Early skin toxicity predicts better outcomes, and early tumor shrinkage predicts better response after cetuximab treatment in advanced colorectal cancer.

Cetuximab-containing treatments for metastatic colorectal cancer have been shown to have higher overall response rates and longer progression-free and overall survival than other systemic therapies. Cetuximab-related manifestations, including severe skin toxicity and early tumor shrinkage, have been shown to be predictors of response to cetuximab. We hypothesized that early skin toxicity is a predictor of response and better outcomes in patients with advanced colorectal carcinoma. We retrospectively evaluated 62 patients with colorectal adenocarcinoma who had unresectable tumors and were treated with cetuximab in our institution. Skin toxicity grade was evaluated on each treatment day. Tumor size was evaluated using computed tomography prior to treatment and 4-8 weeks after the start of treatment with cetuximab.Patients with early tumor shrinkage after starting treatment with cetuximab had a significantly higher overall response rate (P = 0.0001). Patients with early skin toxicity showed significantly longer overall survival (P = 0.0305), and patients with higher skin toxicity grades had longer progression-free survival (P = 0.0168).We have shown that early tumor shrinkage, early onset of skin toxicity, and high skin toxicity grade are predictors of treatment efficacy and/or outcome in patients with advanced colorectal carcinoma treated with cetuximab.

A possible role of chorion protease in shell membrane degradation during development of quail embryos.

In the eggs of the quail Coturnix japonica, the limiting membrane demarcates the shell membrane at the interface with the albumen and decreases in width during the hatching process. This study was done to identify agents that affect the width of this limiting membrane. Zymography tests on extracts from extraembryonic tissues, yolk sacs, or chorioallantoic membranes, or all three, showed proteolytic activities during d 4 to 10 of incubation. Localization experiments on these activities, performed on d 5 eggs, indicated that they were located in an avascular chorion. Electron microscopic analysis showed there were secretory cells specifically located in the avascular chorion. After partial purification of d 5 avascular chorion extracts using QA52 and Sephadex G-200 column chromatography, the proteolytic activity of 20 kDa was isolated. The protease showed a high level of activity toward succinyl-Gly-Pro-Leu-Gly-Pro-4-methylcoumaryl-7-amide. It had an optimal pH of 9 and digested the limiting membrane. These enzymatic activities were inhibited moderately by EDTA and strongly by leupeptin and aprotinin. It was concluded that it is the 20-kDa protease, showing collagenase-like activity produced by the avascular chorion, that affects the limiting membrane.

The crystal polymorphism of calcium carbonate is determined by the matrix structure in quail eggs.

Two calcified structures, the eggshell and sperm-associated body (SB), are present in the eggs of the Japanese quail, Coturnix japonica. X-ray diffractometry showed that calcium carbonates take the form of calcite in the eggshell and aragonite in the SB. The aim of the present study was to identify the factors that determine the morphology of calcium carbonate crystals. The matrix of EDTA-treated eggshell was a meshwork of vesicles, 200 to 500 nm in diameter, connected by fine fibers or fibrous sheets. The matrix of SB cortex was a radiation of rod-shaped projections approximately 130 nm in width. In vitro crystal formation was achieved by adding dissociated matrix substances to test solutions. When eggshell matrix material was added, formation of calcite crystals, which had many vesicular holes on their surface, was observed. When SB matrix material dissociated by sonication was added, rhombohedral calcite crystals formed at protein concentrations of 100 microg/mL or lower, and elongated and bundled crystals formed at concentrations of 150 microg/mL or higher. When SB matrix material dissociated by pipetting was added, aragonite crystals formed. These observations indicate that the matrix structure is the principal factor in determining the crystal polymorphism of calcium carbonate.

The peri-albumen layer: a novel structure in the envelopes of an avian egg.

