Molecular allergen profiling in horses by microarray reveals Fag e 2 from buckwheat as a frequent sensitizer.

BACKGROUND: Companion animals are also affected by IgE-mediated allergies, but the eliciting molecules are largely unknown. We aimed at refining an allergen microarray to explore sensitization in horses and compare it to the human IgE reactivity profiles. METHODS: Custom-designed allergen microarray was produced on the basis of the ImmunoCAP ISAC technology containing 131 allergens. Sera from 51 horses derived from Europe or Japan were tested for specific IgE reactivity. The included horse patients were diagnosed for eczema due to insect bite hypersensitivity, chronic coughing, recurrent airway obstruction and urticaria or were clinically asymptomatic. RESULTS: Horses showed individual IgE-binding patterns irrespective of their health status, indicating sensitization. In contrast to European and Japanese human sensitization patterns, frequently recognized allergens were Aln g 1 from alder and Cyn d 1 from Bermuda grass, likely due to specific respiratory exposure around paddocks and near the ground. The most prevalent allergen for 72.5% of the tested horses (37/51) was the 2S-albumin Fag e 2 from buckwheat, which recently gained importance not only in human but also in horse diet. CONCLUSION: In line with the One Health concept, covering human health, animal health and environmental health, allergen microarrays provide novel information on the allergen sensitization patterns of the companion animals around us, which may form a basis for allergen-specific preventive and therapeutic concepts.

Development of Helicobacter pylori-induced gastric carcinoma in Mongolian gerbils.

Helicobacter pylori is classified by IARC/WHO as a definite human gastric carcinogen, despite "inadequate experimental evidence." To obtain direct evidence concerning this relationship, we investigated the histopathological findings of gastric mucosa using a model of H. pylori infection in Mongolian gerbils. The animals were challenged p.o. with H. pylori ATCC-43504 and sacrificed at 6, 12, and 18 months after inoculation for histological examination. All inoculated animals were infected with H. pylori. Severe infiltration of the lamina propria by polymorphonuclear and mononuclear cells appeared in the lesser curvature of the antrum, with an increase in epithelial cell proliferation, and the infiltration extended to the body. Atrophic gastritis and focal intestinal metaplasia also appeared in the lesser curvature of the antral mucosa at 6 months after inoculation. Intestinal metaplasia became severe, with dysplasia, after that. At 18 months after H. pylori inoculation, two of five infected animals showed three well-differentiated gastric cancers. The uninfected control animals showed no abnormal findings throughout the entire observation period. Here, it was confirmed that H. pylori infection alone causes gastric cancer in an animal model.

Morphological study of recombinant human transforming growth factor beta 1-induced intramembranous ossification in neonatal rat parietal bone.

Recombinant human transforming growth factor beta 1 (rhu TGF beta 1) was injected singly or repeatedly for 3-12 days into the periosteum of the right side parietal bone of neonatal rats under the period of bone growth, and the time course of histological changes of the bone was observed by light and electron microscopy and by enzyme histochemistry. The repeated injections of rhu TGF beta 1 at 200 ng/day increased the thickness of the bone tissue on the treated side, which was about twice the nontreated side value after 12-day injections. On the dermal side, preosteoblasts in the periosteum increased in an early stage of treatment, and thereafter, differentiation into osteoblasts, increase of bone matrix, bone marrow cavity formation, and increase of osteoclasts within the bone marrow cavities were observed. Activation of osteoblasts on the dura mater side was also seen. The single injection of rhu TGF beta 1 at 200 ng resulted only in increased osteoprogenitor cell layers and bone matrix formation in an early stage, and the thickness of the osteoprogenitor cell layers and bone tissue at 12 days after single injection was comparable to the values on the nontreated side. At 1 microgram, however, the osteoblasts were activated, and the osteoprogenitor cell layers and bone matrix formation were markedly increased. At 12 days, the bone tissue thickness on the treated side was about twice the nontreated side value, as in the repeated treatment groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium channel components of action potential in chick skeletal muscle cells developing in culture.

