Highly purified mutant E112K of cholera toxin elicits protective lung mucosal immunity to diphtheria toxin.

We demonstrated that the mutant of cholera toxin (mCT) E112K which was LPS-free supported the induction of protective immunity in mucosal (e.g. lung lavage) and systemic (e.g. serum) compartments when given nasally with vaccine-grade diphtheria toxoid (DT) to mice. Significant DT-specific mucosal IgA antibody (Ab) and serum IgG, IgA and IgM Ab responses were induced when LPS-depleted mCT E112K or native CT (nCT) was co-administered nasally with DT. The analysis of DT-specific Ab-forming cell (AFC) responses supported the Ab titers and significant numbers of DT-specific IgA AFC were present in the lungs, nasal passages and submandibular glands. Furthermore, DT-specific IgG AFC in cervical lymph nodes (CLN) and the spleen were induced in mice administered with DT nasally with either mCT or nCT. The analysis of antigen-specific T cell responses revealed that increased DT-specific CD4+ T cell proliferative and Th2-type cytokine responses were induced in mice nasally-immunized with DT and the LPS-free form of mCT. The neutralization of diphtheria toxin by Abs showed that DT-specific IgG Ab responses in serum and lung lavages of mice immunized with DT and mCT were protective. Furthermore, it was shown that an IgA-enriched fraction of lung lavages possessed diphtheria toxin-specific neutralizing activity. These results are the first demonstration that nasally co-administered mCT E112K can induce DT-specific protective Ab responses in mucosal compartments (e.g. lung lavages and the lungs).

Inhibition of Shiga toxin-induced tumor necrosis factor-alpha production and gene expression in human monocytic cells by CV6209.

Cytokines, in particular TNF-alpha, appear to be necessary to develop the pathological process of Shiga toxin-producing Escherichia coli (STEC) infection. In this study, we investigated whether CV6209, a PAF antagonist, could modulate Shiga toxin (Stx)-induced TNF-alpha production in human monocytic cells. Cells were stimulated by Stx1 or Stx2 (5 ng/ml) with or without CV6209 addition (12-100 microg/ml) for various periods of time. CV6209 significantly suppressed Stx-induced TNF-alpha production in a dose- and time-dependent manner. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that CV6209 suppressed Stx-mediated TNF-alpha mRNA expression. Our results indicated that CV6209 had an important regulatory effect on Stx-induced TNF-alpha production and gene expression.

Nasal immunization with E. coli verotoxin 1 (VT1)-B subunit and a nontoxic mutant of cholera toxin elicits serum neutralizing antibodies.

Escherichia coli O157:H7 produces two forms of verotoxin (VT), VT1 and VT2, which cause hemorrhagic colitis with development, in some cases, of hemolytic uremic syndrome. These toxins consist of an enzymatically active A subunit and pentamers of B subunit responsible for their binding to host cells. We used the secretion-expression system of Bacillus brevis to produce recombinant VT1B and VT2B. The secreted B subunits were purified and sequenced to verify their structure. Receptor-binding showed that rVT1B but not rVT2B bound to Gb3-receptor. When mice were nasally immunized with rVT1B or rVT2B together with a nontoxic mutant of cholera toxin (mCT) or native cholera toxin (nCT) as adjuvants, serum IgG and mucosal IgA antibody responses to VT1B were induced. The VT1B-specific antibodies prevented VT1B binding to its Gb3 receptor. In contrast, poor serum and no mucosal VT2B-specific antibodies but brisk CTB-specific antibody responses were induced by nasal immunization with rVT2B in the presence of mCT or nCT. These results show that nasal immunization with rVTB and mCT as a nontoxic mucosal adjuvant is an effective regimen for the induction of VT1B but not VT2B antibody responses which inhibit VT1B binding to Gb3 receptor.

Protective effect of anisodamine against Shiga toxin-1: inhibition of cytokine production and increase in the survival of mice.

