profile. No CFU were found in spleen or liver of the control mice or in any aerosol background or material. We have shown that viable cells from the commercial product can translocate to spleen and liver of immunocompetent mice in a dose-dependent manner. Furthermore, we discuss the methods of exposure and how bacterial translocation should be taken into consideration when evaluating the safety of novel or reintroduced biopesticides in the future.     

α-Tocopherol succinate protects mice against radiation-induced gastrointestinal injury

The purpose of this study was to elucidate the role of α-tocopherol succinate (α-TS) in protecting mice from gastrointestinal syndrome induced by total-body irradiation. CD2F1 mice were injected subcutaneously with 400 mg/kg of α-TS and exposed to different doses of (60)Co γ radiation, and 30-day survival was monitored. Jejunum sections were analyzed for crypts and villi, PUMA (p53 upregulated modulator of apoptosis), and apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling - TUNEL). The crypt regeneration in irradiated mice was evaluated by 5-bromo-2-deoxyuridine (BrdU). Bacterial translocation from gut to heart, spleen and liver in α-TS-treated and irradiated mice was evaluated by bacterial culture on sheep blood agar, colistin-nalidixic acid, and xylose-lysine-desoxycholate medium. Our results demonstrate that α-TS enhanced survival in a significant number of mice irradiated with 9.5, 10, 11 and 11.5 Gy (60)Co γ radiation when administered 24 h before radiation exposure. α-TS also protected the intestinal tissue of irradiated mice in terms of crypt and villus number, villus length and mitotic figures. TS treatment decreased the number of TUNEL- and PUMA-positive cells and increased the number of BrdU-positive cells in jejunum compared to vehicle-treated mice. Further, α-TS inhibited gut bacterial translocation to the heart, spleen and liver in irradiated mice. Our data suggest that α-TS protects mice from radiation-induced gastrointestinal damage by inhibiting apoptosis, promoting regeneration of crypt cells, and inhibiting translocation of gut bacteria.     

Chemotherapeutic effect of 3-(5-nitrofuryl)-7-(5-nitrofurfurylidene)-3,3a,4,5,6,7-hexahydro-2H-indazole in experimental staphylococcal infection]

Chemotherapeutic activity of 3-(5-nitrofuryl)-7-(5-nitrofurfuryliden)-3, 3a, 4, 5, 6, 7-hexahydro-2H-indazol (compound 26) was studied on albino mice with experimental staphylococcal infection. The animals were contaminated intraperitoneally. The results of culture of the specimens of the organs of the mice killed within various terms and the values of the spleen bacterial index served as the criteria of the effectiveness. Compound 26 was administered in doses of 20, 10 and 5 mg/kg once a day for 3 days. The treatment was started simultaneously with or 24 hours after the contamination. The results showed that compound 26 in a dose 20 mg/kg (0.4 mg/mouse) possessed high chemotherapeutic activity in experimental staphylococcal infection of albino mice and could be recommended for a thorough study as a potential agent for chemotherapy of staphylococcal infection.     

Assessment of mouse strain on bacterial translocation from the gastrointestinal tract

The translocation of indigenous bacteria from the gastrointestinal tract to the mesenteric lymphnodes was compared in ten strains of mice. Indigenous Escherichia coli were cultured from the mesenteric lymphnodes of only two of the six mouse strains examined. Thus, spontaneous translocation of indigenous enteric bacteria across the intestinal barrier did not occur to any significant extent in any of the mouse strains examined. Since bacterial overgrowth in the gastrointestinal tract promotes bacterial translocation, bacterial translocation was tested in ten mouse strains including B10 series after antibiotic-decontaminated and subsequent colonization with streptomycin-resistant E. coli C25. E. coli C25 populated the ceca of the mice at levels of 10(8) to 10(9) per gram and translocated to 90-100% of the mesenteric lymphnodes with mean of 10(1.13) to 10(1.86) per mesenteric lymphnode. However, there were no significant differences between mouse strains as to the translocation incidence or the numbers of viable E. coli C25 per mesenteric lymphnode. Thus, genetic differences between mouse strains did not influence bacterial translocation from the gastrointestinal tract to the mesenteric lymphnodes.     

