In vitro formation of corticospinal synapses in an organotypic slice co-culture.

In order to study biological properties of the corticospinal tract, we have reconstructed this system in an in vitro slice culture preparation. Motor cortex and spinal cord slices, prepared from newborn rats, were co-cultured on pored membranes for 16-24 days. Anterograde labeling with biocytin showed that substantial neural connections had formed between the cortex and spinal cord slices. Retrograde labeling with horseradish peroxidase or 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate demonstrated that the parent cells were located primarily in the deeper layer of the cortex, as is found in vivo. Stimulation of the deep layer of the cortex elicited extracellular postsynaptic responses and intracellular excitatory postsynaptic potentials (EPSPs) in the co-cultured spinal cord that were mediated by the 1-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/ kainate-type glutamate receptor. The intracellular injection of biocytin after EPSPs were recorded showed that one-third of these cells were large stellate cells, which are thought to be motoneurons, while a large portion of the remaining labeled cells were bipolar cells of smaller sizes. Using this reconstructed in vitro preparation, we recorded field EPSPs (fEPSPs) along a 100-microm-interval lattice in the spinal gray matter, which allowed the quantitative evaluation of synapse formation. The fEPSP amplitudes were more than two-fold larger when the forelimb cortex was co-cultured with cervical cord rather than lumbar cord. However, hindlimb cortex did not show this preference. The fEPSP amplitudes were more than twice as large when the dorsal side of the spinal cord was adjacent to the cortex than the ventral side. In summary, we have reconstructed the corticospinal projection and synapses in vitro using cortical and spinal explants. This system allows for an efficient quantitative evaluation of synapse formation and for studies of postsynaptic cells. Our results suggest that synapse formation shows preferences along and perpendicular to the neuraxis of the spinal cord.

Hypertrophic pachymeningitis with anti-neutrophil cytoplasmic antibody (p-ANCA), and diabetes insipidus.

We treated a patient with idiopathic cranial hypertrophic pachymeningitis and elevated serum titer of perinuclear anti-neutrophil cytoplasmic antibody (p-ANCA) reactive against myeloperoxidase. This 67-year-old man showed multiple cranial nerve-palsies, central diabetes insipidus (DI), and an intrasellar mass. DI and intrasellar mass had been present for 3 years, and DI had been well controlled by intranasal desmopressin. His nerve-palsies were most likely caused by thickened dura matter detected by the brain MRI. Granuloma may develop in the sella, and MRI findings in our patient are compatible to it. Corticosteroid and oral cyclophosphamide therapy improved his neurological symptoms and serum p-ANCA level with showing good correlation. DI improved temporally for 2 months. Few other cases of hypertrophic pachymeningitis with elevated p-ANCA have been reported, however the etiology is unknown. As p-ANCA antibodies have been detected in many of vasculitides, microvasculitis may be involved in some cases of idiopathic hypertrophic pachymeningitis.

A severe case of subacute sarcoid myositis.

A 46-year-old woman presented progressive proximal weakness and dysphagia. Her serum creatine kinase and myoglobin levels were markedly elevated. Chest X-rays revealed bilateral swelling of the hilar lymph nodes. Needle electromyography demonstrated active denervation and early recruitment. MRI of her skeletal muscle showed focal high intensities on T1-weighted images that were associated with diffusely increased signal intensities on T2-weighted images. Muscle biopsy revealed infiltration of inflammatory cells associated with non-caseating granulomas, and there was widespread segmental fiber necrosis, where necrotic fibers appeared regardless of these granulomas. Immunohistochemical analysis of the surface markers of the infiltrating cells showed CD68- and CD4-positive cells infiltrating into the central area of the granuloma, while CD8-positive cells infiltrating into the endomysium and the periphery of the granulomas. The characteristic histology of the granuloma confirmed the diagnosis of sarcoidosis. The diffuse muscle pathology was consistent with the patient's severe clinical course.

