Aberrant Collagen Cross-linking in Human Oral Squamous Cell Carcinoma.

Tumor progression is a complex process involving extracellular matrix (ECM) remodeling and stiffening. However, the mechanisms that govern these processes and their roles in tumor progression are still poorly understood. In this study, we performed bioinformatics, immunohistochemical, and biochemical analyses to examine if collagen cross-linking is associated with tumor stage and regional lymph node metastasis (RLNM) in oral squamous cell carcinoma (OSCC). We found that the genes encoding key enzymes for cross-linking are frequently overexpressed in oral, head, and neck cancers. Specifically, the enzymes lysyl hydroxylase 2 (LH2) or lysyl oxidase (LOX) and LOX-like 2 (LOXL2) were significantly upregulated in late-stage tumors and associated with poor patient prognosis. The protein levels of these enzymes in the primary human OSCC were also significantly increased in late-stage tumors and markedly elevated in the RLNM-positive tumors. Notably, while overall LOX/LOXL2-catalyzed collagen cross-links were enriched in late-stage and RLNM-positive tumors, LH2-mediated stable cross-links were significantly increased. To our knowledge, this is the first study to investigate the association of collagen cross-linking and expression of key enzymes regulating this process with OSCC stage. The data indicate a critical role for collagen cross-linking in OSCC tumor progression and metastasis, which may provide insights into development of novel therapeutic strategies to prevent OSCC progression.

Recombinant biglycan promotes bone morphogenetic protein-induced osteogenesis.

The aim of this study was to determine the effects of glutathione-S-transferase-fused recombinant biglycan (GST-BGN) on craniofacial bone regeneration. We recently demonstrated a positive effect of tissue-derived BGN on bone morphogenetic protein 2 (BMP-2) function, which is exerted likely via the BGN core protein. Here, we investigated the effects of GST-BGN lacking any posttranslational modifications on BMP-2 function in vitro and in vivo. In the C2C12 cell culture system, BMP-2-induced Smad 1/5/8 phosphorylation and alkaline phosphatase activity were both enhanced by the addition of GST-BGN. For the in vivo effect, we employed a Sprague-Dawley rat mandible defect model utilizing 1 µg (optimal) or 0.1 µg (suboptimal) of BMP-2 combined with 0, 2, 4, or 8 µg of GST-BGN. At 2 weeks post-surgery, newly formed bone was evaluated by microcomputed tomography and histologic analyses. The results revealed that the greatest amounts of bone within the defect were formed in the groups of suboptimal BMP-2 combined with 4 or 8 µg of GST-BGN. Also, bone was well organized versus that formed by the optimal dose of BMP. These results indicate that recombinant BGN is an efficient substrate to promote low-dose BMP-induced osteogenesis.

Role of glycosaminoglycans of biglycan in BMP-2 signaling.

Recently we have reported that biglycan (BGN) promotes osteoblast differentiation and that this function is due in part to its ability to positively modulate bone morphogenetic protein (BMP) functions. In this study we investigated the role of glycosaminoglycans (GAGs) of BGN in this function using in vitro and in vivo models. C2C12 myogenic cells were treated or untreated with BMP-2 alone or in combination with glycanated, partially glycanated or de-glycanated BGN, and the effects on BMP signaling and function were assessed by Smad1/5/8 phosphorylation and alkaline phosphatase (ALP) activity. Furthermore, the effect of de-glycanation of BGN on BMP-2 induced osteogenesis was investigated employing a rat mandible defect model. The defects were filled with collagen scaffolds loaded with glycanated or de-glycanated BGN alone or in combination with a sub-optimal dose of BMP-2 (subBMP). In in vitro experiments, BMP signaling and function were the greatest when BMP-2 was combined with de-glycanated BGN among the groups tested. In the rat mandible experiments, µCT analyses revealed that the newly formed bone was significantly increased only when subBMP was combined with de-glycanated BGN. The data indicate that the GAG component of BGN functions as a suppressor for the BGN-assisted BMP function.

Structure, formation, mechanical properties, and disposal of the embryo attachment system of an estuarine crab, Sesarma haematocheir.

