Effect of an immunotoxin to folate receptor beta on bleomycin-induced experimental pulmonary fibrosis.

It has been suggested that alveolar and interstitial macrophages play a key role in the pathogenesis of idiopathic pulmonary fibrosis (IPF) by producing proinflammatory and/or fibrogenic cytokines. We showed that inflammatory macrophages expressed folate receptor beta (FRbeta) while resident macrophages in normal tissues expressed no or low levels of FRbeta. In the present study, we examined the distribution of FRbeta-expressing macrophages in the lungs of patients with usual idiopathic pulmonary fibrosis (UIP) and mice with bleomycin-induced pulmonary fibrosis (PF) and tested whether the depletion of FRbeta-expressing macrophages could suppress bleomycin-induced PF in mice. Immunostaining with anti-human or -mouse FRbeta monoclonal antibody (mAb) revealed that FRbeta-expressing macrophages were present predominantly in fibrotic areas of the lungs of patients with UIP and mice with bleomycin-induced PF. Intranasal administration of a recombinant immunotoxin, consisting of immunoglobulin heavy and light chain Fv portions of an anti-mouse FRbeta mAb and truncated Pseudomonas exotoxin A, increased survival significantly and reduced levels of total hydroxyproline and fibrosis in bleomycin-induced PF. In immunohistochemical analysis, decreased numbers of tumour necrosis factor-alpha-, chemokines CCL2- and CCL12-producing cells were observed in the immunotoxin-treated group. These findings suggest a pathogenic role of FRbeta-expressing macrophages in IPF. Thus, targeting FRbeta-expressing macrophages may be a promising treatment of IPF.

Dynamical behavior of the motions associated with the nonlinear periodic regime in a laboratory plasma subject to delayed feedback.

Time-delayed feedback is applied to the motions associated with the nonlinear periodic regime generated due to current-driven ion acoustic instability; this is a typical instability in a laboratory plasma, and the dynamical behavior is experimentally investigated using delayed feedback. A time-delayed autosynchronization method is applied. When delayed feedback is applied to the nonlinear periodic orbit, the periodic state changes to various motions depending on the control parameters, namely, the arbitrary time delay and the proportionality constant. Lyapunov exponents are calculated in order to examine the dynamical behavior.

Hydroxy-Directed, SmI(2)-Induced Conversion of Carbohydrates into Carbocycles.

Polyoxygenated six-membered carbocycles were derived from carbohydrates with complete stereocontrol through hydroxy-directed coupling cyclization induced by SmI(2). For example, the cis-1,3-cyclohexanediol 3 is obtained from the D-glucopyranoside derivative 1 in excellent yield. The coupling cyclization is initiated by single-electron transfer from SmI(2) to the formyl group of the delta-hydroxy aldehyde 2 generated in an equilibrium process.

Immunohistochemical estimation of in-situ cell cycle time in neoplastic epithelial cells in human large intestine: a new cell proliferation index.

In-situ cell cycle time (in-situ Tc) of epithelial cells could be estimated by using a formula; in-situ Tc = cell proliferation rate divided by mitosis rate, on a scale of Tm (cell cycle time in M phase) arbitrary unit (AU), In order to see the nature of in-situ Tc in the adenoma-carcinoma sequence in the human large intestine, the in-situ Tc in 27 cases of adenoma and 71 cases of adenocarcinoma with adenoma components in the human large intestine was estimated by using this formula, counting proliferating cells and mitotic cells in the immunohistochemistry of Ki-67 antigen. C12 antigen was examined as an oncogenic progression indicator in the adenoma-carcinoma sequence. The in-situ Tc tended to shorten in adenoma in accordance with the histological grading of atypia but not in adenoma component. No significant differences in the in-situ Tc was recognized as a whole among adenomas, adenoma components and adenocarcinomas in the mucosa, whereas the in-situ Tc of adenoma components with moderate to severe atypia was significantly longer than that of adenocarcinomas in the mucosa (p = 0.05). The in-situ Tc lengthened in adenocarcinomas invading the submucosa and shortened in adenocarcinomas invading the proper muscular layer. The cases expressing the C12 antigen increased in order of adenoma, adenoma component and adenocarcinoma. The cases expressing the C12 antigen indicated short in situ Tc in the adenomas and adenocarcinomas but not in the adenoma components. Thus, the estimated in-situ Tc is a useful index of the oncogenetic progression, which is different from that detected by the C12 antigen.

