A cationic lipid-formulated plasmid DNA vaccine confers sustained antibody-mediated protection against aerosolized anthrax spores.

DNA vaccines provide an attractive technology platform against bioterrorism agents due to their safety record in humans and ease of construction, testing, and manufacture. We have designed monovalent and bivalent anthrax plasmid DNA (pDNA) vaccines encoding genetically detoxified protective antigen (PA) and lethal factor (LF) proteins and tested their immunogenicity and ability to protect rabbits from an aerosolized inhalation spore challenge. Immune responses after two or three injections of cationic lipid-formulated PA, PA plus LF, or LF pDNAs were at least equivalent to two doses of anthrax vaccine adsorbed (AVA). High titers of anti-PA, anti-LF, and neutralizing antibody to lethal toxin (Letx) were achieved in all rabbits. Eight or nine animals in each group were challenged with 100x LD(50) of aerosolized anthrax spores 5 or 9 weeks after vaccination. An additional 10 animals vaccinated with PA pDNA were challenged >7 months postvaccination. All animals receiving PA or PA plus LF pDNA vaccines were protected. In addition, 5 of 9 animals receiving LF pDNA survived, and the time to death was significantly delayed in the others. Groups receiving three immunizations with PA or PA plus LF pDNA showed no increase in anti-PA, anti-LF, or Letx neutralizing antibody titers postchallenge, suggesting little or no spore germination. In contrast, titer increases were seen in AVA animals, and in surviving animals vaccinated with LF pDNA alone. Preclinical evaluation of this cationic lipid-formulated bivalent PA and LF vaccine is complete, and the vaccine has received U.S. Food and Drug Administration Investigational New Drug allowance.

Mast cells play a partial role in allergen-induced subepithelial fibrosis in a murine model of allergic asthma.

BACKGROUND: Role of mast cells in the development of allergen-induced airway remodelling has not been fully investigated in vivo. OBJECTIVE: To clarify the possible role of mast cells in the development of allergen-induced airway remodelling, we compared their responses of genetically mast cell-deficient mice, WBB6F1-W/Wv (c-kit mutant) and Sl/Sld (c-kit ligand mutant) mice with those of congenic normal mice in a murine model of allergic asthma. METHODS: Mice were sensitized to ovalbumin (OA) with alum, and exposed daily for 3 weeks to aerosolized OA. Twenty-four hours after the last inhalation, bronchial responsiveness to acetylcholine (Ach) was measured, and bronchoalveolar lavage (BAL), and biochemical and histological examinations were performed. RESULTS: In both sensitized mast cell-deficient mice, the degree of bronchial hyper-responsiveness to Ach, the number of inflammatory cells and the level of transforming growth factor-beta1 in BAL fluid, IgE response and goblet cell hyperplasia in the epithelium after repeated allergen provocation were not significantly different from those of congenic mice. In contrast, subepithelial fibrosis, evaluated in the fibrotic area around the airways, observed in congenic mice after repeated allergen challenge was partially attenuated in both mast cell-deficient mice. In addition, the amount of hydroxyproline in the lung of mast cell-deficient mice was significantly lower than that of congenic mice. Furthermore, the decreased fibrotic area and amount of hydroxyproline in W/Wv mice was completely recovered by reconstitution of tissue mast cells with bone marrow-derived mast cells of congenic mice. CONCLUSION: These findings suggest that mast cells play a partial role in the development of allergen-induced subepithelial fibrosis, although airway inflammation, epithelial remodelling and BHR caused by repeated allergen challenge are independent of mast cells, at least in this model.

Time course study on the development of allergen-induced airway remodeling in mice: the effect of allergen avoidance on established airway remodeling.

