Suppression and inactivation of Vibrio vulnificus hemolysin in cirrhotic ascites, a human ex vivo experimental system.

To elucidate the mechanisms underlying the in vivo suppression and inactivation of Vibrio vulnificus hemolysin (VvhA), we used cirrhotic ascites fluid as a human ex vivo experimental system. VvhA expression was suppressed in proportion to the amount of cirrhotic ascites. The expression of vvhA in undiluted cirrhotic ascites could be suppressed further by the addition of glucose, a constituent of cirrhotic ascites. VvhA was readily inactivated in the presence of cirrhotic ascites by a cholesterol-mediated oligomerization and interaction with an undefined constituent(s) of cirrhotic ascites. These results indicate that the expression of vvhA can be suppressed and that any VvhA produced is inactivated by the constituents of cirrhotic ascites. Our results suggest that only a very small portion of the VvhA that is produced in human body fluids may actually contribute to the pathogenesis of V. vulnificus septicemia. It is suggested that cirrhotic ascites could be used as a human ex vivo experimental system for the studies on the in vivo expression and the significance of V. vulnificus virulence factors.

Vibrio vulnificus vulnibactin, but not metalloprotease VvpE, is essentially required for iron-uptake from human holotransferrin.

The roles of metalloprotease (VvpE) and catechol-siderophore (vulnibactin) in the uptake of iron from human transferrins by Vibrio vulnificus have been determined using different experimental conditions and methods. Therefore, in this study, we attempted to elucidate the roles of VvpE and vulnibactin using the same methods and experimental conditions, in an in vitro and a human ex vivo system, and in accordance with the molecular version of Koch's postulates. Neither vvpE mutation nor in trans vvpE complementation affected vulnibactin production, iron-assimilation from human holotransferrin (HT), and bacterial growth in a HT-containing deferrated Heart-Infusion medium (HT-DF-HI) or a HT-containing cirrhotic ascites (HT-CA). In contrast, the mutation of fur gene encoding Fur, a repressor regulating expression of the vulnibactin-mediated iron-uptake system, derepressed vulnibactin production, and facilitated iron-assimilation from HT and bacterial growth in HT-DF-HI or HT-CA. The mutation of vis gene encoding isochorismate synthase required for vulnibactin synthesis abolished vulnibactin production, iron-assimilation from HT and bacterial growth in HT-DF-HI or HT-CA. These results demonstrate that vulnibactin is essentially required for iron-assimilation from transferrin, and that VvpE has no direct effect on facilitating vulnibactin-mediated iron-assimilation from transferrin in vitro or in a human ex vivo system.

Proteases of a Bacillus subtilis clinical isolate facilitate swarming and siderophore-mediated iron uptake via proteolytic cleavage of transferrin.

We isolated a highly serine protease-producing Bacillus subtilis strain (PRY) from a clinical sample and identified it through biochemical testing and ribosomal DNA sequencing. The PRY strain exhibited a robust swarming behavior and was able to digest human transferrin efficiently, concomitantly with the production of catechol-siderophore in the exponential growth phase. The growth of PRY was in proportion to increased iron availability resulting from transferrin destruction. These results suggest that proteases of the B. subtilis PRY strain may play a significant role in the pathogenesis of human infections by facilitating siderophore-mediated iron uptake from transferrin and swarming motility.

Effect of the crp mutation on the utilization of transferrin-bound iron by Vibrio vulnificus.

Cyclic AMP-cAMP receptor protein (CRP) complex plays an essential role in the global regulation of Vibrio vulnificus virulence. We found that growth retardation of V. vulnificus caused by mutation of the crp gene encoding CRP was exacerbated under iron-limited conditions. Accordingly, we investigated the effect of crp mutation on the expression of the vulnibactin-mediated iron-uptake system and the ability of V. vulnificus to utilize transferrin-bound iron, and thus to grow in cirrhotic ascites, a human ex vivo system. The production of vulnibactin was suppressed, and the transcription of the vis and vuuA genes, which encode an enzyme required for vulnibactin synthesis and vulnibactin receptor protein, was also suppressed in the crp mutant. Moreover, the crp mutant could not utilize transferrin-bound iron, and its growth was severely suppressed both on transferrin-bound iron and in cirrhotic ascites. All the defects in the crp mutant were recovered by the in trans complementation of the wild-type crp gene. Putative CRP-binding sequences were found in the regulatory regions of the fur, vis and vuuA genes. These results indicate that crp mutation attenuates the ability to grow on transferrin-bound iron and in a human body fluid by down-regulating the vulnibactin-mediated iron-uptake system.

