A NEW METHOD FOR VITAL STAINING OF CENTRAL NERVOUS TISSUES WITH NEUTRAL RED

A new vital staining method with neutral red has been established where by cerebral ganglion cells can be stained in vivo .     

B-cell repertoire specific for an unfolded self-determinant of mouse lysozyme escape tolerance and dominantly participate in the autoantibody response

We previously found that autoantibodies against mouse lysozyme (ML) were strongly induced in normal BALB/c mice when immunized with mutant ML that has triple mutations rendering the dominant T-cell epitope of hen egg lysozyme (HEL), HEL 107-116. As T cells specific for HEL 107-116 were primed in these mice, the anti-ML immunoglobulin G (IgG) responses would be the result of collaborations between autoreactive B cells specific for ML and T cells specific for HEL 107-116. Serum IgG responses against ML were dominantly focused on the ML 14-69 region, indicating that B cells responding to the epitope escape tolerance. In the present study, we prepared several monoclonal antibodies (mAbs) specific for ML 14-69 and examined their antigen specificities in detail, to characterize the nature of the remaining B-cell repertoire specific for ML. mAbs specific for ML 14-69 interacted weakly with soluble, native ML, but the interactions were strengthened by denaturation of ML. The apparent affinity constants between these mAbs and ML showed an increase, ranging from six- to 80-fold, by denaturation of ML. Therefore, these mAbs were more specific for the denatured determinant than for the determinant in the native structure. These results indicate that a substantial number of autoreactive B cells, specific for the unfolded conformation of ML, escape tolerance and are dominantly involved in the autoantibody response to ML. Our finding provides important information to understand the naturally occurring autoreactive B-cell repertoire in normal mice.     

Significance of fibrils in the formation of the Kimmelstiel-Wilson nodule

Summary The pathogenesis of the nodular lesion in diabetic glomerulosclerosis is described in association with fibrils. Thirteen diabetic patients with glomerular nodular lesions and 9 diabetics without the nodules were examined by electron microscopy using periodic acid-thio-carbohydrazide-silver proteinate staining. In cases of nodular glomerulosclerosis, abundant fibrillar structures mixed with electron-dense material were detected within the nodule and the mesangial matrix. They were also occasionally observed along the subendothelial space of the glomerular capillary walls. On the cross-section, these fibrils, including the lucent periphery, were 34 nm wide. Immunohistologically, collagen V and collagen VI were detected in nodular lesions. In contrast, in cases of the diffuse type of glomerulosclerosis, the widened mesangium was composed of dense material, which resembled the original mesangial matrix. The above fibrils were not detected in the mesangium. These findings suggest that the accumulation of the peculiar fibrils in the glomerular mesangium is a major pathogenic factor in the formation of Kimmelstiel-Wilson nodules.     

Lamivudine and IFN-beta sequential therapy in HBe antigen-positive patients with chronic hepatitis B virus genotype C infection.

Sequential treatment with lamivudine and interferon (IFN) has induced sustained biochemical and virologic responses in the majority of patients with chronic hepatitis B in France. However, the efficacy of sequential treatment in patients with chronic hepatitis B virus (HBV) genotype C infection has not been evaluated. Twenty-four HBe antigen-positive patients were treated with 100 mg lamivudine alone for 16-32 weeks, then with both 6 MU IFN-beta and lamivudine for 4 weeks, and lastly with IFN-beta alone for 20 weeks. Sustained response was achieved in 7 (29%) patients 24 weeks after the end of therapy. No lamivudine-resistant variants emerged in any patient. Hepatitis flare occurred in 3 patients after the withdrawal of lamivudine, but none had decompensation. The patients with sustained response were significantly younger at baseline (p = 0.033) and had a significantly lower HBV DNA level at the start of IFN (p = 0.020) than those without sustained response. In conclusion, the rate of response to sequential therapy with lamivudine and IFN in HBe antigen-positive patients with HBV genotype C infection was lower than the rate reported previously. Patients who were young or who had a favorable virologic response to lamivudine were more likely to have a sustained response.     

Low-grade fibromyxoid sarcoma arising in the big toe

Low-grade fibromyxoid sarcoma (LGFMS) is a rare tumor. Reported herein is a case of LGFMS arising in the big toe. The patient was a 58-year-old man who underwent excision of the tumor. The tumor was well-demarcated. Histologically, there were proliferating spindle-shaped tumor cells arranged in a whorled growth pattern, and the stroma showed hyalinized collagen bundles and a myxoid matrix. Nuclear mitotic figures were conspicuous in part. A large rosette-like structure with hyalinized stroma was found, which is characteristic of LGFMS. The differential diagnosis included tumor occurrence in adults; tending to arise in distal extremities; and having bland fibromyxoid histological features, such as fibroma of tendon sheath, low-grade myxofibrosarcoma and acral myxoinflammatory fibroblastic sarcoma. It was not possible to detect the FUS/CREB3L2 and FUS/CREB3L1 fusion genes from the formalin-fixed and paraffin-embedded tissue, although the histological features of the present case were typical of LGFMS. LGFMS may become more common with time, and unique cases may accumulate.     