The purpose of this study was to investigate whether anisodamine could inhibit Shiga toxin-1 (Stx1)-induced cytokine production and increase the survival of Stx1-treated mice. Human monocytic cells were stimulated by Stx1 (1 to 100 ng/mL) with or without anisodamine addition (1 to 400 microg/mL). For in vivo evaluations, C57BL/6 mice were given a single intraperitoneal injection of anisodamine (1 mg) or saline solution after intraperitoneal injection of Stx1 (2.75 microg/kg). The results showed that anisodamine significantly suppressed Stx1-induced tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-8 production. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that anisodamine suppressed Stx1-mediated TNF-alpha mRNA expression. Further study showed that this TNF-alpha inhibitory effect was via a prostaglandin E2-dependent mechanism. Anisodamine treatment prolonged the survival time of mice and decreased the lethality of Stx1 (94.5% to 44%). Because cytokines, in particular TNF-alpha, contribute to the pathologic process in Stx-producing Escherichia coli (STEC) infection, this study suggested that anisodamine could be a potential drug for treatment of STEC infection.

The cycle of the seminiferous epithelium in the greater Japanese shrew mole, Urotrichus talpoides.

Spermatogenesis and acrosomal formation in the greater Japanese shrew mole, Urotrichus talpoides, were studied by light microscopy. On the basis of acrosomal changes, morphology of spermatid head, nuclear shape, appearance of meiotic figures, location of spermatid and period of spermiation, the cycle of the seminiferous epithelium was classified into 12 stages, and developing spermatids could be divided into 15 steps. The mean relative frequencies of stages from I to XII were 10.9, 8.7, 9.8, 7.3, 8.5, 10.3, 12.5, 8.7, 5.8, 5.4, 5.1 and 7.1%, respectively. Similar to the case in the musk shrew, the spermatid nucleus of the greater Japanese shrew mole remained in the middle region of the seminiferous epithelium and only the acrosome extended towards the basement membrane. The elongation of the acrosome, however, was not prominent. The proacrosomal vesicle first appeared in stage II and then one large and round granule was seen in stage III. The acrosomal vesicle became flattened on the surface of the nucleus in stage IV. Spreading of the acrosomic system has been recognized from stage VII. In stage VII, spermiation occurred. In stage IX, the spermatid nucleus began to elongate. Elongation and condensation of the nucleus were clearly observed in stage X. In stage XII, pachytene spermatocytes divided into diplotene spermatocytes. In stage XII, meiotic figures and secondary spermatocytes were observed.

Protective immunity to Streptococcus mutans induced by nasal vaccination with surface protein antigen and mutant cholera toxin adjuvant.

In this study, mice were immunized nasally with surface protein antigen of Streptococcus mutans serotype c (PAc) and a nontoxic A subunit mutant of cholera toxin (mCT) E112K, as a mucosal adjuvant. Immunization with PAc and mCT elicited significant PAc-specific secretory IgA in saliva and in nasal secretions. Antibody-forming cell (AFC) analysis confirmed the antibody (Ab) titers by revealing significant numbers of PAc-specific IgA AFCs in the submandibular gland and nasal passages. Furthermore, CD4(+) T cells from cervical lymph nodes exhibited significant proliferative responses when restimulated with PAc in vitro. Importantly, mice that were nasally immunized with PAc plus mCT E112K exhibited significantly reduced oral colonization by S. mutans. These results show that nasal administration of PAc and mCT E112K is potentially an effective mucosal vaccine against dental caries and reduces the colonization of S. mutans in the oral cavity.

Caspase-3 activation and apoptosis induction coupled with the retrograde transport of shiga toxin: inhibition by brefeldin A.