In most decapod crustaceans, fertilized eggs extruded from the gonopore attach to ovigerous hairs within the incubation chamber of the female. The attachment is effected by an "embryo attachment system." The three continuous components of this system are the egg envelope, the funiculus, and the investment coat, which wraps around an ovigerous hair. Transmission electron microscopy (TEM) revealed that the embryo of Sesarma haematocheir is enfolded by three distinct envelopes (E1, E2, and E3), whereas the embryo attachment system is composed of only the outermost, single envelope (E1) with two sublayers (E1a and E1b). This envelope (E1) originates from the outer layer of the vitelline membrane (envelope of the ovum) with two sublayers (E1a' and E1b'). The sequence and timing of events in the formation of the embryo attachment system was determined on the basis of observations of female behavior, ultrastructure, and mechanical properties of the membranes. The egg envelope (E1a' + E1b') is not adhesive immediately after extrusion from the gonopore; but 5 min after egg-laying, it becomes adhesive-a change associated with "fusion" of the two sublayers (E1)-and attaches the eggs to the ovigerous hairs from 5 to 30 min after egg-laying. The layer E1a' always binds to an ovigerous hair at specific, electron-dense attachment sites that are distributed longitudinally on the surface of each hair. Plasticity of the egg envelope changes, and the female kneads her eggs by the movement of ovigerous setae; this movement forms the investment coat on the ovigerous hair (10-40 min after egg-laying). Thirty minutes after egg-laying, the egg envelope again divides into two sublayers (E1a and E1b), and the adhesiveness rapidly decreases. The plasticity of the envelope remains, and the funiculus is formed, accompanied by kneading of the eggs (40-90 min after egg-laying). The embryos hatch one month after incubation, and the attachment systems all slip off their ovigerous hairs by the actions of the ovigerous-hair slipping substance (OHSS). This substance appears to act specifically at the attachment sites on the hair, lysing the bond with layer E1a, and thereby disposing of the embryonic attachment system and preparing the hairs for the next clutch of embryos.

Hatching of an estuarine crab, Sesarma haematocheir: from disappearance of the inner (E3) layer to rupture of the egg case.

Hatching of decapod crustaceans is characterized by the sudden rupture of the egg case. This study focused on the following two issues regarding the hatching mechanism of the estuarine terrestrial crab Sesarma haematocheir: (1) dissolution of the egg case, and (2) the site where the egg case breaks. The egg case comprises three layers: the outer two (E1 and E2) layers and the inner (E3) thin layer (0.2 microm in thickness). The outer layers showed no morphological changes upon hatching, but the inner layer (E3) was markedly digested. The digestion of this layer would enable the embryo to absorb ambient water via reverse peristalsis of the intestine, resulting in an increase of the volume. The egg case always ruptured perpendicular to the longitudinal axis of the embryo. In addition, breakage of the egg case occurred at the dorsal thorax of the embryo. The three major organs positioned at this area were (1) a sharp projection (dorsal spine), (2) an assemblage of muscles, and (3) a pair of secretory glands, each of which was about 30 microm in diameter. The dorsal projection is soft before hatching, and it is clear that the egg case does not break with the posterior expansion of this projection. The rupture instead appears to be caused by the expansion of the muscles arranged perpendicular to the body axis. In addition, some (unknown) factor might weaken the egg case just before hatching. The secretory glands may be a kind of rosette gland, but the role that this gland plays at hatching is not known. As a duct comes out from the center and enters the dorsal projection, some active substance may be released at the tip of this projection. However, immunochemical studies are not consistent with this substance being an ovigerous hair stripping substance (OHSS).

High levels of viremia in patients with the Hantavirus pulmonary syndrome.

Hantavirus pulmonary syndrome (HPS) is a rare but acute fulminant disease caused by Sin Nombre virus (SNV). To understand the role of the viral load in the pathogenesis of HPS, the load of virus in the blood of patients with HPS was measured. A quantitative reverse transcription-polymerase chain reaction assay was developed for SNV, because SNV is difficult to grow in cell culture. Thirty-eight samples from 26 patients with HPS were analyzed. Twenty of the 26 initial samples were positive for viral RNA (7 of 9 samples were obtained from patients with fatal cases, and 13 of 17 were obtained from survivors). Mean viral RNA copy numbers were 106.1+/-1.4/mL in positive cases (106.7+/-1.4/mL in fatal cases, 105.8+/-1.3/mL in survivors) and were correlated with peak hematocrit (P<.05) and with the lowest platelet count (P=.05). In 8 survivors who had serial samples obtained, viral RNA copy numbers decreased promptly after resolution of fever.

Infection of human respiratory submucosal glands with rhinovirus: effects on cytokine and ICAM-1 production.