OBJECTIVE AND DESIGN: We carried out a time course study on the development of allergen-induced airway remodeling in a mouse model of allergic asthma. Moreover, we examined the effect of allergen avoidance on the established airway remodeling. MATERIALS AND METHODS: BALB/c mice were sensitized to ovalbumin (OA) with alum, and exposed daily for 3 weeks to aerosolized OA. At each designated point, bronchial responsiveness was measured, and bronchoalveolar lavage and histological examination were carried out. RESULTS: The numbers of inflammatory leukocytes in the airways and the percentage of goblet cells in the epithelium, Th2 cytokine production, IgE production, collagen deposition beneath the basement membrane and bronchial responsiveness to acetylcholine were all markedly increased after repeated antigen challenge for 1-3 weeks. In contrast, after cessation of antigen exposure, goblet cell hyperplasia, inflammatory infiltrates and bronchial hyperresponsiveness were gradually attenuated and had almost resolved 4 weeks after cessation, but subepithelial fibrosis was still observed at this time point. CONCLUSIONS: The present findings demonstrated that epithelial changes following repeated allergen challenge are rapidly induced and recover after the cessation of exposure, but subepithelial fibrosis has a late onset and relatively irreversible changes, and subepithelial fibrosis in contrast to goblet cells hyperplasia did not appear to contribute to bronchial hyperresponsiveness, at least, in this mouse model.

Long-term zinc deficiency decreases taste sensitivity in rats.

The effects of zinc deficiency on taste sensitivity were examined in rats by recording the electrophysiological responses of the chorda tympani (CT) nerve and by use of a preference test. Male 4-wk-old Sprague-Dawley rats were given free access to a diet containing 2.2 (zinc-deficient), 4.1 (low zinc) or 33.7 (zinc-sufficient) mg zinc/kg diet. A fourth group was pair-fed the zinc-sufficient diet (with respect to the zinc-deficient rats). A two-bottle preference test using 0.15 mol/L NaCl and water revealed that NaCl preference was greater in the zinc-deficient and low zinc groups than in the control groups (zinc-sufficient and pair-fed) after 4 d of feeding. In the case of quinine hydrochloride solution (0.01 mmol/L), the preference was greater in zinc-deficient rats than in the other groups after 9 d, and the low zinc rats never showed a preference. Electrophysiological recording indicated that in the zinc-deficient rats, the CT nerve response to 0.20 mol/L NaCl was significantly less than that in the control rats after 21 d of feeding. In the low zinc rats, this response was significantly less than in the control rats after 35 d. The responses to quinine hydrochloride (0.02 mol/L), L-glutamic acid, HCl (0.01 mol/L) and NH(4)Cl (0.25 mol/L) in the zinc-deficient rats were not significantly reduced until d 42. These findings suggest that long-term zinc deficiency decreases taste sensitivity at the level of the CT nerve and that the change in NaCl preference due to zinc deficiency occurs before any change in NaCl taste sensitivity.

Lecithin:cholesterol acyltransferase reduces the adverse effects of oxidized low-density lipoprotein while incurring damage itself.

In this study, we tried to determine (a) whether there is any permanent effect of oxLDL on the LCAT molecule and its activator lipoprotein apoA-I, and (b) the fate of oxLDL after its exposure to LCAT and apoA-I. Purified LCAT and LDL/oxLDL was incubated at 37 degrees C for 1 h, and separated by gel-permeation chromatography. Its activity was significantly less (30% less) after separating from oxLDL compared to that of LDL. Cofactor activity of isolated apoA-I (incubated with LDL/oxLDL) was found affected by oxLDL. These results indicate modification of the LCAT and apoA-I molecule. But LCAT was found more affected compared to apoA-I in terms of LCAT activity. We are also reporting a novel function of LCAT. It was found to reduce the adverse effects of oxLDL, for example, ability to affect the LCAT activity and TBARS value. This ability of LCAT to repair oxLDL was dose-dependent. But there was no change in its REM values or fluorescence intensity.

The effect of allergen-induced airway inflammation on airway remodeling in a murine model of allergic asthma.