Caspase proteolytic activities, such as caspase-3, -2 and -6, of THP-1 human monocytic cells were markedly increased in a time- and dose-dependent manner by treatment with purified Shiga toxin 1 (Stx1) or Stx2. Caspase-3 activation was strictly correlated with internucleosomal DNA fragmentation and chromatin condensation of the cells. In addition, the specific caspase-3 inhibitor, Ac-DEVD-CHO, decreased the percentage of apoptotic cells. The purified B-subunit of Stx1 did not induce apoptosis in THP-1 cells. Caspase-3 activation, DNA fragmentation and chromatin condensation caused by Stx were completely blocked by pretreatment of cells with brefeldin A, an inhibitor of Golgi functions. The findings suggest that Stx1 as well as Stx2 activate caspase-3, which plays a critical role in apoptosis, and that the apoptotic signals rise after Stx is transported to the Golgi apparatus.

The effects of prolonged low-flow sevoflurane anesthesia on renal and hepatic function.

UNLABELLED: We assessed the effects of prolonged low-flow sevoflurane anesthesia on renal and hepatic functions by comparing high-flow sevoflurane with low-flow isoflurane anesthesia. Thirty patients scheduled for surgery of > or =10 h in duration randomly received either low-flow (1 L/min) sevoflurane anesthesia (n = 10), high-flow (6-10 L/min) sevoflurane anesthesia (n = 10), or low-flow (1 L/min) isoflurane anesthesia (n = 10). We measured the circuit concentrations of Compound A and serum fluoride. Renal function was assessed by blood urea nitrogen, serum creatinine, creatinine clearance, and urinary excretion of glucose, albumin, protein, and N:-acetyl-beta-D-glucosaminidase. The hepatic function was assessed by serum aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, and total bilirubin. Compound A exposure was 277 +/- 120 (135-478) ppm-h (mean +/- SD [range]) in the low-flow sevoflurane anesthesia. The maximum concentration of serum fluoride was 53.6 +/- 5.3 (43.4-59.3) micromol/L for the low-flow sevoflurane anesthesia, 47.1 +/- 21.2 (21.4-82.3) micromol/L for the high-flow sevoflurane anesthesia, and 7.4 +/- 3.2 (3.2-14.0) micromol/L for the low-flow isoflurane anesthesia. Blood urea nitrogen and serum creatinine were within the normal range, and creatinine clearance did not decrease throughout the study period in any group. Urinary excretion of glucose, albumin, protein, and N:-acetyl-beta-D-glucosaminidase increased after anesthesia in all groups, but no significant differences were seen among the three groups at any time point after anesthesia. Lactate dehydrogenase and alkaline phosphatase on postanesthesia Day 1 were higher in the high-flow sevoflurane group than in the low-flow sevoflurane group. However, there were no significant differences in any other hepatic function tests among the groups. We conclude that prolonged low-flow sevoflurane anesthesia has the same effect on renal and hepatic functions as high-flow sevoflurane and low-flow isoflurane anesthesia. IMPLICATIONS: During low-flow sevoflurane anesthesia, intake of Compound A reached 277 +/- 120 ppm-h, but the effect on the kidney and the liver was the same in high-flow sevoflurane and low-flow isoflurane anesthesia.

Saponins isolated from Allium chinense G. Don and antitumor-promoting activities of isoliquiritigenin and laxogenin from the same drug.

Investigation of the Chinese crude drug "Xiebai," the bulbs of Allium chinense G. Don (Liliaceae), led to the isolation of 2 saponins, xiebai-saponin I (laxogenin 3-O-beta-xylopyranosyl (1-->4)-[alpha-arabinopyranosyl (1-->6)-beta-glucopyranoside) (1) and laxogenin 3-O-alpha-arabinopyranosyl (1-->6)-beta-glucopyranoside (2), and the aglycone, laxogenin (3), together with 2 chalcones, isoliquiritigenin (4) and isoliquiritigenin-4-O-glucoside (5), and beta-sitosterol glucoside (6). Compounds 1-5 were tested in vitro for their inhibitory effect on the 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated 32Pi-incorporation into phospholipids of HeLa cells. In addition to this, laxogenin (3) was proven to have an antitumor-promoting activity in a two-stage lung carcinogenesis experiment.

