Young C3H mice infected with Toxoplasma gondii are a novel experimental model of communicating hydrocephalus.

The effects of leptomeningeal inflammation on the development of hydrocephalus are less understood than those of obstructing the flow of cerebrospinal fluid (CSF) in animal models. We succeeded in introducing a novel experimental model of hydrocephalus and analysed changes in histopathology and CSF flow in mice infected with an avirulent Fukaya strain of Toxoplasma gondii (T. gondii). Six to 7 week-old male mice were orally inoculated with a brain homogenate containing ten T. gondii cysts. The cerebral ventricles became enlarged in all C3H/HeN and C57BL/6 mice 4 weeks after T. gondii infection, but mildly in BALB/c mice. In addition to the lateral ventricle, the third and fourth ventricles and Sylvian aqueducts were dilated in all mice. Lymphocytes and monocytes infiltrated the subarachnoid space. Indian ink particles required more time to pass from the lateral ventricle to the cervical lymph nodes, although they reached the subarachnoid space. Computed tomography ventriculography demonstrated that the CSF was not obstructed during passage through the ventricular systems, but contrast remained static in the lateral ventricle only in infected mice. These results indicated that the infected mice developed communicating type hydrocephalus without obstructive or mass lesions in the ventricles. The hydrocephalus that arises in mice infected with T. gondii is considered a consequence of leptomeningeal inflammation that blocks CSF circulation at the subarachnoid space, implying that leptomeningeal inflammation is important in other types of hydrocephalus.

Cloning and characterization of a novel gene encoding keratin-like protein from nematode Nippostrongylus brasiliensis.

Nippostrongylus brasiliensis (Nb) is one of the most important parasites in studying Th2 immune response of the host, but little is known about its antigenic structures of the excretory-secretory or structural proteins of the parasite. Here we report cloning and characterization of a novel antigenic gene from cDNA library of Nb adult worm by immunoscreening. The positive clone, KLP-Nb, had an open reading frame of 612 bp that encodes a 203-amino-acid protein and was homologous to 'similar to keratins in a glycine-rich region' of Caenorhabditis elegans. Its expression was confirmed by Northern blotting and IgG enzyme-linked immunosorbent assay. This protein seems to be one of the components of cuticle that covers the nematode body.

Immunologic features of mice with streptozotocin-induced diabetes: depression of their immune responses to sheep red blood cells.

The immune responsiveness in streptozotocin (SZ)-induced diabetic mice was studied using the immune responses to sheep red blood cells (SRBC) as an indicator system. In SZ-diabetic mice, the weights of such lymphoid organs as the thymus and spleen were significantly decreased with time after SZ administration, whereas the weight of liver was markedly increased. In SZ-diabetic mice, the level of delayed type hypersensitivity (DTH) to SRBC was not lower than that in normal controls in most cases, although the level of DTH was significantly depressed, on occasion, in SZ-diabetic mice. In contrast, antibody-forming activity, measured as the number of plaque-forming cells (PFC), was markedly decreased in SZ-diabetic mice. It seems that antibody production is more profoundly depressed than is DTH in SZ-diabetic mice. The transfer of normal thymus and bone marrow cells into SZ-diabetic mice caused only a partial restoration of PFC activity. When normal spleen cells were transferred into diabetic irradiated mice, proliferation of spleen cells and production of splenic PFC was greatly reduced as compared with normal irradiated mice. Treatment with insulin completely reversed such depression in the transfer system. These findings suggest that the chronic insulin-deficient diabetic state caused a depression and delay in the proliferation and differentiation of lymphoid cells.

Synergism of IL-3, IL-5, and GM-CSF on eosinophil differentiation and its application for an assay of murine IL-5 as an eosinophil differentiation factor.