Reduction of GluR2 RNA editing, a molecular change that increases calcium influx through AMPA receptors, selective in the spinal ventral gray of patients with amyotrophic lateral sclerosis.

Enhancement of calcium influx through the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptor is a plausible mechanism underlying selective neuronal death in amyotrophic lateral sclerosis (ALS). The calcium conductance of the AMPA receptor is regulated by the GluR2 subunit that is edited at the glutamine/arginine residue site in the subunit assembly. We investigated the molecular changes of GluR2 mRNA in the spinal cord of ALS cases, those of cases with other neurological diseases, and those of normal cases using reverse transcription-polymerase chain reaction combined with restriction enzyme cleavage. We found that the editing efficiency was significantly lower only in the ventral gray of ALS cases (virtually 0% in 2 cases) than in any spinal region of the disease controls and normal controls. In addition, expression of GluR2 mRNA is lower in the ventral gray of the ALS cases and disease controls than in that of the normal controls. The above molecular changes of GluR2 mRNA in the ventral gray of ALS cases may enhance calcium influx through AMPA receptors, thereby promoting neuronal vulnerability. The decrement of GluR2 mRNA editing efficiency is unique to the ventral gray of ALS cases and may be closely linked to the etiology of ALS.

Generation of 16-fs, 10-TW pulses at a 10-Hz repetition rate with efficient Ti:sapphire amplifiers.

We describe a two-stage Ti:sapphire amplifier laser system that is capable of producing 16-fs pulses of 10-TW peak power at a 10-Hz repetition rate. Thin solid etalons were used to control gain narrowing and gain saturation during amplification. A cylindrical mirror expander was used to permit compensation of the dispersion of the system. An efficiency greater than 90% of the theoretical maximum for conversion of 532-nm pump light to 790-nm radiation is demonstrated.

100-TW sub-20-fs Ti:sapphire laser system operating at a 10-Hz repetition rate.

We developed a compact three-stage Ti:sapphire amplifier laser system that produced peak power in excess of 100 TW for a pulse duration of less than 19 fs and an average power of 19 W at a 10-Hz repetition rate. A final 40-mm-diameter Ti:sapphire amplifier is pumped by a Nd:YAG master-oscillator-power-amplifier system that produces ~7-J output of 532-nm radiation. The spatial beam quality is approximately 2 times diffraction limited for the full amplified compressed output pulse. With f/3 optics, this system should therefore be capable of producing a focused intensity of ~3x10(20) W/cm(2) .

Direct Bonding of Ti:sapphire laser Crystals.

Direct bonding without the use of adhesive materials was demonstrated on a titanium-doped sapphire laser crystal with a bonding surface dimension of 12 mm x 6 mm(2). The bonding surfaces were treated with chemical processes to clean up and to create a hydrophilic (-OH) thin layer for hydrogen bonding. Two different processes of heat treatment performed in succession transformed the hydrogen bonding into direct bonding. The performance of the bonded crystal was tested by laser oscillation with the second harmonics of a Q-switched Nd:YAG laser at a 20-Hz repetition rate. In comparison with a normal laser crystal, there were no differences in output power or spatial profile in an input pumping condition of 30 mJ.

Antigenic differentiation of turkey rhinotracheitis virus strains using monoclonal antibodies and polyclonal antisera.

