Mutants in the ADP-ribosyltransferase cleft of cholera toxin lack diarrheagenicity but retain adjuvanticity.

Cholera toxin (CT), the most commonly used mucosal adjuvant in experimental animals, is unsuitable for humans because of potent diarrhea-inducing properties. We have constructed two CT-A subunit mutants, e.g., serine-->phenylalanine at position 61 (S61F), and glutamic acid-->lysine at 112 (E112K) by site-directed mutagenesis. Neither mutant CT (mCT), in contrast to native CT (nCT), induced adenosine diphosphate-ribosylation, cyclic adenosine monophosphate formation, or fluid accumulation in ligated mouse ileal loops. Both mCTs retained adjuvant properties, since mice given ovalbumin (OVA) subcutaneously with mCTs or nCT, but not OVA alone developed high-titered serum anti-OVA immunoglobulin G (IgG) antibodies (Abs) which were largely of IgG1 and IgG2b subclasses. Although nCT induced brisk IgE Ab responses, both mCTs elicited lower anti-OVA IgE Abs. OVA-specific CD4+ T cells were induced by nCT and by mCTs, and quantitative analysis of secreted cytokines and mRNA revealed a T helper cell 2 (Th2)-type response. These results now show that the toxic properties of CT can be separated from adjuvanticity, and the mCTs induce Ab responses via a Th2 cell pathway.

Examination of quantitative and fractal analysis of sway characteristics of the center of foot pressure movement during a static upright posture. Analysis based on alcohol intake.

AIM: This study aimed to examine the sway characteristics of center of foot pressure (CFP) movement during a static upright posture under the influence of alcohol by using quantitative and fractal analysis. METHODS: Eleven healthy young people participated. They drank in a range of 0.54-1.83 ml/kg of alcohol, standardized by body mass, within 10 min. Blood pressure, heart rate, 2 types of nervous function tests and the CFP movement were measured before and after the alcohol intake. Thirty-six parameters of the CFP movement were used for quantitative analysis. Diffusion coefficient, scaling exponent and critical point coordinates were used in fractal analysis. RESULTS: Quantitative analysis confirmed that the CFP parameters such as distance, velocity and area were significantly changed, although the parameters evaluating cyclical characteristics and center average were not. Fractal analysis confirmed that critical point coordinates existed at time-lag=1.0 before and after the alcohol intake, and the short-term region (ST) and long-term region (LT) could be separated. A diffusion coefficient in the ST was larger than that in the LT, and the scaling exponent was over 0.5 in the ST and under 0.5 in the LT. CONCLUSIONS: Influences of alcohol intake were found in amount of body-sway, but not in the displacement or periodicity of the CFP parameters. Fractal analysis confirmed that CFP movement was dependent on time-series, and its characteristics changed before and after the time-series critical point and was unsteady.

Novel monoclonal antibodies to double stranded DNA that require Ca2+ or Mg2+ for their binding.

Murine monoclonal antibody (MoAb) 2B5 (IgG2aK) was obtained by its binding on a solid phase to double-stranded (ds) DNA from one of the mutant CBA/K1(CBA/K1Jms-1prcg/1prcg) mice which were recently found, in our institute, to develop lymphadenopathy associated with the production of anti-double-stranded (ds) antibodies. MoAb 2B5 was highly specific for dsDNA, as shown by enzyme-linked immunosorbent assay (ELISA). The dsDNA binding of 2B5 was decreased dose-dependently by the chelating agent EDTA, being lost completely with 2.5-5.0 mM EDTA, whereas dsDNA on the solid phase remained intact after incubation with EDTA. Addition of Ca2+ or Mg2+ to antibody in culture supernatant that had lost dsDNA binding activity by dialysis against Ca2+ and Mg(2+)-free buffer restored its binding with dsDNA to the original level, indicating that MoAb 2B5 requires Ca2+ or Mg2+ for its binding with dsDNA. It is unknown whether MoAb 2B5 recognizes new conformational epitopes created in the presence of Ca2+ or Mg2+, but this MoAb should be useful in studies on the modes of interaction of DNA with antibodies and DNA-binding proteins.