In order to elucidate the mechanisms of eosinophil differentiation, we examined the effects of combinations of interleukin 5 (IL-5) with IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) on in vitro differentiation into eosinophils from bone marrow cells of ICR mice. When the amount of added IL-5 was kept constant, IL-3 exhibited dose-dependent production of eosinophils both in the absence and presence of low doses of GM-CSF. In contrast, IL-5 plus GM-CSF showed highly enhanced production of eosinophils, and eosinophil production maximized at a concentration between 10 and 20 U/ml and decreased at higher concentrations. When IL-3 and GM-CSF were kept constant at concentrations of 20 U/ml and 10 U/ml, respectively, the number of eosinophils increased linearly in IL-5-dependent manner in a range from 0.3 to 30 U/ml. These results suggest that IL-3 and GM-CSF act synergistically with IL-5 in in vitro eosinophil differentiation. In addition, we propose a new method for quantifying eosinophil differentiation activity of IL-5.

Hedgehog signaling enhances core-binding factor a1 and receptor activator of nuclear factor-kappaB ligand (RANKL) gene expression in chondrocytes.

Hedgehog signaling is considered to play a crucial role in chondrogenesis by regulation through a network of cytokine actions, which is not fully understood. We examined the effect of hedgehog signaling on the expression of core-binding factor a1 (Cbfa1), a critical transcription factor for the development of bone and cartilage. Primary chondrocytes prepared from the costal cartilage of newborn mice were treated with N-terminal fragment of recombinant murine sonic hedgehog (rmShh-N). Northern blot analysis indicated that Cbfa1 mRNA expression levels in the chondrocyte cultures were elevated by the treatment with rmShh-N. rmShh-N treatment enhanced 1.8 kb Cbfa1 promoter activity in chondrocytes, suggesting the presence of transcriptional control. As Cbfa1-binding site(s) have been located in the promoter of the receptor activator of nuclear factor-kappaB (RANK) ligand (RANKL) gene, we also examined RANKL expression. rmShh-N treatment upregulated RANKL and RANK mRNA expression levels in chondrocytes. Interestingly, RANKL suppressed the hedgehog enhancement of alkaline phosphatase activity in chondrocytes, suggesting the presence of a link between these signaling molecules. We conclude that hedgehog signaling activates Cbfa1 gene expression through its promoter in chondrocytes, and also activates and interacts with RANKL to maintain cartilage development.

Structures of asparagine-linked oligosaccharides of human placental fibronectin.

The asparagine-linked sugar chains of fibronectin purified from human placenta were quantitatively released as oligosaccharides by hydrazinolysis. After N-acetylation, they were converted to radioactive oligosaccharides by NaB3H4 reduction. The radioactive oligosaccharides were fractionated by their charge on an anion-exchange column chromatography. All of the acidic oligosaccharides could be converted to neutral oligosaccharides by sialidase digestion. These oligosaccharides were then fractionated by serial affinity chromatography using immobilized lectin columns. Study of each oligosaccharide by sequential exoglycosidase digestion and methylation analysis revealed the following information as to the structures of the sugar chains of human placental fibronectin: 1) nine sugar chains are included in one molecule; 2) all sialic acid residues are exclusively linked at the C-3 position of the galactose residues; 3) bi-, tri-, and tetraantennary complex-type oligosaccharides with the Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4 (+/- Fuc alpha 1----6)-GlcNac as their cores were found; 4) the bisecting N-acetylglucosamine residue and the Gal beta 1----4GlcNAc beta 1----repeating groups are included in some of the sugar chains.

Detection of bisected biantennary form in the asparagine-linked oligosaccharides of fibronectin isolated from human term amniotic fluid.

Fibronectin purified from human term amniotic fluid contains 10 asparagine-linked sugar chains in one molecule. The sugar chains were quantitatively liberated as radioactive oligosaccharides from the polypeptide moiety by hydrazinolysis followed by N-acetylation and NaB3H4 reduction and fractionated by anion-exchange column chromatography and serial lectin affinity chromatography. The structures of these sugar chains were determined by sequential exoglycosidase digestion in combination with methylation analysis. The results indicated that they are a mixture of bisected and non-bisected bi- and triantennary complex-type sugar chains with and without a fucose on the proximal N-acetylglucosamine residue and with Gal beta 1----4GlcNAc beta 1----, GlcNAc beta 1----, Neu5Ac alpha 2----3Gal beta 1----4GlcNAc beta 1----, and Neu5Ac alpha 2----6Gal beta 1----4GlcNAc beta 1---- groups in their outer chain moieties.