OBJECTIVE AND DESIGN: We examined the effect of airway inflammation on airway remodeling and bronchial responsiveness in a mouse model of allergic asthma. MATERIALS AND METHODS: BALB/c mice were sensitized to ovalbumin (OA), and exposed to aerosolized OA (0.01, 0.1 and 1%). Twenty-four hours after the final antigen challenge, bronchial responsiveness was measured, and bronchoalveolar lavage (BAL) and histological examinations were carried out. RESULTS: Repeated antigen exposure induced airway inflammation, IgE/IgG1 responses, epithelial changes, collagen deposition in the lungs, subepithelial fibrosis associated with increases in the amount of transforming growth factor (TGF)-beta1 in BAL fluid (BALF), and bronchial hyperresponsiveness to acetylcholine. The number of eosinophils in BALF was significantly correlated with TGF-beta1 production in BALF and the amount of hydroxyproline. Furthermore, significant correlations were found between these fibrogenic parameters and the bronchial responsiveness. CONCLUSION: These findings demonstrated that in this murine model airway eosinophilic inflammation is responsible for the development of airway remodeling as well as bronchial hyperresponsiveness in allergic bronchial asthma.

Reduction in carbonic anhydrase activity in the tongue epithelium and submandibular gland in zinc-deficient rats.

We investigated the effects of zinc deficiency on carbonic anhydrase (CA) activity in the tongue epithelium and submandibular gland in rats. Male 4-week-old SD rats were given free access to a diet containing 2.2 (zinc-deficient), 4.1 (low-zinc), or 33.7 (zinc-sufficient) mg zinc/kg diet for 6 weeks. Rats in the fourth group (receiving 33.7 mg zinc/kg) were pair-fed against the zinc-deficient rats. Biochemical analysis at the end of the experimental period indicated that zinc deficiency significantly reduced CA activity in the tongue epithelium and submandibular gland, and the CA activity levels in these tissues seemed to parallel the dietary zinc levels. By enzyme histochemistry, an intensely positive reaction for CA was observed in the middle and basal regions of the taste buds in the circumvallate papilla in the zinc-sufficient and pair-fed (control) rats. The cells in von Ebner's glands also showed a strong positive reaction in control rats, although only a weak reaction product was found in zinc-deficient rats. These results suggest that CA activity is affected by the dietary content of zinc, which is considered to be an indispensable factor for the maintenance of normal taste sensation.

Characteristics of the biotin enhancement of glucose-induced insulin release in pancreatic islets of the rat.

Perifused isolated rat islets were used to show that biotin plus 16.5 mM glucose evoked more insulin secretion than 16.5 mM glucose alone. Whether or not this reinforcement of glucose-induced insulin secretion by biotin is unique was studied by using perifused islets stimulated with 16.5 mM glucose plus 100 microM of one of various components of the vitamin B group. No effect of any of these vitamins was found on glucose-induced insulin secretion. These results indicate that biotin is unique among the members of the vitamin B group in enhancing glucose-induced insulin secretion. Static incubation experiments showed that biotin did not potentiate insulin release when the islets were incubated with an experimental solution containing either no or 2.8 mM glucose. The addition of biotin to 27.7 mM glucose, which is the maximal concentration for stimulating insulin release, did not significantly enhance the effect of the glucose on insulin release (although it did at 16.5 mM glucose). These findings indicate that biotin, by itself, does not stimulate insulin secretion, and does not enhance glucose-induced insulin secretion beyond the ability of glucose itself to stimulate insulin secretion.

Zinc deficiency and taste dysfunction; contribution of carbonic anhydrase, a zinc-metalloenzyme, to normal taste sensation.

The present study was designed to clarify the effect of zinc deficiency on sodium chloride preference, the lingual trigeminal and taste nerves transduction, and carbonic anhydrase (CA) activity of the tongue surface and salivary gland. Male SD rats, 4 weeks old, were divided into four groups, and fed zinc-deficient (Zn-Def), low-zinc (Low-Zn), and zinc-sufficient diets with free access (Zn-Suf) and pair-feeding (Pair-fed). After taking part in the preference tests for 42 days, the rats were provided for the chorda tympani and lingual trigeminal nerves recordings, then finally sacrificed and the tongue and submandibular gland excised to measure CA activity. Sodium chloride preference increased only after 4 days of the feeding of zinc-deficient and low-zinc diets, which means that the taste abnormality appears abruptly in zinc deficieny and even though in marginal zinc deficiency. Reduced CA activities of the taste-related tissues in zinc-deficient group paralleled well with the decreased taste and lingual trigeminal nerves sensitivities.