A unique monoclonal antibody derived from a lupus-prone mouse with multiple bindings to autoantigens associated with rheumatic disease.

Monoclonal anti-poly(ADP-ribose) (MRP-2) was primarily a product of a hybridoma selected by binding to poly(ADP-ribose) from an autoimmune MRL/Mp-lpr/lpr (MRL/1) mouse. Detailed examination revealed that anti-poly(ADP-ribose) monoclonal IgMK antibody bound not only to left-handed Z-DNA and single-stranded (ss) DNA but also to a conformational epitope formed by histone and double-stranded (ds) DNA. A reconstitution study revealed that association of dsDNA with histone H3 plus H4 was essential for their binding to MRP-2 monoclonal antibody. MRP-2 monoclonal antibody acted as a rheumatoid factor (RF). Since some monoclonal or polyclonal human serum antibodies of rheumatoid arthritis (RA) or mixed connective tissue disease (MCTD) have been reported to recognize shared epitopes of denatured IgG and DNA-histone (nucleosomes), this MRP-2 monoclonal antibody with the similar activity derived from a lupus-prone mouse will be useful for the studies on the etiology of autoantibodies associated with RA, MCTD and systemic lupus erythematosus (SLE).

Dorsal skin responses to subchronic ultraviolet B (UVB)-irradiation in Wistar-derived hypotrichotic WBN/ILA-Ht rats.

Dorsal skin responses to a subchronic UVB-irradiation (10kJ/m2/rat /day), were examined in Wistar-derived hypotrichotic WBN/ILA-Ht rats for up to 3 months. Hyperplasia of epidermal cells and hair follicle epithelial cells as well as parakeratosis developed at 1 month and progressed thereafter, resulting in a prominent epidermis thickening and formation of epidermal ingrowths projecting into the dermis. At the same time, the percentage of proliferating cell nuclear antigen (PCNA)-positive epidermal cells significantly increased after I month. In some portions of the hyperplastic epidermis, especially of the epidermal ingrowths, keratinocytes were somewhat pleomorphic and migrated into the dermis. In the upper dermis, edema with capillary congestion, mast cell infiltration and fibroblast proliferation developed at I month, and the intensity of edema and the number of dermal mast cells was most prominent at 3 months. Edema spread to the epidermis, resulting in intercellular edema and subsequent dissociation of epidermal cells. Degeneration of collagen fibers was also detected in the upper dermis, especially beneath the epidermis. In addition, although not significant because of a large individual difference, the serum IgE concentration, showed a tendency to increase after 2 months. The present study clarified the characteristics of the dorsal skin responses to a subchronic UVB-irradiation in rats.

Expression in baculovirus vector system of the nucleocapsid protein gene of rinderpest virus.

The rinderpest (RV) nucleocapsid (NP) gene segment was inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) adjacent to the polyhedrin promoter. The expression of NP protein in Sf9 cells was confirmed by indirect immunofluorescence and by Western blotting analysis with monoclonal antibodies. Recombinant RV-NP protein was purified by ultracentrifugation on a sucrose density gradient, and used as an antigen for an enzyme linked immunosorbent assay to detect anti RV-NP antibody. Both IgM and IgG antibodies against RV-NP were detected in the sera of rabbits infected with the L strain of RV. The pattern of development of IgG anti RV-NP antibody closely correlated with that of virus neutralizing antibody. In rabbits inoculated with recombinant vaccinia virus expressing RV-H gene (RRV-H), anti RV-NP was not detected. The results indicated that the baculovirus vector system can be used for the preparation of the diagnostic antigen of rinderpest as well as to distinguish between natural infection and vaccination with RRV-H.

Leukoencephalopathy with cerebral amyloid angiopathy: a semiquantitative and morphometric study.