<Emphasis Type="Italic">In vivo</Emphasis> studies with a <Emphasis Type="Italic">Candida tropicalis</Emphasis> isolate exhibiting paradoxical growth <Emphasis Type="Italic">in vitro</Emphasis> in the presence of high concentration of caspofungin

We investigated the activity of caspofungin against a Candida tropicalis clinical isolate showing paradoxical growth in vitro. BALB/c mice immunosuppressed by cyclophosphamide were infected intraperitoneally using 10⁷ CFU/mouse. Caspofungin was administered intraperitoneally once daily for 5 days or as a single dose using the following doses: 0.12, 0.25, 1, 2, 3, 5, and 15 mg/kg. The single dose of caspofungin was effective only at 5 and 15 mg/kg concentrations (100% survival). Five-day caspofungin treatment led to 100% survival at doses of 1 mg/kg or higher. Caspofungin treatment significantly decreased the number of viable yeasts in the peritoneal lavage samples as well as in the infected abscesses at doses 1, 3, 5, and 15 mg/kg caspofungin as compared to the untreated control (P<0.001 in all cases), and even to the group treated with 0.12 mg/kg caspofungin (P<0.05 in all cases). At 2 mg/kg caspofungin dose, sterilization of the internal organs was reproducibly incomplete, suggesting that the role of paradoxical growth in the late clinical failure cannot be excluded.     

Paradoxical synergistic effects of tumour necrosis factor and interleukin 1 in murine gut-derived sepsis with Pseudomonas aeruginosa.

The authors evaluated the synergistic effect of tumour necrosis factor (TNF) and interleukin 1 (IL-1) in gut-derived sepsis in mice. After colonization of Pseudomonas aeruginosa strain D4 in the gastrointestinal tract, cyclophosphamide was administered to induce bacterial translocation of the P. aeruginosa and thereby to cause gut-derived sepsis. In this model, treatment either with 8 microg/kg of recombinant human TNF-alpha (rhTNF-alpha) or 2 microg/kg of recombinant human interleukin 1alpha (rhIL-1alpha) solely did not affect the mortality, whereas combined administration of the same doses of rhTNF-alpha and rhIL-1alpha significantly increased the mortality rate in comparison with saline-treated mice. Bacterial counts in liver and blood were significantly higher in rhTNF-alpha and rhIL-1alpha treated mice than in saline-treated mice. Endogenous TNF-alpha and IL-1beta productions were stimulated after combined treatment with rhTNF-alpha and rhIL-1alpha. On the contrary to these adverse effects, combined treatment with 500 microg/kg of rhTNF-alpha and 50 microg/kg of rhIL-1alpha on the day before the administration of cyclophosphamide significantly reduced the mortality from septic infection. We conclude that TNF and IL-1 synergistically affect the mortality of mice after gut-derived sepsis due to P. aeruginosa in mice and the timing of treatment with these cytokines causes both extremes in their effects.     

千斤拔多糖对小鼠免疫功能的调节作用

为研究千斤拔多糖对正常及免疫低下小鼠免疫功能的调节作用,该研究选用SPF级BALB/c小鼠,免疫抑制小鼠采用隔天皮下注射环磷酰胺(40 mg·kg~(-1))5次,通过测定脾脏、胸腺质量及计算脏器指数,采用碳粒廓清法计算单核巨噬细胞吞噬功能,在鸡红细胞免疫后测定小鼠血清溶血素抗体水平,同时观察高、低剂量千斤拔多糖(剂量分别为500、1 000 kg·d~(-1))对正常小鼠及免疫低下小鼠免疫调节的影响。结果表明:千斤拔多糖高、低剂量组对正常及免疫低下小鼠均有不同程度的增加脾脏指数、胸腺指数的作用;千斤拔多糖高、低剂量组对正常及免疫低下小鼠均能提高廓清指数,但没有统计学意义;千斤拔多糖高剂量组对正常及免疫低下小鼠的血清溶血素抗体水平都有显著性提高。这说明千斤拔多糖能有效提高正常及免疫低下小鼠免疫功能的作用。     

Enhanced resistance againstEscherichia coli infection by subcutaneous administration of the hot-water extract ofChlorella vulgaris in cyclophosphamide-treated mice