Diagnosis of Toxoplasma meningoencephalitis in a non-AIDS patient using PCR.

We report herein a rare case of Toxoplasma gondii meningoencephalitis in a non-AIDS patient. Although T. gondii itself was not detected in nucleated cells in peripheral blood and cerebrospinal fluid under the microscope, the polymerase chain reaction method effectively detected the B1 gene of T. gondii in the cells. A serological examination showed increased levels of the IgG but not the IgM antibody to T. gondii, suggesting reactivation of the infection in the brain.

A highly sensitive and selective method for the determination of Leukotriene B4 in human plasma by negative ion chemical ionization/gas chromatography/tandem mass spectrometry.

We have developed a highly sensitive and highly selective method for the determination of Leukotriene B4 (LTB4) in human plasma using negative ion chemical ionization/gas chromatography/tandem mass spectrometry (NICI/GS/MS/MS) analysis. The developed method was summarized as follows. Deuterated LTB4 (d4-LTB4) was added to human plasma samples as an internal standard, and samples were extracted by a Sep-pak C18 column. Extracted LTB4 was derivatized into the pentafluorobenzyl ester of bis-trimethylsilyl ether (PFB-TMS-LTB4) and quantified on the basis of selected reaction monitoring (SRM) at m/z 299 of [M-PFB-2TMSOH]- by NICI/GC/MS/MS analysis, which was the product ion of [M-PFB]-. The detection limit for the quantification of LTB4 in human plasma was 10 pg ml-1, sufficiently sensitive to determine the concentrations of endogenous LTB4 in human plasma. The plasma level of LTB4 measured in healthy male volunteers was 33.85 +/- 33.91 pg ml-1 (mean +/- S.D. in six volunteers). The technique of MS/MS used in this method offers much greater sensitivity and selectivity than single-stage mass spectrometry. The developed method showed good reproducibility with a simple and rapid extraction procedure, and would be useful for examining the relationship between various disease states and the levels of LTB4 in biological fluids.

Simultaneous determination of human neutrophil elastase inhibitor (ONO-5046) and its metabolite in plasma and urine by direct injection column-switching HPLC.

The direct injection method for the simultaneous determination of a human neutrophil elastase inhibitor (ONO-5046) and its metabolite (ONO-EI-601) in plasma and urine has been developed using a column-switching HPLC system. The system was set up with a pre-treatment column, a pre-concentration column and an analytical column which were connected in series via two automatic switching valves. The calibration lines showed a good linearity for concentrations of ONO-5046 and ONO-EI-601 in a range of 156-20000 ng ml-1 in plasma and 1.56-100 micrograms ml-1 in urine, all correlation coefficients being greater than 0.9999. The limit of quantitation of ONO-5046 was 156 ng ml-1 in plasma, that of ONO-EI-601 was 313 ng ml-1 in plasma, and those of ONO-5046 and ONO-EI-601 were 1.56 micrograms ml-1 in urine. The developed method is rapid and sensitive with automated operation, allowing untreated samples to be analysed every 45 min. The application of the present method to the real plasma and urine samples proved to be useful for pharmacokinetic, toxicological and clinical studies.

Identification of human hair stained with oxidation hair dyes by gas chromatographic-mass spectrometric analysis.

This paper describes the gas chromatographic-mass spectrometric (GCMS) analysis of oxidation hair dyes from human hair. Diamines from the dyes were directly extracted from the hair in basic solution and aminophenols were extracted after neutralization. Both extracts were derivatised with trifluoroacetic anhydride and analysed by GCMS. Five components of oxidation hair dyes namely, p-phenylenediamine, toluene-2,5-diamine, o-aminophenol, m-aminophenol and p-aminophenol were clearly identified, whilst no other compounds originating from the hair dyes were detected. The presence and relative amounts of these dye components from hair extracts may assist in the discrimination of human hair especially in cases involving forensic science.