Double ventricular response by a single ventricular extrastimulus to the inner loop of reentry in a patient without apparent heart disease.

In a patient without apparent heart disease, a ventricular extrastimulus delivered from the left ventricular apex where the electrogram was recorded 30 ms after the onset of the QRS complex during VT advanced the second QRS complex, but not the first QRS complex. The morphology of the second QRS complex was the same as that of VT. The postpacing interval was the same as the cycle length of the VT. These findings indicated that the site of stimulation was at the inner loop of the reentry circuit of the VT. A ventricular extrastimulus with a shorter coupling interval advanced the first and second QRS complexes, indicating that the ventricle was activated by antidromic and orthodromic activation from the extrastimulus. Radiofrequency ablation at that site of stimulation terminated the VT and no further VT could be induced.

Sphingosylphosphorylcholine induces cytosolic Ca(2+) elevation in endothelial cells in situ and causes endothelium-dependent relaxation through nitric oxide production in bovine coronary artery.

Sphingosylphosphorylcholine (SPC) increased intracellular Ca(2+) concentration ([Ca(2+)]i) and nitric oxide (NO) production in endothelial cells in situ on bovine aortic valves, and induced endothelium-dependent relaxation of bovine coronary arteries precontracted with U-46619. The SPC-induced vasorelaxation was inhibited by N(omega)-monomethyl-L-arginine, an inhibitor of both constitutive and inducible NO synthase (NOS), but not by 1-(2-trifluoromethylphenyl) imidazole, an inhibitor of inducible NOS (iNOS). Immunoblotting revealed that endothelial constitutive NOS, but not iNOS, was present in endothelial cells in situ on the bovine aortic valves. We propose that SPC activates [Ca(2+)]i levels and NO production of endothelial cells in situ, thereby causing an endothelium-dependent vasorelaxation.

Effect of ageing on blood flow to the bladder and bladder function.

Ischemia, induced by atherosclerosis, is a common cause of disorders in the elderly. Bladder dysfunction in older people may be caused by detrusor ischemia. We compared blood flow to the bladder and detrusor function in vivo and in vitro in young (6-month-old) and aged (24-month-old) male Sprague-Dawley rats. In both young and old rats, blood flow to the bladder measured by a laser Doppler flowmeter decreased as intravesical volume increased and was smaller in old rats than in young rats. Cystometrograms performed under anesthesia showed that old rats had smaller voiding pressure and larger bladder capacity than young rats. In isolated bladders, the pressure increase in response to bethanechol and low frequency field stimulation were impaired by aging. Volume-pressure studies showed that in isolated bladders of old rats compliance was greater and peak response to field stimulation was observed at a larger capacity. These findings indicate that bladders of older rats have a larger capacity with good compliance, but less contractility. Aging changes correlate with a decrease in blood flow to the bladder.

Follow up of women with simple ovarian cysts detected by transvaginal sonography in the Tokyo metropolitan area.

OBJECTIVE: To investigate the management of women with asymptomatic ovarian masses, to determine the appropriate duration of follow up, and to identify diagnostic indicators of growing cysts. DESIGN: Review of women's hospital records. SETTING: Tokyo Metropolitan Cancer Detection Center, Japan. POPULATION: Two hundred and twenty-five pre- and postmenopausal women with a diagnosis of ovarian cyst < 6 cm in diameter and normal serum level of CA125, diagnosed between 1 October 1990 and 25 December 1991. MAIN OUTCOME MEASURE: Change in size of cyst as shown by ultrasound. RESULTS: Seventy-five months after initial diagnosis, 29 (13%) of the masses had progressed, 31 (14%) had persisted, and 165 (73%) had regressed. One hundred and nine masses (48%) had regressed within six months of the initial diagnosis. In univariate analysis transvaginal ultrasonographic assessment of morphology findings, cyst diameter, carcinoembyronic antigen (CEA) and CA19-9 were associated with the prognosis of the cyst. Multivariate regression analysis demonstrated that only the initial serum CA19-9 level and serum CEA level were significant predictors of ovarian masses that regressed (P for trend = 0.004 and 0.02, respectively). CONCLUSION: Simple ovarian cysts in patients with a normal level of CA125 have a low risk for ovarian cancer. Vaginal ultrasound at six months will identify regression of most simple cysts. CA19-9 and CEA at the initial diagnosis are useful parameters to predict future regression of ovarian cysts.