Summary The effects ofChlorella vulgaris extract (CVE-A) on the recovery of leukocyte number and the augmentation of resistance to bacterial infection were examined in CDF1 mice made neutropenic by cyclophosphamide (CY). They were treated intraperitoneally with CY (150 mg/kg) on day 0, and were given CVE-A (50 mg/kg) subcutaneously (s. c.) every other day from day 1 to day 13 after CY treatment. CVE-A accelerated the recovery of polymorphonuclear leukocytes (PMN) in the peripheral blood in CY-treated mice. The number of granulocyte/monocyte-progenitor cells (CFU-GM) in the spleen increased rapidly and highly after the administration of CVE-A in CY-treated mice, in contrast to the absence of change due to CVE-A in the number of bone marrow cells in CY-treated mice. Administration of CVE-A in CY-treated mice enhanced the accumulation of PMN in the inflammatory site and the activity of the accumulated leukocyte cells in luminol-dependent chemiluminescence. The mice became highly susceptible to an intraperitoneal infection withE.coli on day 4 after CY treatment, whereas the mice given CVE-A showed an enhanced resistance againstE.coli infection, irrespective of the timing of challenge. The bacterial number in CY-treated mice increased explosively after inoculation, resulting in death within 24 h. A progressive elimination of bacteria was observed from 6 h in the peritoneal cavity, spleen and liver of CY-treated mice given CVE-A s.c. These results indicate that CVE-A can be used as a potent stimulant of nonspecific resistance to infection in neutropenic mice.     

Alteration of intestinal microbial ecology in mice by recombinant interleuken-2

In addition to its beneficial immunostimulatory properties, interleukin-2 (IL-2) has many significant side effects including gastrointestinal (GI) toxicities. Preliminary studies of the effects of IL-2 on GI bacterial translocation suggested that IL-2 altered intestinal bacterial population levels. In order to further define the effects of IL-2 on intestinal microecology, groups of specific pathogen-free C57BL/6 mice were injected subcutaneously twice daily for 5 days with various dosages of human recombinant IL-2 (up to a maximum of 1.6 mg/kg injection) or an equal volume of sterile buffer. One day after the final injections, IL-2 had significantly (P<.05) increased in a dose-dependent fashion the mean cecal population levels of indigenousEscherichia coli, other gram-negative bacilli, streptococci, and staphylococci. Maximum increases above control cecal population levels were more than 10,000-fold forE. coli and streptococci, 209-fold for gram-negative bacilli other thanE. coli, and 93-fold for staphylococci. These changes were completely reversible, with normal cecal population levels 12 days after the last IL-2 injection. IL-2 did not change the total cecal population levels of strictly anaerobic bacteria. Ileal and cecal structures, as assessed by light microscopy, were not altered by the highest dose of IL-2. When incubated in vivo with 1.6 mg/ml of IL-2, the growth of an indigenous strain ofE. coli was not different from growth in broth alone. Thus, IL-2 treatment caused the intestinal overgrowth of common opportunistic aerobic and facultatively anaerobic bacterial pathogens, without altering total population levels of strict anaerobes or affecting intestinal histology.     

The effect of prednisolone trimethylacetate on the pathogenicity of dermatophilus congolensis to white mice

Albino mice were successfully infected intravenously, subcutaneously, intraperitoneally and on the intact and scarified skin with Dermatophilus congolensis. Various gross lesions were observed. Large subcutaneous nodules were produced in mice that were injected subcutaneously. Lesions involving the internal organs were obtained in mice that had been injected intravenously and intraperitoneally in addition to microabsessation and scab formation on the tails of those that were injected intravenously. The application of D. congolensis on scarified skin resulted in scab formation covering the whole area while its application on non-scarified skin produced isolated lesions. The organism was isolated in pure culture from exudate, pus and scab material obtained from lesions. Severe histopathological changes were noted in sections prepared from the lesions. Premedication of mice with prednisolone trimethylacetate increased the severity of the experimentally induced lesions.     