The present paper describes a novel structure, termed the peri-albumen layer, in the egg-envelopes of the quail Coturnix japonica. It reacts with Alcian blue and exists between the egg white and the shell membrane. Ultrastructurally, it is of fine granular structure and forms a fenestrate sheet, the width of which is 190 nm or less. Isolated materials of the peri-albumen layer include an Alcian-blue-positive polysaccharide of 260 kDa, and three glycoproteins of 160, 108 and 52 kDa. The layer is supplied to an egg when it passes through the magnum-isthmus junction, the normalized length of which is 0.62-0.63 of the oviduct. The mucosa of the junction consists exclusively of a luminal epithelium. It is apparently distinct from the mucosa of the magnum and the isthmus, which consist of a luminal epithelium and tubular glands. The luminal epithelium of the magnum-isthmus junction stains prominently with Alcian blue and consists of alternately distributed ciliated cells and granular cells. Immunohistochemistry with an antiserum raised against the materials of the peri-albumen layer revealed the staining of the peri-albumen layer of the egg, and secretory cells of the luminal epithelium at the magnum-isthmus junction. It was concluded that the materials of the peri-albumen layer are produced by secretory cells at the magnum-isthmus junction of the oviduct.

Changes in shell membranes during the development of quail embryos.

The shell membrane of an avian egg acts as a bag enclosing albumen and water. At its interface with the albumen, a smooth layer of homogeneous, dense material called the limiting membrane demarcates the shell membrane. The present study aimed to investigate changes in the limiting membrane during development of quail embryos that were grown with or without being turned. Sixty-three percent of the embryos were hatched after the eggs were incubated at 39 C and in 60% humidity with automatic rotation around their long axis and with their equatorial side down, whereas the hatch rate decreased to 24% when the eggs were incubated without being turned. The width of the limiting membrane at the equatorial region of turned eggs gradually decreased from 74 nm on Days 0 to 2 of incubation to 35 nm on Day 10 and thereafter. Conversely, water permeability, measured by evaporation through the shell membrane increased from 4 to 5 nL/mm2 per min on Days 0 to 6, to 9 nL/mm2 per min on Day 12 and thereafter. In stationary eggs, the decrease in the width of the limiting membrane on the lower side of eggs was delayed until Day 8 of incubation. The water permeability of the shell membrane in this group was 51% of that of the membrane on the upper side of eggs on Day 8 of incubation. Forty to forty-four nanometers seemed to be the critical width of the limiting membrane at which high water permeation could occur. It was also shown that the albumen hinders water permeation through the membrane. These results show that (1) the limiting membrane is made thin during the development over the whole surface with egg-turning, possibly through digestion of still unknown agents, and (2) this thinning accelerates the rate of water permeation through the membrane.

A study of carboplatin-coated tube for the unresectable cholangiocarcinoma.

Most cases of cholangiocarcinoma have reached an unresectable stage by the time they are discovered despite significant progress of diagnostic modalities. Many of these patients with obstructive jaundice are often treated by biliary drainage using stents to relieve the jaundice. However, the stent patency period is as short as 3 to 9 months because of tumor ingrowth or overgrowth, and mean survival is at most 12 months. Therefore, both continuous relief of obstructive jaundice and local control of the tumor are required in the treatment for advanced cholangiocarcinoma. In this investigation, we developed a new percutaneous transhepatic biliary drainage tube coated with carboplatin (carboplatin-coated tube; CCT). CCT continuously released a fixed amount of carboplatin for 4 weeks and showed an antitumor effect on human cholangiocarcinoma cell line HuCC-T1 in vitro. When CCT was embedded in subcutaneous tumor inoculated in nude mice, a significant reduction of tumor size with no apparent damage to normal adjacent tissue was observed. On the basis of these studies, 5 patients with inoperable cholangiocarcinoma were treated with CCT for 4 weeks. Overall efficacy rate of 5 patients with cholangiocarcinoma was 60% (partial response in 3 and no change in 2). No apparent side effect was observed in these patients. Thus, CCT may provide a new treatment modality for this disease. Randomized controlled trials comparing CCT therapy with palliative stenting are required to confirm these results.

Autoantibodies against the specific epitope of human tropomyosin(s) detected by a peptide based enzyme immunoassay in sera of patients with ulcerative colitis show antibody dependent cell mediated cytotoxicity against HLA-DPw9 transfected L cells.

