Effect of lysed Enterococcus faecalis FK-23 (LFK) on allergen-induced peritoneal accumulation of eosinophils in mice

BACKGROUND: The interest in anti-allergy immunoregulation by lactic acid bacteria has been growing for the last few decades. There is some evidence to suggest that lysed Enterococcus faecalis FK-23 (LFK) could relieve the clinical symptoms of pollinosis. However, the mechanism responsible for this phenomenon remains unknown. OBJECTIVE: To identify the effect of LFK, a lysozyme treated and heat-killed preparation from the lactic acid bacteria Enterococcus faecalis FK-23 strain, on allergen-induced eosinophil accumulation. METHODS: BALB/c mice were sensitized with ragweed pollen extract, and peritoneal accumulation of eosinophils was induced. A total of 60 mg (0.5 mL) LFK was orally administered to the experimental mouse every day during 21 days of the sensitization period. In addition, LFK 4 mg, 25 mg and 60 mg (each 0.5 mL) were also orally administered to a mouse of each group every day for 21 days. Saline was fed in a dose of 0.5 mL/mouse per day for the same duration as a control. RESULTS: Compared with control mice, LFK-treated mice exhibited decreased ragweed pollen allergen-induced peritoneal accumulation of eosinophils (P = 0.013), which showed a tendency to be in a dose-dependent fashion (P = 0.14). CONCLUSION: The results provide laboratory evidence of the role for LFK, a lactic acid bacteria preparation, in combating eosinophil accumulation.     

Effect of water-soluble fraction from lysozyme-treated Enterococcus faecalis FK-23 on mortality caused by influenza A virus in mice

To maintain homeostasis of the immune system is considered important for the prevention of influenza A virus infection. Aberrant systemic inflammation is frequently induced by influenza A virus infection, and the severity of the symptoms is associated with pathogenicity of the virus. Lactic acid bacteria are known to have a positive effect in maintaining the immune system. Furthermore, preparations of a lactic acid bacteria strain, Enterococcus faecalis FK-23 (FK-23), have been reported to exert preferable homeostatic effects on immune diseases such as allergic rhinitis and early asthmatic responses. In this study, we examined the efficacy of the water-soluble fraction of lysed and heat-treated FK-23 (SLFK) against a lethal influenza A virus challenge. Mice were orally administered SLFK from day -7 to day 20, and intranasally infected with influenza virus A/Puerto Rico/8/34 (H1N1) at 10(3) PFU on day 0. The survival rate of SLFK-administered mice after influenza A virus infection was significantly improved compared with that of control mice. In addition, the mRNA expression level of the anti-inflammatory cytokine interleukin-10 (IL-10) in lung tissues was enhanced by the oral administration of SLFK after influenza A virus infection. These observations suggest that the oral administration of SLFK exerts a protective effect against influenza virus infection through the activation of the anti-inflammatory response.     

Stimulatory effects of heat-killed Enterococcus faecalis on cell-mediated immunity in fish

Intracellular bacterial and viral diseases are widespread in the aquaculture industry and cause serious economic losses. Development of effective vaccines and adjuvants that can induce cell-mediated immunity is urgently needed for prevention of these diseases. Here we report the immunostimulatory effects of probiotic bacteria ‘‘E. faecalis’’ in ginbuna crucian carp Carassius auratus langsdorfii. Intraperitoneal injection of heat-killed E. faecalis induced an increase in CD4-1⁺ lymphocytes, CD8α⁺ lymphocytes and macrophages in vivo. Expression of Th1 cytokine genes was enhanced by exposure to the bacteria in vitro. We identified the leukocyte subsets that expressed specific Th1 cytokine genes: granulocytes and macrophages produced IL12 and IFNγrel2, respectively, while lymphocytes produced IFNγs including IFNγ1 and IFNγ2. Finally, expression of Th1 cytokines was also enhanced by intraperitoneal injection of heat-killed E. faecalis in vivo, while expression of Th2 cytokine was unchanged. Together, these findings suggest that heat-killed E. faecalis can induce cell-mediated immunity in fish.     