The action potential was recorded from cultured chick skeletal muscle cells in Na-, Ca-, Cl-free saline containing Ba and tetraethylammonium ions (Ba saline). The action potential consisted of two components: a low-threshold, fast inactivating component and a high-threshold, long-lasting component. Both components of the action potential were dependent on external Ba ions and eliminated by Co ions. It is concluded that both components are generated by inward currents carried by Ba ions through Ca channels. The two Ca channel components of the action potential differed with regard to activation and inactivation potential, presence or absence of fast inactivation, sensitivity to an organic Ca channel blocker, and developmental profile. In addition, the failure of occurrence of one or the other components was observed in some cells. These results could be explained by assuming that two components of the action potential in Ba saline were mediated by the two different Ca channels. Furthermore, there was a tendency for younger cells to have more prominent Ca channel components. This may suggest that Ca channels have some function in the early stages of myogenesis.

Two components of calcium channel current in embryonic chick skeletal muscle cells developing in culture.

The properties of the Ca channel currents in chick skeletal muscle cells (myoballs) in culture were studied using a suction pipette technique which allows internal perfusion and voltage clamp. The Ca channel currents as carried by Ba ions were recorded, after suppression of currents through ordinary Na, K and Cl channels by absence of Na, K and Cl ions, by external TEA, by internal EGTA and by observing the Ba currents instead of the Ca currents. Two components of Ba current could be distinguished. One was present only if the myoballs were held at relatively negative holding potentials below -50 mV. This component first became detectable at clamp potentials of about -50 mV and reached a maximum between -10 and -20 mV. During long clamp steps, it became inactivated completely. The inactivation process of this component at a clamp potential of -30 mV was well fitted to a single exponential with a time constant of about -20 ms. Half-maximal steady-state inactivation was observed at -63 mV. The other component persisted even at relatively positive holding potentials above -40 mV, was observed during clamp pulses to -20 mV and above, and reached a maximum between +10 and +20 mV. This component inactivated very little; a substantial fraction of this component remained at the end of clamp pulses lasting 1 s. The inactivation process of this component at a clamp potential of -10 mV apparently followed a single exponential with a time constant of about 1 s. Half-maximal steady-state inactivation was attained at -33 mV. Both components of Ba current were blocked by Co ions, but organic Ca channel blocker D600 preferentially blocked the high-threshold, slowly inactivating component. The relationship between the current amplitude and the concentration of the external Ba ions was different between the two components. Furthermore, the two components of Ba current also differed in their developmental profile. These findings demonstrate the existence of two distinct types of Ca channels in the early stages of chick muscle cell development.

Regulation by an extract of embryonic chick brain of the densities of voltage-dependent Na+ and Ca2+ channels in embryonic chick skeletal muscle cells during their development in culture.

We studied the chronic effects of a brain extract (BE) prepared from chick embryonic brains on voltage-dependent Na+ channels (VDNCs) and Ca2+ channels (VDCCs) during the development of chick skeletal muscle cells in culture. The maximum rates of rise of Na+ and Ca2+ action potentials were measured electrophysiologically in an attempt to determine the effects of BE on the densities of these channels. The basic culture medium was supplemented with chick transferrin instead of whole-embryo extract and skeletal muscle cells were grown in the absence or in the presence of crude BE or fractionated BE. Long-term inclusion of BE to the culture medium increased the densities of both VDNCs and L-type VDCCs. By contrast, BE apparently decreased the density of T-type VDCCs. Our results indicate that BE contains some protein(s) that has a negative effect on the density of T-type VDCCs of skeletal muscle cells at a less differentiated stage and that this effect of BE is closely associated with subsequent regulation of the densities of VDNCs and L-type VDCCs. Possible roles of the influx of Ca2+ ions through T-type and L-type VDCCs in the control of the densities of VDNCs and L-type VDCCs are discussed.

Pharmacological properties of two types of calcium channel in embryonic chick skeletal muscle cells in culture.