X-gal inhibits the swarming of Vibrio species.

The expressional levels of genes in swarmer cells can be determined by a simple method using X-gal-containing semisolid agars and lacZ-fusion transcription reporter strains of the genes concerned. However, X-gal alone inhibited the swarming of Vibrio, regardless of their ability to digest X-gal. Moreover, X-gal inhibited the growth of V. vulnificus containing functional lacZ. These effects of X-gal itself should be carefully considered when trying to determine the expression levels of genes in swarming cells using X-gal-containing semisolid agar.

Human serum albumin enhances the hemolytic activity of Vibrio vulnificus.

Vibrio vulnificus hemolysin (VvhA) is inactivated in the late growth phase by its oligomerization. Albumin is known to affect the activities of many bacterial toxins. In this study, we investigated the effects of human or bovine serum albumin (HSA or BSA) on the production and activity of VvhA. HSA did not affect V. vulnificus growth and vvhA transcription. However, VvhA hemolytic activity in culture supernatants was significantly higher in the presence of HSA than in the absence of HSA. By Western blot analysis, the oligomerization of VvhA was inhibited and the remaining active VvhA monomer was increased in culture supernatants containing HSA. BSA produced similar results. These findings indicate that both HSA and BSA stabilize VvhA and delay VvhA inactivation by oligomerization, and thus enhance VvhA activity.

Assessment of a bioactive compound for its potential antiinflammatory property by tight junction permeability.

Lactobacillus probiotic strains are proving to be abundant sources of bioactive components, including antiinflammatory components. Lifree was made of fruits fermented by Lactobacillus paracasei, Lactobacillus reuterrii and Saccharomyces cerevisiae. This study was designed to test these compounds in cell assays measuring epithelial barrier function and proliferation in the first instance. Cell proliferation was measured in mouse fibroblasts cells (3T3NIH) and rat intestinal epithelial cells (IEC-6), and tight junction activity in the kidney epithelial cell line (MDCK). Tight junction permeability was assessed by measuring transepithelial electrical resistance (TER) across confluent monolayers, following the addition of Lifree with or without a challenge with EGTA. Lifree promoted tight junction formation and recovery following loss of TER from challenge with EGTA. On the other hand, Lifree did not stimulate cell growth in either 3T3NIH and IEC-6 cells. Lifree stimulates tight junction maintenance and formation, suggesting it may have potential antiinflammatory properties.

Inactivation of Vibrio vulnificus hemolysin by oligomerization but not proteolysis.

Vibrio vulnificus extracellular protease (VvpE) is believed to destroy its hemolysin (VvhA) in the late growth phase, without obvious experimental evidence. So, we attempted to elucidate the mechanism. The hemolytic activity steeply increased with the expression of the VvhA in the early growth phase, and then abruptly declined with the expression of VvpE in the late growth phase. However, the VvhA activity also abruptly declined in a VvpE-deficient mutant. In Western blot, the degradation of VvhA was not observed; instead, the oligomerization of VvhA increased with the concomitant loss of hemolytic activity. These results evidently indicate that the inactivation of VvhA is due to the novel oligomerization of VvhA by unknown mechanism, but not to the destruction of VvhA by VvpE, so that the routine functional assay measuring hemolytic activity cannot reflect the actual production of VvhA.

Staphylococcus aureus siderophore-mediated iron-acquisition system plays a dominant and essential role in the utilization of transferrin-bound iron.