Preparation of ceramic microspheres for in situ radiotherapy of deep-seated cancer

Radiotherapy is one of the most effective treatments for cancers. However, external irradiation provides only small doses to deep-seated cancers, and often causes damage to healthy tissues. It has been reported that 20-30 microm diameter 17Y(2)O(3)-19Al(2)O(3)-64SiO(2) (mol%) glass microspheres are useful for the in situ irradiation of cancers. Yttrium-89 (89Y) in this glass can be neutron bombarded to form the beta-emitter 90Y (half-life=64.1h). When injected in the vicinity of the cancer, such activated glass microspheres can provide a large localized dose of beta-radiation. The Y(2)O(3) content of the glass in the microspheres is limited to only 17 mol%. Chemically durable microspheres with a higher Y(2)O(3) content need to be developed. Phosphorus-31 (31P) with 100% natural abundance can also be activated by neutron bombardment to form the beta-emitter 32P (half-life=14.3d). Chemically durable microspheres containing a high phosphorus content are expected to be more effective for cancer treatment. We prepared pure Y(2)O(3) and YPO(4) microspheres using a high-frequency induction thermal plasma melting technique, and investigated the resulting structure and chemical durability. We successfully prepared smooth, highly spherical polycrystalline Y(2)O(3) and YPO(4) microspheres with diameters in the range 20-30 microm. Both the Y(2)O(3) and YPO(4) microspheres showed high chemical durability in saline solutions buffered at pH=6 and 7. These microspheres are expected to be more effective than the conventional glass microspheres for the in situ radiotherapy of cancer.     

Localization and expression of chondromodulin-I in the rat cornea

The localization and expression in the rat cornea of chondromodulin-I (ChM-I), an inhibitory angiogenesis factor, were examined by immunohistochemistry, Western blot analysis, ribonuclease protection assay, and real-time PCR assay. We found immunoreactivity for ChM-I in the epithelial layer but not the stromal layer or endothelial layer in the cornea, in addition to the positive ChM-I immunoreactivity in other sites in the eye such as the sclera, retina, and ciliary body. The ChM-I immunoreactivity was most intense at the outside of the basal cells and in their cytoplasm while the intensity of the immunoreactivity decreased gradually from the wing cells to the superficial cells in the corneal epithelial layer. No reactivity however, was detected in the Bowman's membrane or conjunctival epithelial cells which had continuity with the corneal epithelial cells. The expression of ChM-I mRNA was demonstrated in the cornea at one-third less intensity than that in the sclera with choroids and retinal pigment epithelium by ribonuclease protection assay and real-time PCR. ChM-I in the corneal epithelial layer may prevent neovascularization and maintain avascularity in the cornea.     

Quantitative analysis for soluble elastin in circulation and cell culture fluids using monoclonal antibody-based sandwich immunoassay

We have newly established 3 distinct murine monoclonal antibodies (MoAbs) against human soluble elastin by using chemically denatured immunogen isolated from human aorta; they are designated as HASG-2, HASG-30, and HASG-61-1. All of these MoAbs were highly reactive with soluble forms of native elastin in normal human serum. HASG-2 and HASG-61-1 MoAbs can recognize soluble bovine elastin as well as human antigen, but HASG-30 cannot. The sandwich enzyme-linked immunosorbent assay (ELISA) for human soluble elastin was developed with HASG-61-1 labeled with peroxidase and HASG-30 immobilized on the microplates. The circulating levels of soluble elastin in human healthy subjects (mean +/- SD; 42.9 +/- 19.9ng/mL; n = 85) could be measured with full accuracy and reproducibility, and gradually increased with aging. The positive correlation between the levels and ages was statistically significant (r = 0.581, p < 0.0001). In addition, we could also determine the concentration of tropoelastin secreted from cultured human dermal fibroblasts accurately by this ELISA. This simple assay can be utilized for the routine clinical laboratory screening of patients with arteriosclerotic vascular diseases or to accurately determine the concentrations of tropoelastin secreted from cultured human cells.     

Histamine downregulates CD14 expression via H2 receptors on human monocytes

Lipopolysaccharide (LPS) binds to LPS-binding protein (LBP) in plasma and is delivered to the cell surface receptor CD14 on human monocyte. LPS is transferred to the transmembrane signaling receptor toll-like receptor (TLR) 4. In the present study, the effect of histamine on the expression of CD14 on human monocytes was investigated. Histamine concentration- and time-dependently decreased the expression of cell surface CD14, whereas histamine did not decrease mRNA for CD14 nor increase soluble CD14 (sCD14). The inhibitory effects of histamine on CD14 expression were antagonized by H2-receptor antagonist, but not by H1 and H3/H4 antagonist. The effects of selective H2-receptor agonists, 4-methylhistamine and dimaprit, on CD14 expression mimicked that of histamine indicating that histamine regulated CD14 expression through the stimulation of H2-receptors. The pretreatment with histamine partially inhibited the LPS-induced TNF-alpha production in human peripheral blood mononuclear cells (PBMC). Such inhibition might be due to the down-regulation of CD14 expression on monocytes by histamine.     

Anionic polymerization of fluorine-containing vinyl monomers, 6. Polymerizations of fluoroalkyl acrylates and methacrylates initiated by organoaluminium compounds

The anionic polymerizations of 2,2,2-trifluoroethyl acrylate ( 1a ), 2,2,2-trifluoroethyl methacrylate ( 1b ), 2,2,2-trifluoro-1-trifluoromethylethyl acrylate ( 2a ), and 2,2,2-trifluoro-1-trifluoromethylethyl methacrylate ( 2b ) were examined with triethylaluminium and diethylaluminium active methylene chelate compounds as initiators. 2b was polymerized with triethylaluminium. Diethyl(ethyl cyanoacetato)aluminium was found to produce polymers of 1a and 1b . Diethyl(acetylacetonato)aluminium and diethyl (dimethyl malonato)aluminium, however, show low reactivity towards these four monomers. The resulting polymers show unimodal molecular weight distribution with number-average molecular weights of 10⁴?10⁵, measured by GPC. The initiation reaction and the copolymerization reactivity with styrene support the anionic polymerization mechanism.