Oral-Derived Bacterial Flora Defends Its Domain by Recognizing and Killing Intruders—A Molecular Analysis Using Escherichia coli as a Model Intestinal Bacterium

Within the same human gastrointestinal tract, substantial differences in the bacterial species that inhabit oral cavity and intestinal tract have been noted. Previous research primarily attributed the differences to the influences of host environments and nutritional availabilities (“host habitat” effect). Our recent study indicated that, other than the host habitat effect, an existing microbial community could impose a selective pressure on incoming foreign bacterial species independent of host-mediated selection (“community selection” effect). In this study, we employed in vitro microbial floras representing microorganisms that inhabit the oral cavities and intestinal tract of mice in combination with Escherichia coli as a model intestinal bacterium and demonstrated that E. coli displays a striking community preference. It thrived when introduced into the intestinal microbial community and survived poorly in the microbial flora of foreign origin (oral community). A more detailed examination of this phenomenon showed that the oral community produced oxygen-free radicals in the presence of wild-type E. coli while mutants deficient in lipopolysaccharides (LPS) did not trigger significant production of these cell-damaging agents. Furthermore, mutants of E. coli defective in the oxidative stress response experienced a more drastic reduction in viability when cocultivated with the oral flora, while the exogenous addition of the antioxidant vitamin C was able to rescue it. We concluded that the oral-derived microbial community senses the E. coli LPS and kills the bacterium with oxygen-free radicals. This study reveals a new mechanism of community invasion resistance employed by established microflora to defend their domains.     

Verapamil contributes to the clastogenic effects of acrylamide, cyclophosphamide, and dioxidine on somatic cells of BALB/C and C57BL/6 mice

The chromosome aberration assay of metaphase bone marrow cells was used to study the clastogenic effects of acrylamide, cyclophosphamide, dioxidine, and their combinations with Verapamil (a calcium antagonist) in male BALB/C and C57BL/6 mice. Verapamil gavage at single (5 mg/kg) and repeated doses (2.5 and 5 mg/kg five times at 24-h intervals) significantly enhanced the clastogenic activity of acrylamide (50 and 100 mg/kg intraperitoneally) in BALB/C mice; in C57BL/6 mice, this effect was only observed when they received Verapamil at doses of 2.5 mg/kg for 5 days. Verapamil administered repeatedly (2.5–10 mg, gavage) significantly increased the clastogenic activity of cyclophosphamide (10 mg/kg intraperitoneally) in C57BL/6 mice. In BALB/C mice, this effect of Verapamil was only observed at a dose of 10 mg/kg (gavage). When injected intraperitoneally at a single dose of 0.1–0.4 mg/kg, Verapamil significantly enhanced the clastogenic activity of cyclophosphamide in mice of both strains. This calcium antagonist produced identical effects when administered to BALB/C mice intraperitoneally (2.5 and 5 mg/kg) and by gavage (5 mg/kg) and to C57BL/6 mice intraperitoneally (5 and 10 mg/kg) and by gavage (2.5 mg/kg). Repeated administration of Verapamil (at all doses tested) promoted the clastogenic effect of dioxidine (100 mg/kg intraperitoneally) on C57BL/6 mice, having no such influence on BALB/C mice. These results demonstrate the co-clastogenic activity of Verapamil in mice and suggest that its specific manifestations depend on the dose, method, and route of drug administration and the genotype of test animals.     

A New Hypothalamic Polypeptide Is a Regulator of Myelopoiesis

The effect of hypothalamic proline-rich polypeptide (PRP) against Pseudomonas aeruginosa infection in BALB/c mice with leukopenia was investigated. Mice were treated with cyclophosphamide (CPA) and were then injected with PRP 24 h after CPA treatment. The lethal doses of P. aeruginosa were injected to mice when the number of peripheral blood leukocytes reached a nadir on day 5 after CPA administration. The administration of PRP significantly increased the survival of infected mice, and had a pronounced protective effect during the period of development of the infection. The number of bacteria in internal organs of PRP-treated mice was significantly lower than that in control mice. In PRP-treated mice, the neutrophil levels in peripheral blood started to increase 7 days after CPA administration and were consistently higher, and they were more mature than those in controls. Our results may indicate the ability of PRP to stimulate recovery of myelopoiesis and enhance mature neutrophil function.