Staphylococcus aureus is known to be capable of utilizing transferrin-bound iron, via both siderophore- and transferrin-binding protein (named IsdA)-mediated iron-acquisition systems. This study was designed in order to determine which iron-acquisition system plays the essential or dominant role with respect to the acquisition of iron from human transferrin, in the growth of S. aureus. Holotransferrin (HT) and partially iron-saturated transferrin (PT), but not apotransferrin (AT), were found to stimulate the growth of S. aureus. S. aureus consumed most of the transferrin-bound iron during the exponential growth phase. Extracellular proteases were not, however, involved in the liberation of iron from transferrin. Transferrin-binding to the washed whole cells via IsdA was not observed during the culture. The expression of IsdA was observed only in the deferrated media with AT, but not in the media supplemented with PT or HT. In contrast, siderophores were definitely produced in the deferrated media with PT and HT, as well as in the media supplemented with AT. The siderophores proved to have the ability to remove iron directly from transferrin, but the washed whole cells expressing IsdA did not. In the bioassay, the growth of S. aureus on transferrin-bound iron was stimulated by the siderophores alone. These results demonstrate that the siderophore-mediated iron-acquisition system plays a dominant and essential role in the uptake of iron from transferrin, whereas the IsdA-mediated iron-acquisition system may play only an ancillary role in the uptake of iron from transferrin.

Asian pear pectin administration during presensitization inhibits allergic response to ovalbumin in BALB/c mice.

OBJECTIVE: A type of respiratory disorder resembling some aspects of human allergic asthma can be induced in mice using ovalbumin. The factors that influence the etiology of asthma are poorly understood even though cytokines are known to play a pivotal role. The purpose of this study was to test the hypothesis whether an administration of Asian pear pectin during presensitization could suppress allergic response to ovalbumin in BALB/c mice. DESIGN: High-dose (100 microg) of pectin-sol was used and values were compared to those from the control. Ovalbumin and aluminum hydroxide were utilized for sensitization while ovalbumin aerosol was used for provocation 2 weeks later. The bronchoalveolar lavage (BAL) and assessment of tracheal smooth muscle responsiveness to electrical field stimulation or acetylcholine were performed 1 day after ovalbumin provocation. Two main cytokines of interferon (IFN)-gamma and interleukin (IL)-5, and serum immunoglobulin E (IgE) were assayed. SETTINGS: Laboratory of the Chosun University Medical School SUBJECT: Male BALB/c mice RESULTS: Antigen dose of 5 microg for sensitization generated TH1 type cytokines in the lungs with a high level of IFN-gamma and a low level of IL-5. In contrast, TH2 type cytokines were produced in splenocytes including a high level of IL-5 and a low level of IFN-gamma. Asian pear pectin-sol administration during presensitization significantly inhibited (p < 0.05) sensitivity of airway smooth muscle to electrical field stimulation and acetylcholine. Further, IFN-gamma production significantly decreased (p < 0.05) in BAL fluids while it significantly increased (p < 0.05) in splenic cells. On the other hand, IL-5 production significantly increased (p < 0.05) in BAL fluids while it was a significant decrease (p < 0.05) in splenic cells. For the histopathologic changes in the lung, pear pectin-sol recovered ovalbumin (OVA)-induced abnormal signs to an almost normal state. As a correlate, IgE production significantly decreased (p < 0.05) in pectin-sol-treated animals compared to the control. CONCLUSIONS: It is possible from these data that BALB/c mice have different susceptibilities to different doses of OVA regulated by pulmonary TH1 and TH2 type cytokines, independent of splenic TH1 and TH2 type cytokines production. These results also indicate that administration of Asian pear pectin-sol in presensitized mice suppresses allergic asthmatic reaction.

Electro-acupuncture reverses nerve growth factor abundance in experimental polycystic ovaries in the rat.