Monoclonal antibodies (mAbs) were prepared against the 8597/CV94 strain of turkey rhinotracheitis virus (TRTV). These mAbs were used to investigate antigenic relationships among three strains (8597/CV94, 1162/92 and CVL14/1 strain) of TRTV, together with polyclonal chicken and rabbit antisera to 8597/CV94 strain, and guinea pig antisera to each of the three strains. Thirty mAbs to the glycoprotein (G:3 clones), fusion (F1:6 clones), phosphorylated (P:6 clones), nucleocapsid (N:12 clones), and matrix (M:3 clones) proteins of viral antigen were obtained by cell fusion. Among these, two mAbs to F1 protein showed virus neutralizing activity. The results of ELISA test indicated that some mAbs only reacted to the 8597/CV94 strain, some reacted to 8597/CV94 and 1162/92 strains, and others reacted to all three viral strains. In neutralization tests with the three virus strains, polyclonal chicken and rabbit antisera against the 8597/ CV94 strain showed the same antibody titers. Results with four neutralizing mAbs including two previously reported mAbs [Ref. 21] indicated the titers of two mAbs (Pn2-2E and Pn3-2F) to 8597/CV94 were much higher than those to the other two viral strains. No differences were observed in the titers of the other two mAbs (Pn01-8E and Pn06-4D) against any viral strains. In cross-neutralization tests with polyclonal guinea pig antisera, there was some variations among viral strains. This work demonstrated that the Japanese isolate 8597/CV94 of TRTV is somewhat different in antigenicity from two British isolates from chickens and turkeys.

A serological survey of turkey rhinotracheitis virus infection in chickens in Japan.

A total of 4,111 chicken sera collected from 1985 to 1995 at 137 farms in 36 prefectures were subjected to an enzyme-linked immunosorbent assay (ELISA) to test for antibodies against turkey rhinotracheitis (TRT) virus. The antibodies to TRT virus were detected in the chicken sera collected from 1988 to 1995, but the antibodies to TRT virus were undetectable in the sera of chickens collected before 1987. The antibody positive rates each year ranged from 2.2% (1988) to 54.2% (1995). Recently, the TRT virus has spread over the flocks of commercial broiler and layer chicken throughout the country. The serological findings indicated that the TRT virus had invaded the chickens before 1988, and has been widespread thereafter in Japan.

Inhibition of cell fusion by neutralizing monoclonal antibodies to F1 protein of turkey rhinotracheitis virus.

Two monoclonal antibodies (mAbs) with neutralizing activities directed to the fusion (F) 1 glycoprotein of the 8597/CV94 strain of turkey rhinotracheitis (TRT) virus inhibited cell fusion by TRT virus in vitro. In contrast, F1-specific mAbs with no neutralizing activity did not inhibit the cell fusion participating in syncytium formation. These findings indicate that the neutralization epitope is the same as or overlaps the fusion active site.

Turkey rhinotracheitis virus isolated from broiler chicken with swollen head syndrome in Japan.

Turkey rhinotracheitis (TRT) virus was first isolated from a commercial broiler chicken with swollen head syndrome (SHS) in Japan. At the same time, Newcastle disease virus (NDV), infectious bronchitis virus (IBV), avian reovirus (ARV), Escherichia coli (E.coli), Morganella morganii, and Proteus mirabilis were also isolated from the same broiler chicken. The presence of antibodies to TRT virus was confirmed in the sera of 34-day-old chickens of the flock with SHS, however the antibodies to TRT virus were undetectable in the sera of 17-day-old chickens. In this investigation, we confirmed avian pneumovirus infection in chickens in Japan, and the virus and other agents may be considered as a cause of SHS.

A case of primary and generalized allergy to human insulin with no history of any prior insulin exposure.

A generalized hypersensitivity reaction against human insulin was demonstrated in a non-insulin dependent diabetic man treated with only human insulin. The patient had no history of previous insulin exposure or atopy. Because of negative reactions to intracutaneous tests of constituents of the formulation and the presence of insulin-specific IgE antibody, this generalized allergic reaction seems to have been caused by the human insulin itself. Although desensitization was not effective, this allergic reaction was improved both by the treatment with oral antihistamines and desensitization. Cases of generalized and primary allergy against human insulin have been rarely reported making this a very rare case.

Single-shot UV autocorrelator that uses a two-photon-induced photoacoustic signal in water.

A single-shot autocorrelation of an ultrashort UV pulse is measured by detecting a two-photon-induced photoacoustic signal in water. A supersonic wave representing the autocorrelation function is detected by a polyvinylidene fluoride piezoelectric film with a spatial resolution of less than 60 microm. The detection sensitivity is higher than that of a multiphoton-induced fluorescence method by approximately 2 orders of magnitude.