Synthesis and enzymatic degradation of optically active depsipeptide copolymers.

This paper describes the synthesis and biodegradation of copolymers of cyclic depsipeptide with epsilon-caprolactone (CL) or lactide (LA). Optically active cyclic depsipeptides, 3,6-dimethyl-2,5-morpholinediones (DMOs), were prepared by the reaction of an amino acid (D-, L-, or DL-alanine) with a hydroxy acid derivative (DL-2-bromopropionyl bromide). These isomers are abbreviated as D-DMO, L-DMO and DL-DMO respectively, according to the names of alanine isomers. Then, we have prepared the copolymers of DMO isomers with CL using tin(II) octylate as a catalyst. The NMR spectra and thermal properties of DMO/CL copolymers revealed that these copolymers exist randomly. The enzymatic degradation of the copolymers has been examined using Rhizopus delemar lipase, cholesterol esterase (from Pseudomonas sp.), and Proteinase K (from Tritirachium album). Cholesterol esterase and Proteinase K show high degradability, while the lipase shows little degradation. Among the enzymes used, only Proteinase K could recognize the isomerism of DMO, resulting in the following order of degradability: copoly(L-DMO/CL) > copoly(DL-DMO/CL) > copoly(D-DMO/CL), i.e. this enzyme has the highest substrate specificity for naturally occurring L-alanine. Further, we have prepared the random copolymers of L-DMO with lactide (L-LA or DL-LA), and evaluated the enzymatic degradation of the copolymers by Proteinase K. The introduction of a small amount (up to c. 10 mol%) of L-DMO unit into LA homopolymers brought about greater degradability compared with LA homopolymers. In particular, L-DMO/L-LA copolymers with high degradability have been obtained without significant decrease in the mechanical and thermal properties of L-LA homopolymer.

[MR imaging appearances of schwannoma: correlation with pathological findings].

Peripheral schwannomas are nerve sheath neoplasms that consist of focal proliferation of Schwann cells. We reviewed the MRI findings in 17 patients with pathologically proved peripheral schwannomas. When compared with the signal intensity of muscle, that of the mass was isointense or hyperintense on T1-weighted images and hyperintense in all 17 tumors on T2-weighted images. All of the masses showed heterogeneous enhancement following the intravenous injection of Gd-DTPA on T1-weighted images. On T1-weighted images, hyperintensity was observed in the tumors that contained predominantly hypercellular Antoni type A tissue, while isointensity was observed in the tumors that contained predominantly hypocellular Antoni type B tissue. Relatively high signal intensity seen on T2-weighted images and Gd-DTPA enhanced T1-weighted images was observed in the tumors that contained predominantly Antoni type B tissue when compared with the signal intensity of tumors that contained predominantly Antoni type A tissue. A capsule was pathologically identified in 15 of 17 tumors. MRI correctly identified the presence of a capsule in 11 of 15 tumors and the absence of a capsule in one of 2 tumors. Thus the diagnostic accuracy was 71% (12/17). The cause of 4 false negative results appeared to be a hemorrhage or cystic change around the peripheral portion of the tumor, and it appeared to be a chemical artifact in one false positive result. Thus the appearance of MRI may suggest the cellular type of schwannoma, Antoni type A or B. However, prediction of the presence or absence of tumor capsule may be relatively difficult with MRI.

Expression of sulfomucins in normal mucosae, colorectal adenocarcinomas, and metastases.

We have examined the expression of specific mucin antigens in tissue sections from 92 cases of colorectal carcinoma, using sulfomucin-specific monoclonal antibody (MAb) 91.9H. The expression of sulfomucins was high in normal mucosae and much lower in primary colorectal carcinoma, in metastatic lesions in lymph nodes or in liver. The intracellular localization of sulfomucins was also different among these tissues. In normal mucosae, MAb 91.9H binding was seen in the supranuclear area, presumably Golgi complexes, the luminal surface, and secretory products. In primary colorectal carcinomas and in their metastatic lesions, MAb 91.9H was preferentially localized in the cell surface and substances attached to the luminal surface of glandular structures. Analysis of the lysates of normal and tumor tissues showed that very-high-molecular-weight components contained the antigenic epitopes. The intensity of MAb 91.9H binding was lower in tumors at advanced stages than in tumors at early stages. These high-molecular-weight components were apparently reactive with MAb FH6 specific for sialyl-Le(X) (s-Le(X) structures. Histological specimens with low levels of MAb 91.9H reactivity often exhibited relatively high levels of MAb FH6 reactivity. These two mucins may have reversed expression during carcinogenesis and carcinoma progression, and this change may be related to metastatic potential.