BACKGROUND AND AIMS: Recent studies suggest that tropomyosin (TM) may act as a putative autoantigen in ulcerative colitis (UC). Recently, we identified, by computer homology analysis, a specific peptide (HIAEDADRK) in human TM that can bind to HLA-DPw9. The aim of this study was to investigate the presence of autoantibodies against this peptide in UC. METHODS: Antibodies were measured by ELISA with a synthetic peptide in 20 healthy volunteers, 48 patients with UC, 26 with Crohn's disease (CD), eight with primary sclerosing cholangitis (PSC), and six with primary biliary cirrhosis (PBC). The functional significance of antibodies was investigated by antibody dependent cell mediated cytotoxicity (ADCC) against DPw9 transfected L cells using a standard (51)Cr release assay. RESULTS: Optical density values (mean (SD)) of sera from patients with UC (1.40 (0. 52)) and PSC (1.65 (0.12)) were significantly higher than those from healthy volunteers (0.32 (0.28)) (p<0.05), CD (0.50 (0.34)) (p<0.05) and PBC (0.14 (0.09)) (p<0.05). Values in UC decreased with clinical improvement. The ADCC activity of UC sera correlated well with antibody titre against this synthetic peptide. CONCLUSIONS: Anti-TM antibody was detected in UC sera by a specific peptide based ELISA with high reproducibility. This peptide may be an antigenic epitope of TM involved in the immunopathogenesis of UC and, perhaps, PSC.

Externalization of tropomyosin isoform 5 in colon epithelial cells.

Ulcerative colitis (UC) is associated with autoantibody response to a cytoskeletal protein, human tropomyosin (hTM) isoform-5 (hTM5). Because hTM5 is an intracellular protein, it may remain inaccessible to the autoantibodies. Therefore, we have investigated the possibility of externalization of hTM5 in colon epithelial cells. Freshly isolated colonic and small intestinal epithelial cells and LS-180 colon cancer cell line were examined for surface expression of hTM5 by flow cytometric analysis using hTM isoform-specific MoAbs. The extracellular release of hTM5 was determined by Western blot and radioimmunoprecipitation analyses. Physical association of hTM5 with a membrane-associated colon epithelial protein (CEP) was examined by co-immunoprecipitation of hTM5 with anti-CEP MoAb, and CEP with anti-hTM5 MoAb. Cell surface expression of hTM5 was observed in colonic epithelial and LS-180 cells but not in small intestinal epithelial cells. LS-180 cells spontaneously released hTM5 as well as CEP into the culture medium that was significantly stimulated by a calcium ionophore, A23187, but inhibited by phorbol-12-myristate-13-acetate, monensin and methylamine. Co-immunoprecipitation experiments revealed that hTM5 forms a complex with CEP. We conclude that hTM5 is externalized in colon but not in small intestinal epithelial cells. The physical association of hTM5 with CEP suggests a possible chaperone function of CEP in the transport of hTM5, a putative target autoantigen in UC.

Analyses of oviductal pars recta-induced fertilizability of coelomic eggs in Xenopus laevis.

The acquisition of fertilizability in coelomic eggs of Xenopus laevis has been shown to be correlated with the physical, biochemical, and ultrastructural alterations of the egg envelope [coelomic envelope (CE)] induced during the passage of eggs through the pars recta portion of the oviduct. However, no direct evidence that the pars recta renders eggs fertilizable has yet been presented. In this study, we show that coelomic eggs are highly fertilizable when they are incubated with continuous shaking for 4 h at 15 degrees C in pars recta extract (PRE) derived from females prestimulated by pregnant mare serum gonadotropin. The PRE from pituitary-stimulated Bufo japonicus was as potent as homologous PRE in rendering Xenopus eggs fertilizable. Incubation of coelomic eggs in PRE for 30 min induced a dramatic increase in the rates of sperm binding to the envelope to a level equivalent to that exhibited by the envelope from uterine eggs (VEs). The CE-to-VE ultrastructural conversion and a 43k-to-41k hydrolysis of the envelope glycoprotein component started 5 min after, and were completed by 15 min after, the start of incubation in PRE and were accompanied by an exposure of a new N-terminal sequence typical to gp41. Thus, the biochemical and ultrastructural conversions and the sperm-binding activity of the envelope induced by PREs, although being prerequisite, were not sufficient to render coelomic eggs fully accessible to fertilizing sperm.