We have studied the effects of organic and inorganic Ca channel blockers on the two-component action potentials (low- and high-threshold components) in cultured chick skeletal muscle cells, which are generated by the T- and L-type voltage-dependent Ca channels (VDCCs), respectively. Nifedipine and D600 effectively blocked the high-threshold component of the action potential, whereas omega-conotoxin and phenytoin had no effect on this component. By contrast, the low-threshold component was insensitive to all of these organic Ca channel blockers. Blocking effects of polyvalent cations were observed with the following rank order of relative potency (mean apparent dissociation constant in microM): La3+ (14.7) greater than Ni2+ (20.7) greater than Cd2+ (51.2) greater than Co2+ (912) for the low-threshold component, and Cd2+ (0.7) greater than La3+ (29.2) greater than Co2+ (431) greater than Ni2+ (1241) for the high-threshold component. Taken together, these findings suggest that the pharmacological properties of the T- and L-type VDCCs in cultured chick skeletal muscle cells may differ from those in other preparations.

Calcium channels in embryonic chick skeletal muscle cells after cultivation with calcium channel blocker.

The effects of chronic treatment with calcium channel blockers were studied on the expression of voltage-dependent calcium channels (VDCCs) in chick skeletal muscle cells developing in culture. Myotubes were treated after 2 days in culture with either 20 microM D600 or 10 microM nifedipine, and measurements were made of the maximum rate of rise (M.R.R.) of the two components of action potential, operated by T- and L-type VDCCs, respectively. Treatment with either blocker reduced the M.R.R. of the action potential component operated by the L-type VDCC throughout the culture period examined. The M.R.R. of the T-type VDCC component, on the other hand, was unaffected by either treatment. The reduction in the M.R.R. of the L-type component in blocker-treated cells is thought to be due to the down-regulation of the expression of L-type VDCC. Thus, it appears that the expression of L-type VDCC in the chick skeletal muscle cells can be regulated by a function of L-type VDCC, which mediate the entry of Ca2+ into the cells. The physiological significance of the L-type VDCC, which expressed prominently early in the development of skeletal muscle cells, for the differentiation of excitability is discussed.

Chronic treatment with D600 enhances development of sodium channels in cultured chick skeletal muscle cells.

We have studied the long-term effects of D600, a blocker of L-type voltage-dependent Ca channels (VDCC), on the development of voltage-dependent Na channels (VDNC) that are sensitive to tetrodotoxin (TTX), by electrophysiological measurements of the maximum rate of rise of the TTX-sensitive Na spike in cultured chick skeletal muscle cells. Chronic treatment with D600 (2-20 microM) caused a dose-dependent increase in the density of VDNC. The density of VDNC was increased by 150-250% when D600 was added to the cultures at 20 microM from the second day of culture onward. Co-treatment with an inhibitor of the transcription of RNA from DNA, alpha-amanitin, or with cycloheximide, an inhibitor of protein synthesis, prevented the up-regulation by D600. Nifedipine, a different type of blocker of L-type VDCC, was also effective in increasing the density of VDNC, and BAY K 8644, an agonist of L-type VDCC, had the opposite effect. It is suggested that the effect of D600 was mediated via a mechanism specific for L-type VDCC that involves regulation of cytosolic levels of Ca2+ and protein synthesis de novo.

Lower eyelid reconstruction with a conchal cartilage graft.

A conchal cartilage graft without skin or mucosa was used as a posterior lining for lower eyelid reconstruction in seven patients. The raw surface of the graft was smooth enough to cause no corneal irritation and was epithelialized from the surrounding mucosa about 3 to 4 weeks after the operation. The lower eyelid reconstructed in this method produced a stable lid margin.

Symmetrical lipomatosis of the tongue presenting as macroglossia. Report of two cases.

Symmetrical lipomatosis in the oral cavity is extremely rare. Two cases of symmetrical lipomatosis presenting as macroglossia are presented. Glossectomy was performed in order to reduce the size of the tongue and for diagnosis. Because of their multiplicity, non-encapsulation and invasiveness, the lesions were diagnosed histopathologically as symmetrical lipomatosis.

[Acute toxicity study of cefpirome sulfate in mice and rats].