A piperidine amino acid, 2,4,5-piperidinetricarboxylic acid from Clitocybe acromelalga.

A new piperidine amino acid, 2,4,5-piperidinetricarboxylic acid (11) was isolated from the poisonous mushroom, Clitocybe acromelalga. The structure determination and its biogenetic potential are discussed.

Paraneoplastic vasculitic neuropathy: immunohistochemical studies on a biopsied nerve and post-mortem examination.

We studied a patient with paraneoplastic vasculitic neuropathy (PVN) associated with a carcinoma of the common bile duct. Immunohistochemical analysis of the biopsied sural nerve showed that the cellular infiltrates in the vascular lesions were composed primarily of CD8-positive T lymphocytes and macrophages. Pathogenic significance of the T-cell-mediated immunological reaction was suggested. Post-mortem examination revealed the absence of systemic vasculitis, which may be a characteristic feature of PVN. The patient responded to immunosuppressive treatment. We discuss the efficacy and the risk of immunosuppressive therapy for PVN.

Epstein-Barr virus and gastric remnant cancer.

BACKGROUND: Carcinoma arising in the gastric remnant many years after partial gastrectomy for benign disease, referred to as gastric remnant cancer (GRC) is well known, and many causal explanations have been proposed. Elsewhere, Epstein-Barr virus (EBV) involvement has been demonstrated in a small but significant fraction of gastric cancers, and evidence has been presented suggesting that, in positive cases, EBV may have played a causal role. The present report is concerned with EBV involvement in GRC in particular. METHODS: Paraffin sections from 48 cases of GRC were studied by EBER-1 in situ hybridization. RESULTS: Thirteen cases (27.1%) showed uniform hybridized signals restricted to the carcinoma cells in contrast to no hybridization in the normal mucosa, intestinal metaplasia, or hyperplastic epithelium. The prevalence of EBV involvement in GRC was significantly higher (P < 0.0001) than in gastric carcinomas from 1825 nonremnant cases; the difference remained highly significant even when the comparison was restricted to nonremnant cancers arising in the cardia and middle stomach, for which EBV-positive rates were highest. CONCLUSION: The EBV may play an important role in the carcinogenesis of GRC.

The structure of a new dipeptide from the mushroom Clitocybe acromelalga.

From the poisonous mushroom Clitocybe acromelalga, a new glutamate-containing dipeptide was isolated. Its structure was deduced to be N-(gamma-aminobutyryl)-L-glutamic acid (10) based on spectral data and was confirmed by synthesis.

Midkine, a retinoic acid-inducible growth/differentiation factor: immunochemical evidence for the function and distribution.

Midkine (MK) is a heparin-binding growth factor specified by a retinoic acid responsive gene. A rabbit was immunized against recombinant MK produced in L cells, and the resulting antibody was affinity purified using MK-glutathione S-transferase fusion protein as a ligand. The MK-specific antibody was used to investigate the function and distribution of MK. MK of the same size as the recombinant MK produced in L cells (13 kDa) was strongly detected in a 13-day rat embryo. Weak expression was observed in the brains of a 19-day embryo, neonates, and adults. In the 13-day mouse embryos, high levels of MK were detected on the surfaces of brain cells, as well as in basement membranes and in the epithelial cells of the intestine, the jaw, and the rib. Nerve cells from the brains of 13-day or 19-day rat embryos extended neurites about twofold more efficiently on MK-coated dishes than on poly-L-lysine-coated dishes. Furthermore, anti-MK antibody inhibited neurite extension not only on MK-coated dishes, but also on poly-L-lysine-coated dishes. These results suggest that MK is an endogenous neurite outgrowth factor involved in the development of the central nervous system. Anti-MK antibody was also found to inhibit growth of Wilms' tumor cells, which secreted MK into culture medium. Thus, overproduction of MK is involved in enhanced growth of Wilms' tumor cells.