To investigate changes in caliber of vessels in leukoencephalopathy with cerebral amyloid angiopathy (CAA) we performed a histological and morphometric study of cerebral arteries in this disease. We histologically examined changes in cortico-leptomeningeal arteries in five cases of leukoencephalopathy with CAA and compared their morphometrically determined wall-to-lumen ratio [(external diameter-internal diameter) x 0.5/internal diameter] with those of amyloid-negative arteries to estimate stenotic changes. Additionally, we compared wall-to-lumen ratios of medullary arteries in brains with CAA and white matter lesions (WML) (CAA(+)/WML(+), n = 5), subcortical arteriosclerotic encephalopathy without CAA (CAA(-)/WML(+), n = 7), and neither CAA nor white matter lesions (CAA(-)/WML(-), n = 5). Amyloid-positive arteries had thinned walls and dilated lumens. The external diameter and the wall-to-lumen ratio for amyloid-positive arteries was smaller than for amyloid-negative arteries in CAA(+)/WML(+) brains. There was no significant difference in the external diameters among the three groups. The wall-to-lumen ratio for medullary arteries in CAA(-)/WML(+) brains was significantly greater than for CAA(+)/WML(+) and CAA(-)/WML(-), but there was no significant difference between CAA(+)/WML(+) and CAA(-)/WML(-). Amyloid deposition causes degeneration of the tunica media, resulting in thinning of the wall and dilation of the lumen. The tunica media of small arteries is important in regulation of cerebral blood flow with degeneration causing impairment of cerebrovascular autoregulation in response to blood pressure. This impairment may lead to white matter lesions.

Presence of anti-ovalbumin IgE antibody in the sera of laboratory-reared squirrel monkeys (Saimiri sciureus) fed quail eggs.

In this study, we examined serum anti-ovalbumin (OVA) IgE and IgG antibodies in laboratory-reared squirrel monkeys (Saimiri sciureus), that were fed a boiled quail egg everyday. We found that 36 of 95 monkeys (38%) possessed specific IgE and 44% (42/95) had specific IgG against OVA. These antibody titers seemed to increase with age. There was, however, no apparent correlation between the anti-OVA IgE and IgG antibody titers.

Development of spontaneous tongue calcification and polypoid lesions in DBA/2NCrj mice.

Calcified tongue lesions were observed in 4- to 8-week-old DBA/2NCrj mice, the highest frequency being reached at 6 weeks of age. Calcification was initially observed in the superficial longitudinal muscles and these lesions were distributed segmentally under the dorsolateral mucous epithelium in the middle portion of the lingual body. After calcification, marked inflammation was observed in the same lesion. Finally, the swollen tissue elevated the covering mucous epithelium and produced polypoid lesions.

Mouse strain difference in bile duct lesions induced by swine serum injections.

The mouse strain difference in bile duct lesions was studied on male A/J, BALB/c, C57BL/6, C3H/He, DBA/2 and DDY mice 4 weeks old given intraperitoneal injections of swine serum (0.05 or 0.2 ml per mouse) twice a week for 4 weeks. The hepatic lesions were restricted to the portal tract. Biliary epithelial cells showed hypertrophy and hyperplasia, and eosinophilic and homogeneous or needle-shaped material appeared in the cytoplasm of such hypertrophied epithelial cells and in the ductular lumen. Around these damaged biliary epithelia, eosinophil leukocyte and plasma cell infiltration with proliferation of collagen fibres was commonly detected. These changes became more apparent with increasing size of bile duct. Such histopathological characteristics of hepatic lesions were essentially the same in all strains, but the severity showed a clear strain difference: the lesion was marked in the DDY, A/J and BALB/c strains, moderate in C3H/He and slight in C57BL/6 and DBA/2. A high production of anti-swine-serum antibodies associated with a marked increase in the number of mouse IgG-producing lymphocytes in the spleen was detected in the strains showing the marked hepatic lesions.

[A case of hereditary motor and sensory neuropathy with vocal cords palsy and diaphragmatic weakness].