An experimental model of death from anaphylactic shock with compound 48/80 and postmortem changes in levels of histamine in blood.

The present study was made on an experimental animal model of a death from anaphylaxis, in which postmortem changes in levels of histamine and 1-methylhistamine, in whole blood were measured. Instead of the usual immunological method administering compound 48/80, a degranulating agent of mast cell and the effect closely resembling the immuno-reaction, resulted in reliable death in a short time. The animals that died rapidly after the injection of compound 48/80, were found to have large increases in levels of histamine and 1-methylhistamine soon after the administration. These results were similar to the results of injecting histamine exogenously. On the other hand, the animals that died after a longer time showed no increases in levels of those amines within about 24 h, but 24 hours after death histamine levels were only increased tremendously without rise in 1-MHA levels. These phenomena closely resembled those in the control animals that were treated with overdoses of Nembutal.

Demonstration of oxidation dyes on human hair.

This paper describes a method of selected ion monitoring (SIM) analysis which can demonstrate the staining of human hair with oxidation hair dyes. Hair samples were decomposed with NaOH-Na2S2O4 solution by heating (100 degrees C, 30 min) in a stream of nitrogen. Basic and neutral ether extracts from the reaction mixture were trifluoroacetylated with trifluoroacetic anhydride in ethyl acetate and were then analyzed by SIM. The minimum lengths of a single hair for the detection of the 5 components of oxidation hair dyes acting as indicators were 1 mm for toluene-2,5-diamine, 2 mm for p-phenylenediamine, 20 mm for p-aminophenol, 50 mm for m-aminophenol and 100 mm for o-aminophenol. This method was applied to practical cases and the results were good.

Marked eosinophilia in interleukin-5 transgenic mice fails to prevent Trichinella spiralis infection.

In order to study the role of eosinophils in the host defense against Trichinella spiralis infection, worm recovery after infection with T. spiralis was compared between interleukin-5 transgenic (IL-5 Tg) mice with a constant high level of peripheral eosinophils and nontransgenic C3H/HeN mice. No significant difference in the recovery of muscle larvae or adult worms in the small intestine, fecundity of female adult worms, or infectivity of newborn larvae was observed between nonimmunized C3H/HeN and IL-5 Tg mice or C3H/HeN and IL-5 Tg mice immunized with somatic antigen of T. spiralis. However, a significant difference was observed in the fecundity of female adult worms and recovery of muscle larvae between nonimmunized and immunized IL-5 Tg mice or C3H/HeN mice. These results demonstrate that having more eosinophils does not improve immunity against the various aspects of T. spiralis infection.

Experimental model of vertigo induced by detached otoconia.

Isolated posterior semicircular canals (psc) of bull frogs were used for a model of positional vertigo. Induced ampullary nerve action potentials were recorded. When the cupula was removed and the saccular otoconia were dropped onto the cilia, excitatory, and inhibitory potentials were evoked by changing the psc positions into canal-down and canal-up, respectively. When the otoconia were allowed to stay on the cupular surface, canal-down and canal-up positions also evoked excitatory and inhibitory responses, respectively. When the otoconia were inserted in the canal and were allowed to move, changing the psc positions likewise evoked responses. However, when one end of the canal was closed, these responses disappeared, indicating the efficacy of canal plugging.

A study on temperature dependent vestibular potential: effect of long lasting thermal stimulus and aminoglycoside agent.

Effect of temperature changes on the vestibular receptor was studied using isolated posterior semicircular canals (PSC) of bull frogs. Cupula was removed from the crista. PSC was placed in the inner compartment of the double chamber. Warm or cool water was filled in the outer compartment to change the temperature of the inner compartment. Changes of the spontaneous discharge were recorded from the ampullary nerve. When cooled, the discharge temporarily increased, followed by a gradual decrease. When warmed, it temporarily decreased and then increased, forming response curves of a mirror image. After addition of streptomycin, the temperature dependent response disappeared. These results suggest that the semicircular canal receptor is activated by direct temperature effects.