To further understand the early biochemical events that occur in infected surface epithelium, we developed for the first time a model in which a respiratory submucosal gland cell population can be infected with rhinovirus (RV). Viral infection was confirmed by demonstrating with PCR that viral titers in supernatants and lysates from infected cells increased with time. Infection by RV14 upregulated the expression of intercellular adhesion molecule-1 (ICAM-1) mRNA, the major RV receptor, on submucosal gland cells, and it increased production of interleukin (IL)-1alpha, IL-1beta, IL-6, IL-8, tumor necrosis factor-alpha, and granulocyte-macrophage colony-stimulating factor in supernatants. Antibodies to ICAM-1 inhibited RV infection of submucosal gland cells and decreased the production of cytokines after RV infection. Both IL-1alpha and IL-1beta upregulated ICAM-1 mRNA expression and increased susceptibility to RV infection, whereas other cytokines failed to alter ICAM-1 mRNA expression. Furthermore, neutralizing antibodies to IL-1alpha and IL-1beta significantly decreased the viral titers in supernatants and ICAM-1 mRNA expression after RV infection, but a neutralizing antibody to tumor necrosis factor-alpha was without effect. These findings suggest that respiratory submucosal gland cells play an important role in the initial stages of inflammation and provide useful insights into the pathogenesis of RV infection.

High-yield reassortant influenza vaccine production virus has a mutation at an HLA-A 2.1-restricted CD8+ CTL epitope on the NS1 protein.

Current influenza virus vaccines are prepared using high-yield reassortant virus strains obtained from a mixed infection of the new virus strain and a prototype high-yielding virus strain. The high-titered reassortant virus strain used as vaccine seed virus possesses the recent virus HA and NA and contains the internal genes from the high-growing prototype parent. We established a human CD8(+) cytotoxic T cell (CTL) line, 10-2C2, which recognizes an HLA-A2.1-restricted influenza A virus H1, H2, H3 cross-reactive T cell epitope on amino acids 122-130 of the NS1 protein, and unexpectedly we observed that there was decreased lysis of target cells infected with the A/Texas/36/91 (H1N1) vaccine virus strain compared to the lysis of target cells infected with the prototype A/PR/8/34 (H1N1) virus. RT-PCR results showed that the A/Texas vaccine virus strain contained a quasispecies. Approximately 50% of viral RNA of the NS1 gene had a nucleotide substitution that resulted in the N --> K amino acid change at the sixth position of the nonamer peptide. Current influenza vaccines are inactivated and do not contain the NS1 protein; however, future influenza vaccines may include live attenuated vaccines and with this mutation a live virus would fail to induce a CD8(+) CTL response to this epitope in individuals with HLA-A2.1, a very common allele, and potentially have reduced efficacy.

Human CD8+ and CD4+ T lymphocyte memory to influenza A viruses of swine and avian species.

Recently, an avian influenza A virus (A/Hong Kong/156/97, H5N1) was isolated from a young child who had a fatal influenza illness. All eight RNA segments were of avian origin. The H5 hemagglutinin is not recognized by neutralizing Abs present in humans as a result of infection with the human H1, H2, or H3 subtypes of influenza A viruses. Subsequently, five other deaths and several more human infections in Hong Kong were associated with this avian-derived virus. We investigated whether influenza A-specific human CD8+ and CD4+ T lymphocytes would recognize epitopes on influenza A virus strains derived from swine or avian species, including the 1997 H5N1 Hong Kong virus strains. Our results demonstrate that adults living in an urban area of the U.S. possess influenza A cross-serotype reactive CD8+ and CD4+ CTL that recognize multiple epitopes on influenza A viruses of other species. Bulk culture cytotoxicity was demonstrated against avian and human influenza A viruses. Enzyme-linked immunospot assays detected precursor CTL specific for both human CTL epitopes and the corresponding A/HK/97 viral sequences. We hypothesize that these cross-reactive CTL might provide partial protection to humans against novel influenza A virus strains introduced into humans from other species.

Effects of rhinovirus infection on hydrogen peroxide- induced alterations of barrier function in the cultured human tracheal epithelium.