Effects of lysed Enterococcus faecalis FK-23 on allergen-induced serum antibody responses and active cutaneous anaphylaxis in mice

BACKGROUND: Our previous studies have presented evidence that lysed Enterococcus faecalis FK-23 (LFK), a lysozyme and heat-treated probiotic product, can inhibit allergen-induced local accumulation of eosinophils in mice. OBJECTIVE: The purpose of this experimental study was to evaluate the influence of orally administrated LFK on the host immune responses. METHODS: BALB/c mice were sensitized subcutaneously, and challenged intraperitoneally by cedar pollen allergen. Blood and spleen samples were collected after oral administration of LFK 60 mg/day for 21 days. The serum levels of total and allergen-specific IgE and IgG2a antibodies and the production of IL-4, IL-5 and IFN-gamma generated by allergen-stimulated cultured splenocytes were determined. Additionally, the effect of LFK on active cutaneous anaphylaxis (ACA) induced by ovalbumin (OVA) challenge in mice was measured after 28 days LFK treatment. RESULTS: No significant differences in serum immunoglobulin levels, as well as in cytokine production of splenocytes were observed between LFK-treated and control mice (P>0.05). There was, however, an increasing tendency of allergen-specific IgG2a level in mice after LFK treatment for 21 days compared with controls (P=0.060). Furthermore, the serum ratio of specific IgE to IgG2a was found to be significantly decreased in the LFK group (P=0.005). In addition, a significant inhibition of OVA-induced ACA reaction was observed in mice that had been fed for 28 days with LFK compared with control mice (P=0.008). CONCLUSION: These results suggest that LFK shows an anti-inflammatory effect, which may be part of the mechanism for protection against IgE-mediated allergy.     

Oral administration of Enterococcus faecalis FK-23 suppresses Th17 cell development and attenuates allergic airway responses in mice

Evidence is increasing that oral administration of probiotics can attenuate asthmatic responses both in murine models and clinical trials. T-helper 17 (Th17) cells, a subset of CD4+ T cells have been implicated as having an important role in the development of several allergic disorders, but the relationship between oral administration of probiotics and Th17 development has not been well studied. BALB/c mice were given lysed Enterococcus faecalis FK-23 (LFK) orally for 28 days. After sensitization by subcutaneous injection of ovalbumin (OVA) on Days 14 and 21 and 1% OVA inhalation on Days 25, 26 and 27, they were challenged with a 5% OVA aerosol on Day 28. Twenty-four hours later, airway resistance and accumulation of inflammatory cells in bronchoalveolar lavage fluid (BALF) and lung tissues were determined. Ιnterleukin (IL)-17-expressing CD4+ lymphocytes isolated from lung, spleen and lamina propria of the intestine were detected by flow cytometry. The expression of IL-6 and TGF-β mRNA was assessed by real-time PCR. Increases in airway hyperresponsiveness, and numbers of total leukocytes and mast cells in BALF induced by OVA challenge were significantly suppressed by oral administration of LFK. The increased percentage of IL-17-expressing CD4+ cells from lung, spleen and intestine in OVA-challenged mice was reduced following LFK treatment. We conclude that the oral administration of LFK suppresses the asthmatic response and that this is associated with attenuation of Th17 cell development.     

Single administration of enterococcal preparation (FK-23) augments non-specific immune responses in healthy dogs

Influence of a single administration of heat-killed Enterococcus faecalis FK-23 preparation (FK-23) on non-specific immune responses was evaluated in healthy dogs. The dogs received a single administration of FK-23 at a dose of 100 mg/kg perorally and thereafter a complete blood count (CBC) , leukocyte differential count, phagocytic activity of peripheral blood neutrophils and a lymphocyte blast transformation (LBT) test were assessed. Although FK-23 had no effect on the CBC or leukocyte differential count, treatment of the drug caused a 1.4-fold increase in neutrophil phagocytosis compared to FK-23 non-treated healthy dogs. LBT activities of phytohemagglutinin (PHA) and pokeweed mitogen (PWM) were also activated to twice the control levels and that of concanavalin A (Con A) was increased to one and a half times by the FK-23 treatment. Thus, a single administration of FK-23 appears to augment host resistance through stimulation of the non-specific immune responses in vivo.     