Percutaneous absorption of biotin in healthy subjects and in atopic dermatitis patients.

The study was designed to test the ability of sequential applications of biotin-containing ointment to increase serum biotin levels. Twenty atopic dermatitis patients (mean age, 20.5 yr) and 11 healthy subjects (mean age, 25.5 yr) volunteered to participate in this study. The diagnosis of atopic dermatitis was established dermatologically. Seven grams per day of ointment containing 0.3% biotin and 1-4 g per day of steroid ointment were both applied sequentially. The healthy subjects applied only biotin ointment. The biotin concentration was determined microbiologically. Before biotin treatment, the average serum biotin level was significantly lower in atopic dermatitis patients than in healthy subjects. The percutaneous application of biotin-containing ointment caused a significant increase in the serum biotin concentration in both healthy subjects (from 41.5 +/- 10.0 to 50.2 +/- 9.2 nmol/L) and in atopic dermatitis patients (from 27.9 +/- 17.4 to 50.7 +/- 21.6 nmol/L), especially in patients whose initial level was low, and also could be effective in regulating the atopic allergic response involving eosinophils. In conclusion, biotin appears to be readily absorbed through both normal and dermatitis-affected human skin.

Effects of dietary biotin on enhanced sucrose intake and enhanced gustatory nerve responses to sucrose seen in diabetic OLETF rat.

We used the sucrose preference test and taste nerve recording to investigate the effect of dietary biotin on the abnormal sucrose taste sensitivity and preferences seen during the course of diabetes mellitus. For this, we used Otsuka Long-Evans Tokushima fatty (OLETF) rats. The chorda tympani nerve (CT nerve) response to sucrose (> 1 M) was of greater relative magnitude in OLETF rats than in non-diabetic control (Long-Evans Tokushima Lean, LETO) rats, but the responses to other basic taste stimuli (such as HCl, quinine-HCl and L-glutamic acid) did not differ between the two groups. In behavioral experiments using a two-bottle preference test, solution intake for sucrose (> 50 mM) was higher in OLETF rats than in LETO rats. The neural responses to sucrose (1.5-2 M) in OLETF rats were lower when given a biotin-high diet (BH-OLETF) than when given a biotin-basal diet (BB-OLETF), but this was not true of the other basic tastes. However, there were no significant differences between BH-OLETF and BB-OLETF rats in terms of sucrose solution intake. These findings suggest that the enhanced sugar sensitivity observed in OLETF rats is probably the result of a genetic difference between OLETF and LETO rats, though the discrepancy can be modified by the dietary biotin level.

Capsaicin modifies responses of rat chorda tympani nerve fibers to NaCl.

Single-fiber preparations of the rat chorda tympani (CT) nerve were used to study the mechanism of action of capsaicin on salt-taste transduction. Capsaicin selectively suppressed the responses to NaCl of the CT nerve fibers (N-fibers) that are sodium-specific (insensitive or poorly sensitive to potassium). Among the more broadly responsive, cation-sensitive fibers (E-fibers) there are two subtypes, both of which responded to capsaicin but in different ways ('enhanced' type and 'suppressed' type). In both N- and E-fibers, 5% ethanol (the vehicle for capsaicin) slightly reduced the response to 100 mM NaCl. The suppressive effect of capsaicin on the response of the N-type fibers to 100 mM NaCl was significantly stronger than the effect of 5% ethanol. The suppression lasted for at least 20 s after the simultaneous application of 100 p.p.m. capsaicin-100 nM NaCl. These results indicate that 100 p.p.m. capsaicin can modify the response of CT fibers to NaCl. The observed effect of capsaicin on gustatory fibers could be the net result of opposite suppressive and enhancing processes in the taste buds cells and excited intra- or extragemmal trigeminal nerve endings.