Polycystic ovary syndrome (PCOS) remains one of the most common causes of anovulation in women of reproductive age. There is some evidence that nerve growth factor (NGF) is involved in the pathogenesis of PCOS. Therefore, seeking the pathogenesis of PCOS is important for controlling fertility. In traditional Oriental Medicine, acupuncture has been used for the function of ovaries. The present study was designed to determine whether electro-acupuncture (EA) could affect experimentally induced polycystic ovary (PCO) in the rat. The two acupoints Sp-6 and E-128 were stimulated to test for efficacy in the protein expression of NGF. Polycystic ovaries were induced by a single injection of estradiol valerate (4 mg i.m.). During the experimental period of 8 weeks, some of the rats were treated with EA twice weekly; this group was compared with a vehicle-treated control group and an estradiol-injected group not subjected to EA. At day 60, the protein expression of NGF was examined by immunohistochemistry in the ovaries, the adrenal glands and some parts of the brain. The estradiol treatment induced a clear PCO appearance, and was associated with a robust increase in NGF expression in the ovaries, the adrenal glands and the brain. EA treatment partly reversed the NGF abundance, particularly in the ovaries, but not in the brain. Our data show that EA affects the NGF involvement in ovarian dysfunction.

Effect of apitherapy in piglets with preweaning diarrhea.

This study was designed to examine the therapeutic effect of honeybee (Apis mellifera L.) venom in piglets with bacterial diarrhea Comparison between bee venom- and drug-treated groups was our main concern in the present study. Preweaning piglets were assigned to treated and non-treated control groups. In the treated group, 47 piglets were acupunctured with the worker honeybee once a day for three consecutive days. Two acupoints, GV-1 (Jiao-chao) and ST-25 (Hai-men), were selected for apitherapy. In the control group, 44 piglets were intramuscularly injected with a standard dose of a known antibacterial drug, colistin sulfate (300,000 IU/kg of body weight), and an antidiarrheal drug (berberine, 2 ml/kg) once a day for three consecutive days. At post-treatment, 90.9% of the control piglets and 93.6% of piglets in the treated group recovered from bacterial diarrhea. Bee acupuncture therapy did not show any side effects such as allergy, intoxication, hemorrhage or infection. It is concluded that bee venom therapy was effective in controlling bacterial diarrhea in preweaning piglets.

The effect of herbal medicine on nerve growth factor in estradiol valerate-induced polycystic ovaries in rats.

A type of polycystic ovary resembling some aspects of human polycystic ovarian syndrome (PCOS) can be induced in the rat with a single injection of long-acting estradiol valerate. Among several theories behind the development of polycystic ovaries (PCO), the involvement of the sympathetic nervous system draws much attention, and herbal medicine is known to relieve the abnormal symptoms of PCO. Two herbal formulas, Changbudodam-Tang (cang fu dao tan tang) and Yongdamsagan-Tang (long dan xie gan tang), were used in the present study. The administration of herbal medicine was done every other day for 60 days. The morphological changes of ovaries from herbal medicine treatment were compared to those from an oil-treated control group and an estradiol valerate-injected group. This study also examined the possible hypothesis of neurogenic participation in terms of nerve growth factor (NGF) in the pathology of ovarian dysfunction. The nerve growth factor was analyzed in the central nervous system and ovaries by immunohistochemistry. The main findings of the present study were: (1) PCO were fully developed in rats with a single intramuscular injection of estradiol valerate, (2) PCO resulted in the expression of NGF in the ovaries and the brain tissues, and (3) herbal medicine administration significantly decreased the elevated NGF staining in the ovaries without affecting the brain tissues significantly.

Tocolytic Effect of Morphine via Increased Metabolic Clearance of Oxytocin in the Baboon.

Morphine is known to inhibit nocturnal uterine contractions in several animal models, and oxytocin is known to be a primary causative factor of uterine contractions. The purpose of the present study was to determine the tocolytic effect of morphine in relation to the pharmacokinetics of oxytocin, after a bolus injection of oxytocin. The metabolism of oxytocin was investigated during the third trimester in baboons. Four animals were placed on a tether system with venous and arterial access, including continuous uterine monitoring. Plasma oxytocin levels were determined by radioimmunoassay after extraction with petroleum ether/acetone. Morphine consistently increased the metabolic clearance rate of oxytocin in all four animals (p < 0.05) and this was in accordance with suppressed uterine contractions. We conclude that morphine could be used as an inhibitor of nocturnal uterine contractions, and that this is caused by the morphine induced increased metabolic clearance rate of oxytocin.