Effects of lysedEnterococcus faecalis FK-23 on experimental allergic rhinitis in a murine model

In the current study,we sought to investigate whether lysed Enterococcus faecalis FK-23(LFK),a heat-killed probiotic preparation,attenuated eosinophil influx into the upper airway and had immunomodulatory activity in a murine allergic rhinitis model.Eighteen BALB/c mice were divided into three groups;the ovalbumin(OVA)-sensitized/challenged group,which received saline orally for 6 weeks(OVA group),the OVA-sensitized/challenged group,which received LFK orally for 6 weeks(LFK-fed group),and the non-sensitized group,which received saline for 6 weeks(saline control group).Nasal rubbing and sneezing were monitored during the study.After the final challenge,interleukin(IL)-4,interferon(IFN)-γ,and OVA-specific IgE levels in the sera and splenocyte culture supernatants were determined,eosinophilic infiltrate into the upper airway was quantified,and splenic CD4+CD25+ regulatory T cells(Tregs) were examined by flow cytometry.We found that nasal rubbing was significantly reduced in LFK-fed mice compared to the OVA group on d 27 and 35,and sneezing was significantly inhibited by LFK administration for 35 d.LFK-fed mice had significantly less eosinophil influx into the nasal mucosa than the OVA group.There were no significant differences between the LFK-fed group and OVA group in the serum and splenocyte culture supernatant levels of IL-4,IFN-γ,and OVA-specific IgE.Interestingly,the LFK-fed mice had a significantly greater percentage of splenic CD4+CD25+ Tregs than OVA group.Our results indicate that oral administration of LFK may alleviate nasal symptoms,reduce nasal eosinophilia,and increase the percentage of CD4+CD25+ Tregs in experimental allergic rhinitis.     

Effect of CCL2 antisense oligodeoxynucleotides on bacterial translocation and subsequent sepsis in severely burned mice orally infected with Enterococcus faecalis

Severely burned mice are susceptible to sepsis stemming from Enterococcus faecalis translocation due to the impaired generation of M1 macrophages (M1MΦs) in local translocation sites. In our previous studies, CCL2 has been characterized as a major effector molecule on the burn-associated generation of M2MΦs, an inhibitor cell type for resident MΦ conversion into M1MΦs. In this study, we tried to protect burned mice orally infected with E. faecalis utilizing CCL2 antisense oligodeoxynucleotides (ODNs). We show that M2MΦs in mesenteric lymph nodes (MLNs) were not demonstrated in burned mice treated with CCL2 antisense ODNs. M1MΦs were not induced by heat-killed E. faecalis from resident MΦs transwell-cultured with mesenteric lymph node macrophages (MLN-MΦs) from burned mice, while M1MΦs were induced by the same antigen from resident MΦs transwell-cultured with MΦs which were isolated from burned mice treated with CCL2 antisense ODNs. Bacterial growth in MLNs was shown in burned mice orally infected with a lethal dose of E. faecalis. However, after the same infection, sepsis did not develop in burned mice treated with CCL2 antisense ODNs. These results indicate that bacterial translocation and subsequent sepsis are controlled in burned mice orally infected with a lethal dose of E. faecalis by gene therapy utilizing CCL2 antisense ODNs.     

Effect of orally administered beta-glucan on macrophage function in mice

The effect of orally administered SSG, a beta-1,3-glucan obtained from the culture filtrate of the fungus Sclerotinia sclerotiorum IFO 9395, on the function of peritoneal macrophages in CDF1 mice was examined. Oral administration of SSG (20, 40, 80 or 160 mg/kg, daily for 10 consecutive days) enhanced the acid phosphatase activity of peritoneal macrophages. The greatest enhancing effect was observed at 80 mg/kg of SSG. Relatively long periods of administration (more than 10 consecutive days) were needed to induce significant enhancing effects. Phagocytic activity, candidacidal activity, hydrogen peroxide (H2O2) production and interleukin-1 (IL-1) production of peritoneal macrophages were also enhanced after the administration of SSG by the oral route (80 or 160 mg/kg). However, the durations of the activated state after completion of administration differed depending on the activity. Enhanced activity of lysosomal enzyme (acid phosphatase) was also shown in peritoneal macrophages taken from C3H/HeJ mice, which is a nonresponder strain to bacterial lipopolysaccharide (LPS). These results demonstrate that SSG given by the oral route can activate peritoneal macrophages in mice.     