Acute toxicity of cefpirome sulfate (CPR) was examined in 6-week-old mice and rats and immature (5-day-old) rats. The LD50 values of CPR (mg/kg) were as follows: (1) mice: intravenous, 2420 (95% confidence limits, 2122-2758) for males and 2400 (2181-2640) for females; intraperitoneal, 3850 (3407-4351) for males and 4200 (3889-4536) for females; and oral, 16200 (14781-17755) for males and 18500 (17290-19795) for females. (2) 6-week-old rats: intravenous, 1900 (1784-2023) for males and 2080 (1953-2215) for females; intraperitoneal, 6550 (6179-6943) for males and 5800 (5311-6334) for females; subcutaneous, more than 10000 for both sexes; and oral, more than 8000 for both sexes. (3) 5-day-old rats: subcutaneous, between 1750 and 2500 for males and 2080 (1651-2621) for females. Major changes in general health conditions observed in 6-week-old mice and rats were decreased spontaneous activity, lying prone, tremor, respiratory changes (slow or deep respiration, gasping), clonic or clonic-tonic convulsions. In the 6-week-old rats dosed subcutaneously, vocalization, writhing and cutaneous changes at the injection site (dark reddening or blackening, swelling, exfoliation, depilation, induration) were also observed. In the 5-day-old rats dosed subcutaneously, the changes noted were slow respiration, writhing, cyanosis, and dark reddening and swelling of the skin at the injection site. After administration, transient depression of body weight gain or loss of body weight was observed in the mice and rats except the rats dosed orally. These changes disappeared at 7 days after administration at latest, and all surviving animals showed favorable body weight gain thereafter. Necropsies revealed hemorrhage under meninges in the brain in many of the mice and rats which died. Other findings included subcutaneous changes at the injection site in the 6-week-old and 5-day-old rats dosed subcutaneously (dark reddening, retention of dark red fluid, retention of red, white or dark red gelatinous material) and changes in the peritoneal cavity in the 6-week-old rats dosed intraperitoneally (red or dark red spots on the serous membrane, reddening of adipose tissues).

[Antigenicity study of cefpirome sulfate].

Immunological properties of cefpirome sulfate (CPR) were examined. The immunogenicity and challenging ability of CPR were examined in guinea pigs by active systemic anaphylaxis (ASA) and homologous 4-hr passive cutaneous anaphylaxis (PCA) tests. The animals given CPR alone intraperitoneally for sensitization and their sera were negative for ASA or PCA reactions, like the results with reference substances, ceftazidime (CAZ) and cephalothin sodium (CET). When each antibiotic plus Freund's complete adjuvant (FCA) was used for sensitization, ASA reactions were observed with CPR, cephaloridine (CER), CET, and cefazolin sodium (CEZ), and PCA reactions, with CPR and CET. CPR had the ability to challenge the ASA and PCA reactions. CER and CET also showed the ability to challenge ASA or PCA reactions, though at low incidences. The cross-reactivity of CPR with commercially available antibiotics was examined by heterologous PCA test and by passive hemagglutination test and its inhibition test. The antiserum used was from rabbits immunized with each antibiotic-ovalbumin conjugate plus FCA, and the antigen was each antibiotic-bovine serum albumin conjugate. CPR cross-reacted markedly with cefotaxime sodium (CTX) having the same side chain at position 7 and showed weak, unidirectional reactions with CAZ and CET. In the in vitro direct Coombs test, the positive reactions noted with CPR were stronger than those with latamoxef sodium, equal to those with CEZ and slighter than those with CTX, CET and benzylpenicillin potassium. In conclusion, in the safety evaluation of CPR, its antigenic potential may not be a problem, like the cases of other antibiotics.

[Local irritancy study of cefpirome sulfate].