Cysteine proteinase plays a key role for the initiation of yolk digestion during development of Xenopus laevis.

In electrophoretic analyses, extracts of Xenopus laevis neurulae exhibited activities digesting yolk proteins maximally at pH 4.8. These activities were completely inhibited by a mixture of pepstatin A and Z-Phe-Phe-CHN2, thus being identifiable as cathepsin D and cysteine proteinase. The electrophoretic profiles of yolk proteins cleaved by embryonic extracts changed at gastrula stages; the profile before stage 13 was the same as that given by cathepsin D treatment and the profile at stage 13 was a combination of the profile given by cathepsin D treatment and that given by cysteine proteinase treatment. Quantitative measurement of enzyme activities showed that the cathepsin D activity that was preserved from the beginning of development increased from stages 13 to 25 and decreased thereafter, whereas the cysteine proteinase activity appeared at stage 13, gradually increased until stage 35 and strongly increased thereafter. Immunoblot analyses showed that the 43 kDa form of cathepsin D was processed to its 36 kDa form, presumably by cysteine proteinase. This change can explain the increase of cathepsin D activity at stage 13 and thereafter. Immunofluorescent staining with the antibody against cysteine proteinase occurred in mesodermal and ectodermal cells other than neural ones at stages 13-24, and in the endodermal cells at stages 24-36. Faint staining in the neural ectoderm persisted from stages 18 to 36. Immunoelectron microscope observation showed that what stained was the superficial layer of yolk platelets. All these results indicate that cysteine proteinase plays a key role in the initiation of yolk digestion during embryonic development.

Purification and properties of embryonic cysteine proteinase which participates in yolk-lysis of Xenopus laevis.

The study reported here aimed to purify a cysteine proteinase from neurula embryos of Xenopus laevis, since this enzyme was thought to be involved in yolk-lysis in developing embryos. The purification procedure consisted of fractionation of an embryonic extract by means of 30-90% ammonium sulfate, chromatography on diethylaminoethyl cellulose and carboxymethyl cellulose, gel filtration on Sephadex G-75 and affinity chromatography on concanavalin A-agarose. The purified enzyme had a molecular weight of 30 kDa according to both SDS-PAGE and Sephadex G-75 gel-filtration and an optimum pH of 5.5, and it preferentially cleaved the synthetic substrate, Z-Phe-Arg-MCA. Its activity was inhibited by Z-Phe-Phe-CHN2, a specific cathepsin L inhibitor, as well as by leupeptin and E-64. The NH2-terminal amino acid sequence of the enzyme was similar to that of chicken cathepsin B. These characteristics indicate that the purified enzyme is a member of the cysteine proteinase family. The antibody raised against the purified enzyme specifically stained a 30 kDa protein of neurula embryo extracts on immunoblot tests. The enzyme effectively digested Xenopus yolk proteins when the NaCl concentration in test solutions was 0.2 M. It was also confirmed that cysteine proteinase inhibitors inhibited yolk-lysis by the enzyme.

Tropomyosin isoforms in intestinal mucosa: production of autoantibodies to tropomyosin isoforms in ulcerative colitis.