Effect of orally administered oestrogens on gonadotrophin levels in post-menopausal women

The gonadotrophin-suppressing effect of 4 peroral oestrogen regimens (A: 2.5 mg piperazine oestrone sulphate; B: 1.25 mg piperazine oestrone sulphate + 5.0 mg oestriol; C: 2.0 mg oestradiol valerate; D: 10.0 mg oestriol; all administered daily) was studied in 7 post-menopausal women, in the absence, and then in the presence of daily oral doses of 250 micrograms of levonorgestrel. A randomized, complete cross-over design was used, and from each volunteer 80 blood samples were drawn during a period of 252 days for the assay of immunoreactive follicle stimulating hormone (FSH), luteinizing hormone (LH) and bioactive LH levels. All 4 formulations significantly diminished pretreatment gonadotrophin levels. Combination with levonorgestrel enhanced the suppressive effects. Subsequent placebo administration for a week restored gonadotrophin levels to those found previously during oestrogen administration, but not to pretreatment levels. Regimen D exhibited the relatively weakest and regimens A and B the strongest suppression, both in the absence and in the presence of levonorgestrel. Treatment with all oestrogen formulations decreased, and the addition of levonorgestrel increased the ratio of bioactive (B) to immunoreactive (I) LH. Seven days of placebo administration abolished the effect of levonorgestrel, but not that of the various oestrogens on the B/I ratios.     

Differential effects of orally versus parenterally administered qinghaosu derivative artemether in dogs.

Artemether (AM) is an antimalarial drug derived from artemisinin (Qinghaosu), an extract of the herb Artemisia annua L., sweet wormwood. Its antiparasitic effect is that of a schizontocide and is explained by rapid uptake by parasitized erythrocytes and interaction with a component of hemoglobin degradation resulting in formation of free radicals. It has been shown to exhibit a high clinical cure rate. Previous animal safety studies with Qinghaosu derivatives revealed dose-dependent neurotoxicity with movement disturbances and neuropathic changes in the hindbrain of intramuscularly treated dogs, rats and monkeys. Such effects have not been seen in man. The objective of our present studies was to compare the effects of high levels of AM administered to dogs p.o. versus i.m. In a pilot study 20 mg/kg/day of AM was given i.m. to groups of 3 male Beagle dogs for 5 and 30 days, respectively. Clinical signs of neurotoxicity were noted in some individual dogs from test day 23 on. One dog had to be sacrificed pre-term. Hematologic findings indicated a hypochromic, microcytic anemia. Microscopic examination demonstrated neuropathic changes only at 30 days, but not at 5 days. The animals had neuronal and secondary axonal damage, most prominent in the cerebellar roof, pontine and vestibular nuclei, and in the raphe/paralemniscal region. The affected neurons showed loss of Nissl substance, cytoplasmic eosinophilia, shrinkage of the nucleus and in advanced stages scavenging by microglia. In a subsequent experiment, AM was administered to groups of 4 male and 4 female dogs, respectively, at 8 daily doses of 0, 20, 40 and 80 mg/kg i.m., or 0, 50, 150 and 600 mg/kg p.o. Neurologic signs were seen at high i.m. doses only. In most animals they were inconspicuous and consisted of reduced activity with convulsions seen in single dogs shortly before death. Neuronal damage occurred in all animals at 40 and 80 mg/kg following i.m. treatment. At 20 mg/kg minimal effects occurred in 5/8 dogs only, indicating that this level was close to tolerated exposure. No comparable lesions were observed after oral administration. Both i.m. and p.o. exposure at high dose levels was associated with a prolongation of mean QT interval of ECG, suggesting slowing of repolarization of the myocardium. Individual data indicated that in 1 of 4 females at 80 mg/kg i.m. this prolongation was above the 25% level considered as threshold for concern. After intramuscular administration pharmacokinetics indicated peak plasma levels of AM at 2 to 4 hours post-dose, slow elimination and a tendency to accumulate after repeated administration. Only low levels of the major metabolite, dihydroartemisinin (DHA), were found. AM levels in the cerebrospinal fluid (CSF) were < 10% of plasma levels. After oral administration AM concentrations were considerably lower than after i.m. administration. The concentration of DHA was high on day 1 but almost nil on day 7 indicating its fast inactivation in dogs. Two hours after the 8th oral administration neither AM nor DHA was detected in CSF which may explain the absence of neurotoxicity in dogs after oral administration of AM.     