As a series of safety studies of cefpirome sulfate (CPR), its local irritancy was examined in rabbits after the following treatments: intracutaneous injection (single), application into the conjunctival sac of the eye (single), intramuscular injection (single, 7-day repeated), and intravenous injection (8-day repeated). In addition, the hemolysis test was carried out with human blood. When CPR was injected intracutaneously at a high concentration of 20%, its irritating effects were only equal to or slightly stronger than those of distilled water for injection and Na2SO4 solution. The same concentration of the compound applied into the conjunctival sac had little irritancy to the eye. In the single intramuscular administration experiment, muscular changes caused by 10% CPR were comparable to those by 0.75% acetic acid, slightly severer than those by physiological saline, and slighter than those by 6% acetic acid at 2 days after administration, but at 7 days, the changes were apparently slighter than those by 0.75% acetic acid. CPR is classified under Grade 3 according to the draft guidelines for local tolerability studies issued by the Ministry of Health and Welfare of Japan. The repeated intramuscular administration experiment showed the following results. Muscular changes caused by 10% CPR were comparable to those by physiological saline and slighter than those by 0.75% acetic acid, 5% cefotetan (CTT) and 20% cephalothin sodium (CET) at 2 days after the last administration. At 7 days, the changes were slightly severer than those by physiological saline, but slighter than those by the other control solutions. Microscopically, a tendency toward recovery was marked. In the vascular irritancy experiment, the 10% CPR group showed thrombus macroscopically on and after day 5 of dosing. Microscopy at the end of the 8-day administration period revealed thrombus and organized thrombus. In the 20% CET group, thrombogenesis was slighter than that in the 10% CPR group, while perivascular changes were severer. The changes caused by 5% CTT (thrombogenesis, perivascular changes) were somewhat severer than those by 10% CPR. In the hemolysis test, 10% CPR solution applied to fresh blood from adult men caused to hemolysis when determined by the macroscopic or spectrophotometric method.

[Combined valve and coronary artery bypass surgery].

Between January 1997 and November 1999, 31 patients underwent combined valve and coronary artery bypass grafting (CABG) surgery in our institute. There was no hospital death and clinical symptom improved in all patients after surgery. Mean follow-up was 16.3 months. The actuarial survival rate was 96.8% at 1 year. There were 4 late deaths (CHF in 3, arrhythmia in 1). Tepid blood cardioplegia was effective for those operations. Operation of CABG combined with valve surgery or LV surgery could be safely performed. We therefore considered that complete correction of the lesion with not only CABG but also combined valve surgery or LV surgery was the procedures of choice in patients with complicated lesions.

[Difference between horizontal enlargement and vertical enlargement of the internal auditory meatus in acoustic neuroma].

Twenty-one patients with acoustic neuroma were investigated. The vertical diameter of the internal auditory meatus was measured by polytomography, the horizontal by target imaging CT. The ratio of the abnormal side to the normal side was calculated in each dimension. Two dimensional ratios were compared with each other in order to investigate the difference between horizontal and vertical enlargement of the internal auditory meatus. The results were as follows: 1) Horizontal enlargement of the internal auditory meatus was greater than that of the vertical one in 15 cases (75%). Vertical enlargement was greater in 6 cases but the difference between the two dimensions was not significant. 2) With horizontal enlargement, the posterior wall of the internal auditory meatus was more extended or destroyed than that of anterior wall. 3) With enlargement of the posterior wall, the bony eminence forming the inner side of the porus was predominantly extended or destroyed in every case.

[Bone mineral density of the internal auditory meatus by quantitative computed tomography].

The bone mineral density of the internal auditory meatus was investigated by means of quantitative computed tomography in 20 normal subjects (40 ears). Investigated portions of the internal auditory meatus were the porus anterior and posterior and the fundus anterior and posterior. Two other portions of the ear, the bony vestibule and lateral wall of the mastoid, were also investigated. The bone density values (calcium carbonate equivalent value) for each portion were analyzed statistically. The following results were obtained: 1) There was no significant difference between the right and left values in any portion. 2) The highest mean value was found in the fundus posterior, the lowest in the porus anterior. There was a significant difference between the values of the fundus and porus. Bone hardness generally correlates with bone density. Thus, the bone hardness of the porus of the internal auditory meatus was appraised to be lower than that of the fundus. These results suggest that this is one of the factors promoting enlargement of the internal auditory meatus in acoustic neuroma.

[Selection of antibiotics and planning of eradication for H. pylori infection].