BACKGROUND & AIMS: Autoantibodies against tropomyosins (TMs) have been reported in ulcerative colitis (UC). In this study the hTM isoforms (hTM1-5) present in intestinal epithelial cells and in smooth muscle were investigated, and the immunoreactivity against hTMs by immunoglobulin G (IgG) produced in vitro by colonic mucosal lymphocytes (LPMCs) from patients with UC, Crohn's disease (CD), and controls was examined. METHODS: TMs were extracted from colonic and jejunal epithelial cells and smooth muscle, and hTM isoforms were identified using isoform-specific monoclonal antibodies by enzyme-linked immunosorbent assay and transblot analysis. The immunoreactivity of IgG produced by colonic LPMCs was analyzed against the recombinant hTM isoforms. RESULTS: The major hTM isoforms present in colonic and jejunal epithelial cells are hTM5 and hTM4, whereas intestinal smooth muscle contains the hTM1-3 isoforms. The IgG synthesized in vitro by LPMCs from UC (n = 19) recognized hTM5 and hTM1, more significantly (P < 0.04 to <0.001) when compared with CD (n = 12) and controls (n = 17). However, IgG produced by LPMCs from CD did not show such anti-hTM reactivity. Mucosal anti-hTM IgG mainly belonged to the IgG1 subclass. CONCLUSIONS: Intestinal epithelial cells and smooth muscle have distinct hTM isoforms. Patients with UC, and not CD, show mucosal autoantibody response against hTM isoforms, particularly hTM5 and hTM1.

Changes in the chorion and sperm entry into the micropyle during fertilization in the teleostean fish, Oryzias latipes.

Specific antibodies against the major chorionic glycoproteins (ZI1-2 and ZI3) of unfertilized eggs were used to analyze the differences in the chorion and its surrounding constituents before and after fertilization. The glycoproteins in the inner layers of the chorion and its surrounding material were specifically stained by both of the antibodies. Thirty and 60 min after activation, the thickness of the chorion's inner layers was already reduced and the micropylar canal was closed. At the same time, the broadly diluted mucous area (DMA) of glycoproteins on the outermost layer of the chorion in unfertilized eggs was modified to a thin, compact layer. When unfertilized eggs were treated with trypsin, the inner third portion of the micropylar canal closed and the glycoproteins in the DMA were digested. The incidence of sperm entry into the micropyle of these eggs was extremely reduced. These results suggest that in medaka eggs, the chorionic glycoproteins in the DMA on the chorion surface, which have an affinity for spermatozoa, play an important role in sperm guidance into the micropyle.

Degenerative hairlets on the vestibular sensory cells in mutant bustling (BUS/Idr) mice.

The bustling mouse (BUS/Idr: bus) is a mutant mouse strain which exhibits deafness, bustling/hyperkinetic behaviour and functional disorders seemingly related to the vestibular system. This phenotype develops in homozygous (bus/bus) mice and has been shown from cross experiments to be genetically induced by a single autosomal recessive gene. We previously detected, with light and electron microscopy, post-natal degeneration of the inner ear sensory cells in homozygotes. In the present study, we examined, by electron microscopy, the development of pathological changes in the sensory epithelia of the macula acustica and crista ampullaris of homozygous mice of various ages, paying special attention to the detailed morphology of the sensory hairlets. The homozygous mice exhibited specific pathological changes: a decrease in the number of hairs; disarrangement of the kinocilium-stereocilia pattern; and, fused and/or very large stereocilia. Homozygotes also frequently exhibited apical cytoplasmic herniation, or bleb of hair cells, as well as a degenerated kinocilium in the sensory epithelium. Heterozygotes showed similar changes, but to a lesser degree and frequency. As for the vestibular organs, similar pathological changes had developed at day, 17 of gestation. These pathological findings and onset suggest that the BUS mouse may be a mutant mouse strain distinct from other reported strains which display similar behaviour, and may be a useful animal model for the study of human degenerative vestibular disorders.

Vitellogenesis-related ovary cathepsin D from Xenopus laevis: purification and properties in comparison with liver cathepsin D.

Cathepsin D was purified from ovaries of Xenopus laevis by both QAE-cellulose and pepstatin-Sepharose chromatography and then characterized and compared with Xenopus liver cathepsin D. Ovary cathepsin D appeared predominantly as a 43-kilodalton (kDa) molecular mass, as revealed by SDS-polyacrylamide gel electrophoresis, whereas the liver enzyme was obtained exclusively as a 36-kDa protein. The purified 43-kDa ovary enzyme cleaved vitellogenin limitedly to produce yolk proteins at pH 5.6. The specific activity of ovary cathepsin D was five to six times lower than that of the liver enzyme, as measured by hemoglobin-hydrolysis at pH 3, but the ovary enzyme was shown to be superior to the liver enzyme in terms of vitellogenin-cleaving activity, as examined at pH 5.6. Ovarian enzyme preparations contained variable amounts of 36-kDa species; this form was considered to be an autolytic product of the 43-kDa form arising during purification, because it was not detected in oocyte extracts but was generated by incubation of the purified 43-kDa enzyme alone in an acid solution. The conversion of the 43-kDa form by hepatic factors was accompanied by a marked increase in hemoglobin-hydrolytic activity.