Effect of orally administered oestrogens on circulating oestrogen profiles in post-menopausal women

The effect of 4 peroral oestrogen regimens without and with levonorgestrel (LNG) supplementation (250 micrograms/day) was studied in 7 post-menopausal women. The regimens were: A: 2.5 mg piperazine oestrone sulphate/day; B: 1.25 mg piperazine oestrone sulphate + 5.0 mg oestriol/day; C: 2.0 mg oestradiol valerate/day and D: 10.0 mg oestriol/day. A randomized complete cross-over design was employed and systemic blood levels of progesterone (P), oestrone (E1), oestradiol (E2), oestriol (E3), oestrone sulphate (E1S), oestradiol sulphate (E2S) and oestriol sulphate (E3S) were analyzed. All subjects were consistently post-menopausal, as indicated by low P, E2, E1S and E3S and high FSH and LH levels. The pretreatment and post-treatment levels of E2 and E2S as well as those of E3 and E3S were invariably below detection limit (60 pmol/l and 220 pmol/l, respectively). Treatment with regimens A, B and C resulted in consistently detectable levels of E2, E2S and E3S, but not of E3, and in grossly elevated E1 and E1S levels. Treatment with regimen D gave rise to very high E3S levels. The oestrogen profiles produced by regimens A and C were almost identical, with the exception of higher E1 and lower E3S levels following the administration of regimen A. Combination with LNG did not modify the levels of unconjugated oestrogens, but in certain cases it seemed to diminish those of oestrogen sulphates. A comparison with the results of a preceding study (Aedo A-R et al., Effect of orally administered oestrogens on gonadotrophins levels in post-menopausal women. Maturitas 1989; 11:) reveals that the gonadotrophin-suppressing effect of all 4 regimens still persisted in the post-treatment period at a time when the levels of all oestrogens analyzed returned to those of the pretreatment period.     

Enhancement of chronic Trypanosoma cruzi myocarditis in dogs treated with low doses of cyclophosphamide.

An enhancement of chronic myocarditis was obtained in dogs chronically infected with Trypanosoma cruzi protozoa soon after they were submitted to treatment with low doses of cyclophosphamide (50 mg/sq m bs three times a week for 3 weeks). Such treatment did not cause immunodepression. Myocarditis varied in intensity, but was quite severe and diffuse in some animals, with focal fibrinoid, coagulative, and lytic necrosis and invasion of disintegrating myocardial fibers by the mononuclear inflammatory cells. Untreated infected controls exhibited mild focal myocarditis, usually represented by accumulation of lymphocytes in the interstitial connective tissue. It is suggested that the administration of low doses of cyclophosphamide interfered with the immunologic suppressor network that is thought to maintain the chronic indeterminate (or latent) phase of T cruzi infection.     

Effect of systemic and orally administered iota-carrageenan on ovalbumin-specific antibody response in the rat

Iota-carrageenans are high molecular weight polygalactans commonly used in the food industry. Their immunomodulating effects were examined on the reaginic and IgG ovalbumin-specific responses after systemic or oral administration to Brown Norway rats. Intraperitoneal injection of ovalbumin with either iota-carrageenan or alum induced both a reaginic and IgG response. Reaginic antibodies were detected in the primary response with alum, but only in the secondary response with iota-carrageenan. Oral tolerance to ovalbumin was similarly induced whether the protein was given alone or admixed in solution with iota-carrageenan. These results confirmed the systemic adjuvant action of iota-carrageenan described earlier, and showed that this effect did not take place when orally administered.     

Characteristics of polyarthritis in rabbits by hyperimmunization with attenuated Enterococcus faecalis.