Mouse heparin binding protein-44 (HBP-44) associates with brushin, a high-molecular-weight glycoprotein antigen common to the kidney and teratocarcinomas.

Heparin binding protein-44 (HBP-44) is a heparin binding protein of 44 kDa, found by cDNA cloning using antibodies against teratocarcinoma glycoproteins [Furukawa, T. et al. (1990) J. Biochem. 108, 297-302]. The N-terminal sequence analysis reported in this publication establishes the structure of its mature form. Immunohistochemical staining revealed that HBP-44 was located in the tubular brush border of the kidney. HBP-44 formed a complex with brushin, a high molecular weight (450 kDa) glycoprotein antigen common to the kidney and teratocarcinoma, but not with OR8 antigen, another antigen (350 kDa) of the same category. Brushin was shown to be the mouse counterpart of rat Heymann nephritis antigen, called gp330. The association between HBP-44 and brushin was revealed not only by co-precipitation upon indirect immunoprecipitation, but also by ligand blotting with HBP-44-maltose binding protein fusion protein. Calcium ion stabilized the association. Disulfide bonds in brushin seemed to be necessary for the complex formation, since reductive cleavage of the bonds resulted in failure of the protein to associate with HBP-44 in a ligand blotting experiment. Association of HBP-44 with brushin occurred both in teratocarcinoma cells, in which these molecules are mainly located in extraembryonic endoderm cells, and in the kidney, suggesting that the complex has an unknown common function in the renal tubular brush border and the extraembryonic endoderm.

Immunohistochemical study of mucin carbohydrates and core proteins in hepatolithiasis and cholangiocarcinoma.

The expression of mucin carbohydrates [Tn, sialosyl-Tn(STn), and T antigens] and core proteins [MUCI-apomucin-related antigen (ARA) and MUC2-ARA] was examined immunohistochemically in tissues from 40 patients with hepatolithiasis and 26 patients with intrahepatic bile-duct carcinoma. Tn and STn antigens were expressed in most of the carcinomas, and were also often expressed in the atypical bile-duct epithelium of the patients with hepatolithiasis or carcinoma, whereas they were rarely or never expressed in the normal bile duct, suggesting that they are effective tumor markers. T antigen was less useful as a marker for intrahepatic bile-duct carcinoma or the atypical epithelium, because it was expressed in normal bile-duct of some cases. Regarding the expression of ARAs in the carcinomas, non-invasive bile-duct cyst adenocarcinomas with favorable prognosis either expressed no MUCI-ARA with [DF3(-), MUSEII(-) and 139H2(-)] staining pattern or expressed MUCI-ARA with [DF3(-), MUSEII(+) and 139H2(+)] staining pattern. However these tumors often expressed MUC2-ARA with [anti-MRP(+) and CCP58(+)] staining pattern. In contrast, most invasive non-papillary cholangiocarcinomas with poor prognosis expressed MUCI-ARA with [DF3(+), MUSEII(+) and 139H2(+)] staining pattern, but expressed no MUC2-ARA with [anti-MRP(-) and CCP58(-)] staining pattern. These results suggests that different apomucins are produced by bile-duct cystadenocarcinomas and cholangiocarcinomas with differing prognosis. Furthermore, expression of Tn and STn antigens is a useful indicator of malignancy in the intrahepatic duct.

Reduction of alpha-tocopherolquinone to alpha-tocopherolhydroquinone in rat hepatocytes.

The contents of alpha-tocopherolhydroquinone (TQH2), alpha-tocopherolquinone (TQ) and alpha-tocopherol (Toc) in isolated rat hepatocytes and liver homogenates were determined by HPLC under anaerobic conditions, because TQH2 easily autoxidizes to TQ under aerobic conditions. The viable hepatocytes were used for the determination without homogenization. The hepatocytes contained 3.1, ND and 5.0, 3.1-9.0, and 31.3-63.2 nmol of TQH2, TQ and Toc/g liver, respectively. However, TQH2 was not detected in liver homogenates because endogeneous TQH2 autoxidizes to TQ during preparation of homogenates under aerobic conditions. The homogenates contained 2.0-23.5 and 36.5-54.9 nmol of TQ and Toc/g liver, respectively. Addition of TQ showed that TQ was reduced and converted into TQH2 in isolated hepatocytes. The TQH2 formation from TQ was also observed in liver homogenates in the presence of either NADPH or NADH. The formation was further analysed and confirmed by HPLC and mass spectrometry. The formation of TQH2 was also found to occur in mitochondria, microsomes and cytosol. The specific activity of NADPH-dependent TQ reductase activity was in the order of mitochondria greater than or equal to microsomes greater than cytosol. Furthermore, NADPH-cytochrome P450 reductase was found to catalyse TQH2 formation from TQ.