To investigate whether rhinovirus infection impairs epithelial barrier functions, human rhinovirus 14 (HRV-14) was infected to primary cultures of human tracheal epithelial cells and experiments were performed on Day 2 after HRV-14 infection. Hydrogen peroxide (H2O2; 3 x 10(-)4 M) increased electrical conductance (G) across the epithelial cell sheet measured with Ussing's chamber methods. Exposure of the epithelial cells to HRV-14 had no effect on H2O2-induced increases in G and [3H]mannitol flux through the cultured epithelium in the control condition, but it markedly potentiated H2O2- induced increases in both parameters in IL-1beta (100 U/ml) pretreated condition. However, pretreatment with TNF-alpha (100 U/ml) was without effect. IL-1beta enhanced the intercellular adhesion molecule-1 (ICAM-1) expression assessed by immunohistochemical analysis and susceptibility of epithelial cells to HRV-14 infection. An antibody to ICAM-1 inhibited HRV-14 infection of epithelial cells and abolished H2O2-induced increases in G and [3H]mannitol flux in IL-1beta-pretreated epithelial cells with HRV-14 infection. These results suggest that rhinovirus infection may reduce barrier functions in the airway epithelium in association with upregulation of ICAM-1 expression.

A case of multicentric Castleman's disease associated with renal amyloidosis and pure red cell aplasia.

This is the first reported case of multicentric Castleman's disease (MCD) associated with renal amyloidosis and pure red cell aplasia (PRCA). Inguinal lymph node biopsy showed follicular hyperplasia with prominent germinal centers and plasma cell proliferation in the interfollicular areas. Renal biopsy specimens revealed intraglomerular amyloid deposits, defined as AA amyloidosis. Since amyloid deposits were not present in the gastric and rectal mucosal tissues, his renal disorder was found to be an unusual secondary amyloidosis associated with MCD. Following treatment by plasma exchange, there was progressive deterioration of anemia due to PRCA detected by bone marrow aspiration. Subsequently he was successfully treated with steroid pulse therapy not only for anemia but also for renal function.

Dipeptidase inhibitor and epithelial removal potentiate leukotriene D4-induced human tracheal smooth muscle contraction.

We investigated the role of epithelium in smooth muscle contraction induced by leukotriene D4 (LTD4) in isolated human trachea. The contractile response to LTD4 was potentiated by an inhibitor of dipeptidases L-cysteine and by removal of the epithelium. Both L-cysteine (3 x 10(-3) M) and removal of the epithelium shifted the concentration-response curves to LTD4, to lower concentrations by 0.7 and 0.6 log units, respectively. Incubation of cultured or isolated human tracheal epithelial cells with LTD4 resulted in the formation of LTE4, which was completely blocked by pretreatment with L-cysteine (3 x 10(-3) M). The isolated and cultured human tracheal epithelial cells contained microsomal dipeptidase (MDP) activity. Immunohistochemical study indicated MDP protein was present in the epithelium and endothelial cells of submucosal microvessels in the human trachea. These results suggest that the epithelium modulates the contractile response to LTD4 in human trachea by dipeptidases degrading LTD4.

Rhinovirus infection of primary cultures of human tracheal epithelium: role of ICAM-1 and IL-1beta.

Exacerbations of asthma are often associated with respiratory infection caused by rhinoviruses. To study the effects of rhinovirus infection on respiratory epithelium, a primary target for respiratory viruses, human rhinovirus (HRV)-2 and HRV-14 were infected to primary cultures of human tracheal epithelial cells. Viral infection was confirmed by showing that viral titers of supernatants and lysates from infected cells increased with time and by polymerase chain reaction. HRV-2 and HRV-14 infections upregulated the expression of intercellular adhesion molecule-1 (ICAM-1) mRNA, the major rhinovirus receptor, on epithelial cells, and they increased the production of interleukin (IL)-1beta, IL-6, IL-8, and tumor necrosis factor (TNF)-alpha in supernatants. Antibodies to ICAM-1 inhibited HRV-14 infection of epithelial cells and decreased the production of cytokines after HRV-14 infection, but they did not alter HRV-2 infection-induced production ofcytokines. IL-1beta upregulated ICAM-1 mRNA expression and increased susceptibility to HRV-14 infection, whereas other cytokines failed to alter ICAM-1 mRNA expression. Furthermore, a neutralizing antibody to IL-1beta significantly decreased viral titers of supernatants and ICAM-1 mRNA expression after HRV-14 infection, but a neutralizing antibody to TNF-alpha was without effect. Immunohistochemical studies revealed that both HRV-14 infection and IL-1beta increased ICAM-1 expression on cultured epithelial cells. These findings imply that HRV-14 infection upregulated ICAM-1 expression on epithelial cells through increased production of IL-1beta, thereby increasing susceptibility to infection. These events may be important for amplification of airway inflammation after viral infection in asthma.