Assessment of right artrial mapping and P wave-triggered signal-average in patients with paroxysmal atrial fibrillation.

To assess right atrial mapping and P wave-triggered signal-averaged electrocardiogram (ECG) in patients with paroxysmal atrial fibrillation (PAF), this study examined right atrial electrograms using atrial mapping and parameters by P wave-triggered signal-averaged ECG in 39 patients without sick sinus syndrome. Subjects were divided into those with PAF (n = 13; 60+/-13 years old) and a control group (n = 26; 49+/-19 years old). The total number of abnormal right atrial electrograms per patient was significantly greater in the PAF group (3.2+/-1.9) than in the control group (1.1+/-0.9; P < .001). The longest duration of right atrial electrogram in the PAF group tended to be greater than that in the control group (P = .06). The filtered P wave duration was significantly longer in the PAF group than in the control group (144+/-21 vs 125+/-14 ms [P < .002]). The values of the root mean square of P wave-triggered signal-averaged ECG 15 ms from the onset (RMSi 15) and 20 ms from the offset (RMSe 20) were significantly lower in the PAF group (1.1+/-0.4 microV, 1.4+/-0.5 microV) than in the control group (1.9+/-1.1 microV [P < .02], 2.1+/-0.9 microV [P < .01]). The total number of right atrial electrograms in patients with RMSi 15 of < or =1.5 microV was significantly greater than in patients with RMSi 15 of >1.5 microV (2.2+/-1.8 vs 1.3+/-1.3 [P < .05]). Thus, the total number of abnormal right atrial electrograms per patient, the total filtered P wave duration, RMSi 15, and RMSe 20 may be good indices of PAF in patients without sick sinus syndrome. RMSi 15 may reflect the total number of the abnormal right atrial electrograms per patient.

Selective effects of genistein, a soybean isoflavone, on B-lymphopoiesis and bone loss caused by estrogen deficiency.

Genistein, an isoflavone abundantly present in soybeans, has structural similarity to estrogen, suggesting that genistein may act as a phytoestrogen. To examine the possible role of genistein in hemopoiesis and bone metabolism, female mice were either sham-operated or ovariectomized (OVX), and selected OVX mice were administered genistein for 2-4 weeks (0.1-0.7 mg/day) or 17beta-estradiol (E2; 0.01-0.1 microg/day) s.c., using a miniosmotic pump (Alza Corp., Palo Alto, CA). In OVX mice, uterine weight declined but was completely restored by E2 administration. In contrast, genistein did not demonstrate a reversal of the OVX-induced uterine atrophy. The number of bone marrow cells markedly increased, 2-4 weeks after OVX, and most of these were B220-weakly positive pre-B cells. The increased B-lymphopoiesis was completely restored, not only by E2 but also by genistein administration. In OVX mice, the trabecular bone volume of the femoral distal metaphysis, measured by microcomputed tomography scanning and dual-energy x-ray absorptiometry, was markedly reduced; and genistein restored this, as did E2. These results indicate that genistein exhibits estrogenic action in bone and bone marrow, to regulate B-lymphopoiesis and prevent bone loss, without exhibiting estrogenic action in the uterus. Phytoestrogens may be useful for preventing bone loss caused by estrogen deficiency in females.