A case of hereditary motor and sensory neuropathy (HMSN) type 1 (Charcot-Marie-Tooth disease (CMT)) is reported with vocal cords palsy, deafness, diaphragmatic weakness, and cerebellopontine atrophy. A 42-year-old man was admitted to our hospital in April, 1991 with marked respiratory distress. He had been diagnosed as having CMT 14 years previously. On admission to our hospital, he revealed dyspnea with marked stridor during inspiration. Physical examination showed marked use of respiratory accessory muscles with thoracoabdominal paradox in the supine position. Neurologic examination revealed tonic pupils, mild bilateral weakness of facial muscles, deafness, mild bulbar palsy, severe wasting and weakness in both proximal and distal muscles of the arms and legs, areflexia, distal loss of all sensory modalities. Pes cavus and hammer toe were present. Movement of upper extremities was ataxic. No hypertrophic changes were noted in his peripheral nerves. Peripheral nerve conduction study showed undetectable both sensory and motor action potentials. Electromyography showed evidence of denervation, more marked in distal muscles. Auditory brain stem response was undetectable. Chest radiographic film showed a normal-sized heart with marked elevation of both hemidiaphragm. Laryngofiberscopy confirmed the presence of bilateral vocal cord paralysis without tumor formation, inflammation or anomaly. The vocal cords lay near the midline and did not show any movement during respiration. Moderate cerebellopontine atrophy was confirmed on MRI scan. A sural nerve section showed severe decrease of myelinated fibers, and onion bulbs. Diagnosis of HMSN type 1 was made by clinical, electrodiagnostic, and sural nerve sections study.(ABSTRACT TRUNCATED AT 250 WORDS)

[A case of hereditary motor and sensory neuropathy with pyramidal tract sign, optic nerve atrophy and mental retardation].

The patient was a 61-year-old man who suffered from gait disturbance since childhood. He also had mental retardation. Gait disturbance was slowly progressive. His mother, sister, brother and son of his sister suffered from gait disturbance. On neurological examination, he showed mental retardation, optic nerve atrophy and neural deafness. He also showed severe muscle atrophy and weakness of bilateral lower limbs associated with pes cavus. Muscle tonus of lower limbs and patellar tendon reflex were increased bilaterally. Achilles tendon reflex was absent. Babinski and Chaddock signs were positive. Superficial and deep sensations were almost normal. There were no cerebellar signs. Blood chemistry was normal. On nerve conduction studies, motor nerve conduction velocity of the upper limbs was normal and that of the posterior tibial nerve was decreased; right 36.0m/sec, left 29.7m/sec. Sensory nerve conduction velocity of the median nerve was slightly decreased; right 36.5m/sec, left 45.2m/sec and sural nerve did not respond to electric stimuli. On sural nerve biopsy, the density of myelinated fibers was severely decreased. Onion bulb formation was not observed. We classified this case as hereditary motor and sensory neuropathy (HMSN) type II based on nerve conduction studies and findings from sural nerve biopsy. HMSN with pyramidal tract sign has been classified as type V and HMSN with optic nerve atrophy as type VI. This case had characteristic symptoms as type V and VI. Histopathological findings of HMSN type V and VI have not been established yet. This case might provide an important clue for classification of HMSN.

[An association of Shy-Drager syndrome with moyamoya disease--a case report].

A 58-year-old female was admitted to our hospital because of orthostatic syncope, and difficulty in urination and walking for 2 years. At age 35, she suffered from cerebral hemorrhage and was diagnosed as Moyamoya disease (MD). Neurological examination on admission revealed severe orthostatic syncope so that she could not stand. At that time, her blood pressure and heart rate were 104/74 mmHg and 78/min in supine position and 52/48 mmHg and 52/min in 60 degrees head-up position. She also had severe pollakisuria, cerebellar ataxia and dysarthria. Cranial nerves, motor strength and deep reflexes were normal. ECG, chest X-ray, and EEG were normal. Aschner, Czermak, and cold pressor tests revealed no response. Urodynamic study revealed autonomic bladder. MRI showed only enlarged fourth ventricle because of atrophy of the pons. Bilateral CAG revealed "Moyamoya" vessels in the cerebral basal regions. Cerebral blood flow (CBF) was measured by 133Xe inhalation method. Mean arterial blood pressure changed from 134 mmHg to 126 mmHg in 45 degrees head-up tilting and CBF decreased from 47.5 mg/100 g/min to 37 ml/100 g/min position. Though there was no relationship between SDS and MD, each one shows dysautoregulation of CBF, it is supposed that a severe orthostatic syncope attack was resulted from synergism of both effects.

Simultaneous measurements of shifts of the center of gravity caused by ventilation and cardiac motions towards three body axes.