Suppressive effect of recombinant human Cu, Zn-superoxide dismutase on lung metastasis of murine tumor cells.

The inhibitory effect of recombinant human Cu++Zn++superoxide dismutase (rhSOD) on metastasis of tumor cells in the mouse was investigated. In an experimental pulmonary metastasis model employing Meth A cells as inoculum, significant inhibition of metastasis was obtained by intravenous pre- and post-administration of rhSOD. An inhibitory effect of rhSOD was also observed in a spontaneous pulmonary metastasis model with 3LL cells as the inoculum. rhSOD was not observed to have any significant effects on the platelet-aggregating activity of tumor cells, the adhesiveness of tumor cells to vascular components (endothelial cells, laminin and type-IV collagen), or the growth of tumor cells either in vitro or in vivo. However, rhSOD suppressed invasion of Meth A and 3LL cells into Matrigel (an artificially reconstituted basement membrane of collagen, laminin and heparan sulfate) in the presence of hypoxanthine and xanthine oxidase, in vitro producers of superoxide. Thus, the present study shows that rhSOD is able to inhibit both experimental and spontaneous pulmonary metastasis, possibly through the suppression of tumor cell invasion into the extracellular matrix.

A role for the fyn oncogene in metastasis of methylcholanthrene-induced fibrosarcoma A cells.

Expression of various oncogenes (ras, myc, erbB2, src, fyn, yes and sis) in a high-metastatic clone (MH-02) derived from a murine methylcholanthrene-induced fibrosarcoma A (Meth A) was compared with those of its parent clone (ML-01) by Northern blot analysis. Two oncogenes, fyn, belonging to the tyrosine-kinase family, and sis, belonging to the cellular-growth-factor family, were found to have higher signals (3.6-fold and 1.8-fold respectively) in MH-02 than in ML-01 cells. To explore the possibility that higher expression of these oncogenes is involved in enhanced metastasis of the MH-02 clone, ML-01 was transfected by a fyn vector and the metastatic potential of the transfectant was examined. Mice administered fyn-transfected ML-01 cells had significantly increased metastatic nodules in the lung, as compared with those whose ML-01 cells were transfected with control vector without the fyn gene. The result indicates that the fyn gene is one of the factors governing the metastatic potential of Meth A cells.

Augmented expression of a type IV collagen-binding protein in a highly metastatic murine fibrosarcoma clone.

The adhesive properties of highly and weakly metastatic murine sarcoma (Meth A) clones were investigated. A highly metastatic clone, MH-02, preferentially adhered to type IV collagen-coated plastic dishes and to bovine pulmonary arterial endothelial cell-coated plastic dishes as compared to a weakly metastatic clone, ML-01. Pretreatment of MH-02 and ML-01 cells with antisera against MH-02 cells resulted in almost equivalent adhesiveness to type IV collagen. Preincubation of 125I-radiolabeled tumor cells with the antisera against MH-02 significantly reduced the arrest of MH-02 cells in the lung, but ML-01 cells were not affected. The number of pulmonary metastatic nodules of MH-02 cells was reduced to the same level as that of ML-01 cells by preincubation of the tumor cells with the antisera in an experimental metastasis experiment. These results indicated that the high metastatic ability of MH-02 can be attributed to its preferential adhesiveness to type IV collagen. The type IV collagen-binding proteins of MH-02 and ML-01 were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. Among several proteins which bound to type IV collagen, expression of a protein with a molecular weight of 29 kD was significantly greater in MH-02 than in ML-01. These results suggest that the greater adhesion of highly metastatic MH-02 cells to type IV collagen is due to enhanced expression of the type IV collagen-binding 29 kD protein.