Induction of cytokines in a human colon epithelial cell line by Shiga toxin 1 (Stx1) and Stx2 but not by non-toxic mutant Stx1 which lacks N-glycosidase activity.

Stx1 and Stx2 produced by Shiga toxin-producing Escherichia coli are cytotoxic due to their N-glycosidase activity on 28S rRNA. In this study, we have shown that proinflammatory cytokine mRNAs, especially IL-8, were induced by Stx1 and Stx2 in Caco-2 cells. A non-toxic mutant of Stxl which lacks N-glycosidase activity did not induce cytokine mRNAs. IL-8 production at the protein level was enhanced by Stx1 and Stx2, but not by the mutant Stx1. These results demonstrate that Shiga toxins induce expression and synthesis of cytokines in Caco-2 cells and their N-glycosidase activity is essential for the induction.

Evaluation of inner ear histology and auditory brainstem response in Wriggle Mouse Sagami.

Wriggle Mouse Sagami (WMS) is a spontaneous mutant strain with neuroepithelial defects. These animals are characterized by abnormal movements linked to an autosomal recessive gene. To determine the association between inner ear histology and hearing ability, we assayed these characteristics in mice homozygous and heterozygous for the mutation, as well as in wild-type animals. In homozygotes, the cochlea and saccule degenerated 3 months after birth. Beginning at 3 months of age, and progressing in an age-dependent manner, the organ of Corti disappeared and the number of spiral ganglion cells decreased, starting at the basal turn and moving toward the apical turn. The sensory epithelium became atrophic in the saccule. Three-month-old heterozygotes demonstrated degeneration in the cochlea, not in the saccule. No obvious auditory brainstem evoked response (ABR) was observed at any frequency in homozygotes aged 1 month and older. In contrast, the heterozygotes retained some hearing acuity until the age of 1 month, after which they became deaf. These findings suggest that WMS mice may provide a good model that will be useful in identifying deafness genes in humans.

Effects of sevoflurane anesthesia on plasma inorganic fluoride concentrations during and after cardiac surgery.

PURPOSE: Sevoflurane metabolism results in the production of inorganic fluoride, which is known to be nephrotoxic. Since marked changes in body temperature and hemodynamics in cardiac surgery affect sevoflurane metabolism, plasma inorganic fluoride concentrations may differ in this situation compared with other types of surgery. We therefore measured plasma inorganic fluoride concentrations during and after sevoflurane anesthesia in patients undergoing cardiac surgery. METHODS: Sixteen patients undergoing coronary artery bypass grafting or valve replacement were premedicated with 5-10 mg midazolam and 0.5 mg scopolamine injected intramuscularly. Anesthesia was induced with 5-10 mg midazolam, 0.5-1 mg fentanyl, and 0.12-0.15 mg.kg(-1) vecuronium. Following tracheal intubation, anesthesia was maintained with oxygen, sevoflurane, and fentanyl. At the onset of cardiopulmonary bypass (CPB), sevoflurane was discontinued, and additional fentanyl, midazolam, and pancuronium were administered. Plasma inorganic fluoride concentrations were measured before anesthesia, immediately before and after CPB, and at 0, 2, 6, 12, 24, and 48 h after anesthesia. RESULTS: The individual maximum plasma inorganic fluoride concentration was 19.2 +/- 7.2 micromol.l(-1) (mean +/- SD; range, 9.2-36.7). The mean plasma inorganic fluoride concentrations increased during anesthesia, but the rate of increase decreased after the initiation of CPB. Concentrations peaked at 2 h after anesthesia and decreased thereafter. The concentrations in three cases continued to increase 2 h after anesthesia. CONCLUSION: The plasma inorganic fluoride concentrations observed in patients undergoing cardiac surgery were below nephrotoxic levels. However, the decrease in mean fluoride concentration after anesthesia was slower than that in the previous study in general surgical patients.