Menaquinone-4 accumulation in various tissues after an oral administration of phylloquinone in Wistar rats.

The distributions of phylloquinone (PK) and menaquinone-4 (MK-4) in various tissues were assessed after the oral administration of phylloquinone. Wistar rats were fed a vitamin-K-deficient diet for nine days, fasted for 24 h and then given phylloquinone orally at 4 mg/kg body weight. Rats were sacrificed 0, 6, 12 and 24 h after the administration, and an analysis was made of the vitamin K analogues in the plasma, liver, brain, testis, kidney and spleen. The phylloquinone concentration in plasma and the tissues reached a peak 6 h after the oral administration of phylloquinone. By contrast, the concentration of MK-4 peaked in the liver, plasma, kidney and spleen at 12 h, and in brain and testis at 24 h. This data suggests that the ingested phylloquinone was probably converted into MK-4 within the tissues themselves, rather than via hepatic metabolism. The evidence for this is that, after phylloquinone administration, (i) in each of the tissues, the MK-4 concentration increased much more slowly than that of phylloquinone, and (ii) the MK-4 concentration in the plasma and liver reached only much lower levels than those seen in other tissues.

Active inhibitory effect of nafamostat mesylate against the elevation of plasma myeloperoxidase during hemodialysis.

Plasma levels of myeloperoxidase (MPO) were compared between hemodialysis (HD) sessions using heparin and those using nafamostat mesylate (NM) as an anticoagulant by an enzyme immunoassay established in our laboratory. MPO levels were markedly elevated during the entire HD procedure with heparin. In contrast, MPO levels were scarcely elevated during the HD with NM. On the other hand, polymorphonuclear leukocyte-elastase was markedly elevated during both of these HD procedures. These observations indicated that NM selectively attenuated MPO elevation in vivo during HD. This inhibitory effect of NM was further investigated ex vivo. Blood samples from a normal subject were collected with heparin alone, NM alone and a mixture of heparin and NM. Each sample was then circulated in a closed circuit composed of a dialyzer with a cuprophane membrane. MPO levels with heparin alone were shown to markedly rise in the closed system. In contrast, levels of MPO in the blood samples mixed with NM were not elevated even in the presence of heparin. These ex vivo results indicate that NM has an active inhibitory effect on the elevation of plasma MPO induced by granulocyte activation through a dialysis membrane. Our results demonstrate that clinical use of NM as an anticoagulant serves to selectively suppress MPO elevation considered as a consequence of granulocyte activation during HD.

A high biotin diet improves the impaired glucose tolerance of long-term spontaneously hyperglycemic rats with non-insulin-dependent diabetes mellitus.

The Otsuka Long-Evans Tokushima Fatty (OLETF) rat, serving as a spontaneously diabetic model with non-insulin-dependent diabetes mellitus (NIDDM), exhibits impaired glucose tolerance (IGT) at about 16 weeks of age. In this study, we investigated whether or not biotin, a water-soluble vitamin, improved the IGT of OLETF rats. To this end, we administered diets containing one of three levels of biotin, a high-biotin diet (BH), a normal-biotin diet (BN) and a basal-biotin diet (BB), to OLETF rats up to 24 weeks of age. An oral glucose tolerance test (OGTT) was performed four times between 13 and 22 weeks of age. The administration of a BH corrected the IGT of OLETF rats. Upon further investigation, we found that insulin secretion in the OLETF-BH rats was decreased to a significant extent, signaling that the hyperinsulinemia typical to the OLETF-BH rats had clearly improved. Body weights were significantly lower in the OLETF-BH group than in the other OLETF groups, even though the OLETF-BH rats showed a significantly higher average daily food intake. The body weight gain of the OLETF-BH rats followed the same tendency as the control-LETO (Long Evans Tokushima Otsuka) rats (LETO-BB and LETO-BN). These results demonstrate that a high-level biotin diet can improve the glucose handicap in NIDDM rats.

Establishment and molecular characterization of a novel leukemic cell line with Philadelphia chromosome expressing p230 BCR/ABL fusion protein.