A method is described for the simultaneous measurements of three dimensional shifts of the gravitational center of the body caused by ventilatory and cardiac motions. The measuring system consists fundamentally of three load cells which support an aluminum die-casting platform. Measurement of the shift towards the X axis (front-to-back) was achieved by double integration of the outputs of load cells in order to convert acceleration into a shift of gravity. The records of a volunteer subjects measured with ECG and ventilatory flow showed two cardinal oscillations; one coinciding with the heat rate and the other with the ventilatory frequency. This became clear when recorded through either a low-pass or high-pass filter. Animal who breathed a SF6-O2 mixture into one side of the lungs or those with partial occlusion of a bronchus showed distinct changes in the pattern of the Y axis (right-to-left). These preliminary experiments convinced us that the system can serve as a non-invasive monitoring system of cardiopulmonary functions.

[Effects of Perilla frutescens extract on anti-DNP IgE antibody production in mice].

Perilla frutescens is a Chinese herbal medicine. In this study, we prepared an extract of Perilla frutescens (PFE) and examined its effects on anti-DNP antibody responses in mice. The mice were immunized with DNP-ovalbumin in Alum adjuvant. To examine the effects of PFE on primary antibody responses, PFE was intraperitoneally injected the day before primary immunization. Anti-DNP IgE antibody production was found to be markedly suppressed by PFE injection. Then, we examined the effects on secondary antibody responses. PFE was injected only the day before secondary immunization. Anti-DNP IgE production was markedly suppressed, but IgG response was not so affected. These results suggest that the immunosuppressive effects of PFE are preferentially on IgE production and that PFE may be useful for the suppression of IgE antibody in certain allergic disorders.

[Effects of Celosia argentea and Cucurbita moschata extracts on anti-DNP IgE antibody production in mice].

We have already reported that the Perilla frutescens extract (PFE) suppressed anti-DNA IgE antibody production in mice. In this study, we prepared extracts of Celosia argentea L. (CAE) and Cucurbita moschata Duch (CME), which are Chinese herbal medicines like Perilla frutescens, and examined the effects on anti-DNP antibody responses in mice. To examine the effects of CAE & CME on primary antibody responses, CAE & CME were intraperitoneally injected the day before primary immunization of DNP-ovalbumin. Anti-DNP antibody production was markedly suppressed. Then, we examined the effects on secondary antibody responses. CEA & CME were injected only the day before secondary immunization. Anti-DNP IgE production was markedly suppressed, but IgG responses were not affected. It was also found that mitogenic activity occurred in CAE & CME dose dependently in vitro. These effects of CAE & CME were superior to that of PFE. These results suggest that CAE & CME may be more useful than PFE for the suppression of IgE antibody in certain allergic disorders.

[Feelings of well-being and depression in relation to social activity in normal elderly people].

In order to clarify the influence of social activities on the feeling of well-being or depression in the elderly, we studied these relationships in 2 groups with different activities. Group I consisted of 26 subjects (mean age, 77.2 years) living in a retirement house and exposed to relatively few social stimuli. Group II consisted of 47 subjects (mean age, 75.6 years) who were living in their own homes and were confirmed to be socially active. The Morale Scale and the Zung Self-Rating Depression Scale (SDS) were used to evaluate feelings of well-being or depression. The morale scale in group I was significantly lower than in group II. In particular, there was a significant difference in the factor related to aging. The SDS score of group I was significantly higher than that of group II. The depressive state incidence was significantly higher in group I than in group II in all subjects. There was a highly significant correlation between the morale scale score and the SDS score. These results indicate that group I is less satisfied and more depressed than group II. We conclude that social environmental factors are extremely important for the quality of life of elderly people.

[Influence of social environments on brain aging].

To investigate influence of social activity on normal brain aging, we studied the social activity score, cognitive functions, self-rating depression scale, cerebral blood flow (CBF), MRI and motor function in the normal elderly people living in different social environments. There was no difference in risk factors for stroke, MRI findings and CBF between the two groups. However, the subjects living in a home for elderly showed significantly lower social activities than those living with families. Cognitive functions and motor function were lower, and SDS was higher in subjects living in retirement house than those living with families. The social environment including social activities closely related to life style may significantly influence brain aging with regard to silent brain infarctions or risk factors for stroke.