There is general agreement that H. pylori should be eradicated in patients with peptic ulcers. But the optimal therapeutical regimen to be used still remains a matter for many investigations. An increase in the prevalence of antibiotic-resistant H. pylori strains has been reported recently. The recommended drugs for the eradication in Japan are clarithromycin (CAM) and amoxicillin (AMPC) because metronidazole (MNZ) is anti-parasites drug in Japan. A total of 392 H. pylori strains in the last twelve years were tested for sensitivity to CAM, MNZ, and AMPC. The Primary resistance of H. pylori to CAM, MNZ, and AMPC were found in 10.2%, 26.5%, and 0.3% strains, respectively. The resistant strains to CAM were gradually increasing in the last few years. The eradication therapies which do not increase antibiotics resistant strains after eradication failure were reported. The recommendation for eradication in patients with peptic ulcer disease includes those with bleeding ulcers. The pretreatment with proton pump inhibitors (PPI) does not influence the success of PPI-based triple therapy in eradicating H. pylori.

[A case of hypertrophic cardiomyopathy with right ventricular outflow obstruction manifested during three-year follow-up study].

A case is reported of a 55-year-old female with idiopathic hypertrophic cardiomyopathy, which was accompanied with outflow obstruction in the right ventricle developed during the previous 3 years without lesion of the left ventricle. In 1984, she was admitted to our hospital to be examined for cardiac murmurs and abnormal electrocardiogram including ST depression and inverted T. The findings of echocardiography and cardiac catheterization revealed non-obstructive hypertrophic cardiomyopathy. She had been treated with sympathetic beta-blockade and calcium antagonist for 3 years until she complained of dyspnea on exertion, and she was readmitted to our hospital in 1987. Echocardiographic findings showed protrusion of the ventricular septum toward the right ventricle and systolic turbulent flow along the right ventricular outflow tract (by pulsed Doppler technique). A pressure gradient of 20 mmHg across the protrusion was detected by the examination of the cardiac catheter. However, neither protrusion nor pressure gradient was observed in the left ventricular outflow tract as well as that in 1984. Idiopathic hypertrophic cardiomyopathy has been described as involving both ventricles, and outflow obstruction is the usual hemodynamic finding in the left ventricle. However, right ventricular outflow obstruction is the usual hemodynamic finding in the left ventricle. However, right ventricular outflow obstruction with the left ventricular outflow tract intact has been very rare. In addition, in this case, the change of hemodynamic characteristics from non-obstructive to obstructive hypertrophic cardiomyopathy, and the development of these changes only in the right ventricle were observed during the last 3 years.

[A case of beriberi heart--with special reference to the rapid effect of fursultiamine on hemodynamics].

A 33-year-old man was admitted to Kushiro City General Hospital on February 27, 1989, because of palpitation, shortness of breath and anasarca. Eight months previously he had noted the onset of pretibial edema, which had progressed to anasarca. He had had a meal only once a day for nine months. Physical examination revealed a blood pressure of 114/46 mmHg and pulse rate of 80/min. The 3rd sound was audible. No rales in the chest and no hepatosplenomegaly were noted. Ascites, pretibial edema and anasarca were present. Vibration sensation was diminished, and the deep tendon reflexes were absent in the legs. The blood thiamine level on the 4th day of hospitalization decreased to 2.9 micrograms/dl. The red cell transketolase activity and TPP effect on the 10th hospital day were 0.76 IU/gHb and 11%, respectively. A chest roentogenogram showed pulmonary congestion and cardiomegaly (CTR 61.3%). The electrocardiogram showed non-specific T wave changes. On the echocardiogram, remarkable pericardial effusion and diffuse hypertrophy of the left ventricular wall were observed. In addition, the left ventricular wall motion showed a hyperkinetic state. On the basis of these findings, the diagnosis of beriberi heart was made. The hemodynamic study performed on the 10th hospital day showed a remarkable high cardiac output (CO) of 10.7 l/min and an extremely reduced total peripheral resistance (TPR) of 352 dynes.sec.cm-5. 15 min after intravenous administration of Fursultiamine 100 mg, CO decreased to 7.24 l/min and TPR increased to 848 dynes.sec.cm-5. Following the administration of Fursultiamine 75 mg, po/day, his symptoms and abnormal findings of clinical examination data rapidly improved.(ABSTRACT TRUNCATED AT 250 WORDS)