Dynamics of the bilayer-water interface of phospholipid vesicles and the effect of cholesterol: a picosecond fluorescence anisotropy study.

The motion of the head group of phospholipid molecules in the bilayer structure was investigated by a picosecond fluorescence anisotropy technique using a newly synthesized fluorescent phospholipid, dipalmitoyl-L-alpha-phosphatidyl-(3-p-methoxyphenyl)umbelliferone (DPPU). In this phospholipid, a coumarin derivative is attached covalently to the phosphate moiety. The motion of the acyl chain of the phospholipid was also investigated by the same method using 1-palmitoyl-2-(3-diphenylhexatrienyl)-propanoyl-L-alpha-phospha tid ylcholine (DPHpPC). From fluorescence anisotropy decay the wobbling diffusion rate (Dw) of DPPU and DPHpPC in DPPC vesicles at 45 degrees C was calculated to be 2.7 x 10(9) s-1 and 5.1 x 10(7) s-1 using the wobbling-in-cone-model. The range of the motion was calculated as the cone angle (theta c), which is half of the angle of the cone in which the fluorophore can diffuse. The cone angle of the coumarin skeleton of DPPU in DPPC vesicles at 45 degrees C was 64 degrees, which was larger than that of the DPH skeleton of DPHpPC, 40 degrees. These results indicate that the motion of the head group is much faster and wider than that of the acyl chain. When cholesterol was added to the DPPC vesicles, the range of motion of the acyl chain decreased, but that of the head group increased. These facts show that cholesterol restricts the motion of the acyl chain but enhances that of the head group in the phospholipid bilayer.

Adsorption of plasma proteins onto polymer latices.

In this article we review the adsorption of plasma proteins onto polymer latices on the basis of our experimental data. First, the surface characteristics of the latices were examined. Hydrophilic polymer layers (water-soluble polymer layers) were found to exist on the surfaces of copolymer latices, e.g., a polyacrylamide (polyAAm) layer existed on the surface of the styrene/acrylamide copolymer [P(St/AAm)] latex. These diffuse layers strongly affected the protein adsorption, that is, the amount of plasma proteins adsorbed onto copolymer latices (viz. P(St/AAm) and styrene/2-hydroxyethyl methacrylate copolymer [P(St/HEMA)] latices), particularly in the alkaline pH region, was much smaller than that onto a hydrophobic polystyrene (PS) latex. The protein adsorption was also studied as a function of pH, ionic strength and electrolyte concentration. Further, the adsorbability of heat- and urea-denatured albumins was investigated. A higher affinity of denatured components for polymer latices was observed compared with that of the native components.

Analysis for the molecular motion of phospholipid bilayer with picosecond fluorometry.

The viscosity and the molecular motion of phospholipid molecule in biological and artificial phospholipid bilayers were studied using picosecond fluorescence depolarization method with rod-like fluorophore, DPH. From the relationship between the viscosity in the lipid bilayer and the free space of phospholipid acyl-chain, it is concluded that the viscosity is determined mainly by the range of wobbling motion of the acyl-chain. Motion of polar head group was also measured by the same method with a newly synthesized fluorescent phospholipid, dipalmitoyl-phosphatidyl-umbelliferone. The rate and the range in the motion of head group were faster and larger than those of acyl-chain and gave the viscosity of head group layer to be 0.03 poise, which was about one tenth of that of acyl-chain layer in the liquid crystalline phase. This fact indicates that the head group layer would not resist the lateral diffusion of molecules in membrane and that the lateral diffusion rate of molecules could be estimated from the viscosity in the acyl-chain layer.