The cell line AR230 was established from the peripheral blood mononuclear cells of a patient with chronic myeloid leukemia and t(9;22) translocation bearing a variant type of BCR/ABL rearrangement. AR230 expresses a BCR/ABL fusion protein with a molecular mass of 230 kilodaltons (kDa) due to the insertion of 180 amino acids encoded by 3' exons of BCR (b4 to c3). An immune complex kinase assay showed that the 230-kDa BCR/ABL protein ahd autophosphorylation activity. Immunoprecipitation analysis revealed a stable complex of GRB2 and 230-kDa BCR/ABL proteins, indicating that the Ras activation pathway is involved in the process of transformation. AR230 expressed another short transcript consisting of a BCRc2/ABL junction, which is associated with a stop signal shortly after the junction. To our knowledge, this is the first cell line expressing a 230-kDa fusion product of BCR/ABL. AR230 will be useful for studying the biological function of divergent BCR/ABL proteins.

The effect of dietary protein levels on the responses of the taste nerve to sodium chloride in spontaneously hypertensive rats (SHRs).

The present study was undertaken to clarify the effect of dietary protein level on the response of the taste nerve to NaCl solutions in spontaneously hypertensive rats (SHRs). The results showed that the taste sensitivity to NaCl in SHRs fed a 5% whole-egg protein diet for 3 weeks was significantly lower than in those fed a 15% protein diet. This observation suggests that chronic feeding of a low-protein diet causes an impairment of salt-taste reception or subsequent transduction.

Surgical procedure and results of repair of massive tears of the rotator cuff.

One hundred eighty-seven patients (189 shoulders) were treated surgically between 1970 and 1992 for massive rotator cuff tears using either a tendon-to-tendon repair or the McLaughlin procedure. The age of the patients ranged from 20 to 86 years; 95% of them were 45 years or older. The average followup was 6 years 9 months. Excellent or good functional results were attained in 93% of patients. Thirty-three percent of those who underwent tendon to tendon repair complained of pain after overuse compared with only 18% who had the McLaughlin Procedure.

Enhancement of gustatory nerve fibers to NaCl and formation of ion channels by commercial novobiocin.

Single fibers of the rat chorda tympani nerve were used to study the mechanism of action of the antibiotic novobiocin on salt taste transduction. In the rat, novobiocin selectively enhanced the responses of sodium-specific and amiloride-sensitive chorda tympani nerve fibers (N type) without affecting more broadly responsive cation-sensitive and amiloride-insensitive fibers (E type). In the presence of amiloride, novobiocin was ineffective at enhancing the response of N-type fibers toward sodium chloride. Novobiocin also increased the conductance of bilayers formed from neutral lipids by forming nonrectifying ion channels with low conductance (approximately 7 pS in 110 mM NaCl), long open times (several seconds and longer), and high cation selectivity. Amiloride did not alter either the conductance or kinetics of these novobiocin channels. These observations suggest that even though novobiocin is able to form cation channels in lipid bilayers, and possibly in cell membranes as well, its action on the salt-taste response is through modulation of existing amiloride-sensitive sodium channels.

Urinary mercury monitoring of university staff and students occasionally exposed to mercury vapor.

To monitor the exposure to mercury (Hg) vapor among university staff members and students who occasionally handle elemental Hg in laboratory experiments, urine samples were collected at health examinations conducted by the Health Service Center, University of Tokyo, for six years. Geometric mean of urinary Hg concentrations of 343 samples collected from 234 subjects was 1.61 micrograms Hg/g creatinine (Cr), with the range of 0.30 to 9.31 micrograms Hg/g Cr. Elevated urinary Hg levels, i.e. 3 micrograms Hg/g Cr or higher, were found only among the subjects who worked in several laboratories. This urinary Hg level is judged to correspond to 1-2 micrograms/m3 of air Hg concentration in working areas. The contribution of dental amalgam fillings to urinary Hg excretion, though it exists, was concluded to be small from the result of multiple regression analysis.