Functional model of benign paroxysmal positional vertigo using an isolated frog semicircular canal.

Bull frogs posterior semicircular canals (psc) wee used to simulate the condition of benign paroxysmal positional vertigo (BPPV). The psc was isolated in frog Ringer's solution, and the saccular otoconia were used as a responsible material to stimulate the cupula. When the otoconia were placed on the cupular surface to mimic the condition of cupulolithiasis, the psc ampullary nerve action potentials instantaneously changed according to the direction of the gravity produced by otoconia. When the otoconia were dropped into the canal to mimic the condition of moving otoconia in the canal, the action potentials changed together with the otoconial flow after a latent period. Both cupulolithiasis and moving otoconia are possibly valid mechanisms of BPPV, since they effectively stimulate the cupula. However, moving otoconia with a latent period would better explain clinical features of BPPV.

Direct influence of temperature on the semicircular canal receptor.

Effect of thermal stimulus on the vestibular receptor was studied using the isolated frog semicircular canal. The posterior (PSC) and lateral semicircular canals (LSC) were placed in the horizontal plane in frog Ringer's solution. The ampullary nerve was sucked into a glass suction electrode to record compound potentials. The steel thermal probe was positioned next to the ampullary surface to give thermal stimuli. When the PSC ampulla was cooled, the spontaneous discharge markedly increased. When the PSC ampulla was warmed, the discharge decreased. When the LSC ampulla was cooled, the discharge increased in the same manner as in the PSC. Also, warming of the LSC decreased the discharge in the same manner as in the PSC. The cupula was removed from the crista in order to eliminate the effect of volume change of the endolymph. The results were comparable to those with intact cupula in both the PSC and LSC. Cooling increased, while warming decreased the discharge. These responses are possibly due to a mechanism other than mechanical volume change, because the PSC and LSC yielded the same type of responses. Direct temperature reaction of the vestibular hair cell was suggested.

[Clinical effects of CS-1170 on respiratory tract infections. II: Mainly on the use of 2 g piggy bag alone (author's transl)].

The following results have been obtained in our trial of CS-1170, in 2 g piggy bag, on the respiratory tract infections: 1) CS-1170 was found to be effective in all the cases treated, including 6 cases in which CS-1170 was used alone. The cases treated with CS-1170 included those cases in which no other antibiotics had been used, as well as 5 cases in which other penicillins and cephalosporins were found to be ineffective or not adequately effective. The results indicate the usefulness of CS-1170 in the treatment of the respiratory tract infections. 2) Slight elevation of GOT and GPT was observed in one case as the side effect of CS-1170. No abnormality was found in other laboratory tests. The incidence of side effects of CS-1170 is believed to be not higher than those of other cephalosporins.

[Clinical trial of netilmicin in infections of respiratory organs and studies on its penetration into pleural fluid II. (author's transl)].

To 10 cases with respiratory infections, 200 mg, twice daily, of netilmicin was administered without other antibiotics and the following results were obtained. 1) Netilmicin was administered to 2 cases of pneumonia and 8 cases of bronchitis for 7 to 30 days, and 4 remarkably effective and 6 effective cases were observed, that is, netilmicin was effective in all cases. 2) Abnormal laboratory test values were found in 2 cases; 1 case showed slightly elevated creatinine value, and 1 case showed slightly increase GTP value, and these values were normalized rapidly without any treatment after discontinuation of netilmicin administration. Netilmicin 100 mg was intramuscularly injected to 15 patients with pleural effusion to see the time-course distribution of the drug to serum and to pleural fluid by determining the concentration of netilmicin. 1) Netilmicin concentrations in serum reached the peak at 30 minutes after the intramuscular injection and it gradually decreased, while in the pleural fluid, it reached the peak at 3 hours after the injection, and the peak value in the pleural fluid in average was 2.63 +/- 1.98 micrograms/ml, and it was still detectable at 24 hours after the injection. 2) The ratio of netilmicin concentrations in the serum and pleural fluid at the peak was 31.7 +/- 23.4%, and distribution of netilmicin into the pleural fluid was considered to be high enough.