To produce polyarthritis and rheumatoid factor like substance (RFLS), rabbits were hyperimmunized intravenously with 0.02% thimelosal (TMS)-treated Enterococcus faecalis (E. faecalis) as a persistent bacterial flora. Swelling of knee joints occurred at a rate of 41% (27/66), and of shoulder joints at a rate of 25% (17/66) while that of elbow joints occurred at a rate of 4.5% (3/66). On culturing of knee joint fluids, no colonies appeared while 2/4 fluid specimens from the shoulder joints gave positive colonies for 78 days after the first immunization; thereafter, no colonies appeared. On histological examination, in early stages, acute inflammatory reactions with degenerative changes of synovial tissue was observed. In later stages, chronic inflammatory changes, proliferation of synovial cells with pannus formation, destruction of articular cartilage and subchondral bone were observed. RFLS titer showed bi-phasic peaks at 11 days and 41 days after the first immunization. A high incidence of polyarthritis, particularly knee joints, occurred. Thus, hyperimmunization with attenuated E. faecalis as a normal intestinal flora may provide an animal model of chronic polyarthritis.     

Effect of orally administered monoclonal antibody on persistent Cryptosporidium parvum infection in scid mice.

Scid mice, persistently infected after exposure to 10(7) Cryptosporidium parvum oocysts, were treated daily for 14 to 17 days with 0.4 mg of monoclonal antibody (mAb) 17.41 administered by the oral route. Mice receiving mAb 17.41 shed significantly fewer (P < 0.005) C. parvum oocysts than scid mice receiving isotype control mAb. Intestinal (but not gastric) infectivity scores were also reduced for scid mice treated with mAb 17.41 (P < 0.01).     

Effect of hospitalization on the antibiotic resistance of fecal Enterococcus faecalis of surgical patients over time

The prevalence of antibiotic resistant Enterococcus faecalis was determined in fecal samples of 263 patients admitted to the surgical wards of three university-affiliated hospitals on admission, at discharge, and at 1 and 6 months after discharge. A slight increase in the prevalence of antibiotic resistance of E. faecalis was found at discharge for the antibiotics tested compared to those on admission, vancomycin excepted. At 6 months after discharge, the prevalence of resistance for amoxicillin (0%), ciprofloxacin (3%), erythromycin (47%), and oxytetracycline (60%) decreased to the level on admission (respectively 0%, 8%, 45%, and 64%). Gentamicin resistance was the same at discharge (10%) as 1 month later (12%), but decreased 6 months after discharge (8%) to the level on admission (7%). In conclusion, hospitalization resulted in the study population in a slight increase in the prevalence of resistant fecal E. faecalis isolates at discharge, which decreased again (slowly) to the level on admission 6 months after discharge. Thus, the influence of hospitalization on the prevalence of antibiotic resistance in the extramural situation disappears between 1 and 6 months after discharge in this population.     

Molecular epidemiology of unilateral amyloid arthropathy in broiler breeders associated with Enterococcus faecalis

Although symmetrical polyarticular amyloidosis has been described extensively in brown layers, spontaneous unilateral amyloid arthropathy has not been described previously in chickens. Birds from nine flocks of broiler parent stock (PS) had unilateral lameness associated with severe swelling of the left hock joint and the caudal aspect of the metatarsus. Gross pathology was restricted to the left hock joint and the left digital flexor tendons in almost all cases, suggesting an association with administration of Marek's disease vaccine. Amyloid deposits were found in 83% (25/30) of affected joints by histological examination of Congo red stained sections. Systemic amyloidosis, involving mainly the liver and spleen, was found in 59% (10/17) of birds. Enterococcus faecalis was isolated from joints in 77% (23/30) of cases and Staphylococcus aureus was isolated from the joint in one case (1/30). Thirty-five E. faecalis isolates from joints, tendons and blood samples from birds in five affected PS flocks were compared using pulsed-field gel electrophoresis (PFGE) to separate genomic fragments after digestion with Sma I. All but one isolate had identical or closely related restriction endonuclease digestion (RED) patterns that were very similar to a known arthropathic and amyloidogenic E. faecalis isolate. A further 30 E. faecalis isolates from seven grandparent stock (GPS) flocks and two isolates from two unaffected PS flocks of the same genetic background were analysed by PFGE. Among these isolates, 11 originating from four GPS flocks had RED patterns identical to or closely related to the reference amyloid-inducing strain. Moreover, one E. faecalis isolate from amyloidotic joints of brown layers housed in California, USA was included in the analysis and appeared to be identical to the reference strain. This study showed that the E. faecalis isolates involved in these outbreaks of unilateral amyloid arthropathy in broiler breeders belonged to the same clone as that responsible for outbreaks in brown layers.