Effect of genetic risk factors and disease progression on the cerebrospinal fluid tau levels in Alzheimer's disease.

OBJECTIVE: This study was undertaken to gain insights into the clinical utility of measuring cerebrospinal fluid tau protein (CSF-tau) to aid in the diagnosis of Alzheimer's disease (AD). SETTING: AD patients from Tohoku University Hospital, Sendai Japan were sampled. SUBJECTS AND METHODS: CSF-tau levels were examined by sandwich enzyme-linked immunosorbent assay in a total of 62 patients carrying different alpha 1-antichymotrypsin (ACT) and presenilin-1 (PS-1) polymorphic alleles. Further, the CSF-tau levels were followed up on two occasions during the progression of the disease in 17 AD patients. RESULTS: There was no evident gradient for tau protein in CSF. Neither the ACT/A allele nor the PS-1/1 allele affected the CSF-tau levels. Although CSF-tau levels changed to a variable extent over time, the CSF-tau levels were significantly increased (P < .01) during the follow-up period. Three of the AD patients demonstrated decreasing values, whereas 14 patients showed increasing values. Finally, these temporal changes in CSF-tau levels were not influenced by the apolipoprotein E epsilon 4, ACT/A or PS-1/1 alleles during the progression of AD. CONCLUSION: Regardless of the mechanisms leading to the degeneration of neurons in AD, our findings provide further evidences that monitoring CSF-tau levels may provide useful information about AD irrespective of the background of genetic risks and disease progression.

Molecular defects of the coproporphyrinogen oxidase gene in hereditary coproporphyria.

Hereditary coproporphyria (HCP) is an acute hepatic porphyria, and is an autosomal dominant disorder but with a variable degree of clinical expression. Molecular cloning, sequencing and expression of the defective gene for coproporphyrinogen oxidase (CPO) in a patient with HCP were carried out. Enzyme assays revealed that CPO activity in EBV-transformed lymphoblastoid cells from the proband and one of her sisters was approximately 50% of normal. Nucleotide sequence analysis of CPO cDNAs isolated from the proband's cells demonstrated 3 base substitutions which accompanied 3 different amino acid substitutions. An A514-->C transition causing an Asn172-->His substitution occurred in one allele, while two other transitions, G265-->A and G580-->A, caused Gly89-->Ser and Val194-->Ile substitutions, respectively, in the other allele. The A514-->C and the G580-->A transitions were shown to be genetic polymorphisms. Transfection of CPO cDNA into E. coli demonstrated that cDNA with the G265-->A transition produced a protein with less than 5% of normal enzyme activity. These findings indicate that the G265-->A transition, involving the highly conserved glycine residue at the 89th position, is responsible for the CPO defect in the patient and accounts for the partial deficiency of CPO activity in this pedigree. This mutation is different from three other mutations reported in patients with HCP. Molecular defects in the porphyrias including HCP are highly heterogeneous.

[Rhinovirus infection and expression of adhesion molecules in human tracheal epithelium].

Rhinovirus infection has attracted attention because it can lead to acute exacerbations of chronic inflammatory airway diseases such as bronchial asthma and chronic bronchitis. We established a culture system and inoculated human rhinovirus to human tracheal epithelial cells, and found that infection was augmented by up-regulation of intercellular adhesion molecule-1, which is the receptor for this virus. We also found that human airway epithelial cells infected with rhinovirus were susceptible to a chemical oxidant (H2O2) released by inflammatory cells, which would contribute to acute exacerbations of inflammatory airway diseases. Finally, we found that anti-ICAM-1 antibodies or dexamethasone can inhibit the infectivity to rhinovirus by suppressing ICAM-1, and diminish susceptibility to oxidants in the cultured human tracheal epithelium.

[Tau protein in cerebrospinal fluid--a potential marker of Alzheimer's disease].

Levels of the microtubule-associated protein tau in cerebrospinal fluid (CSF-tau) were measured in samples from 87 patients with Alzheimer's disease (AD), 114 patients with non-AD neurological diseases, and 22 normal control subjects, by sandwich enzyme-linked immunosorbent assay. The CSF-tau level was significantly higher in patients with AD than in patients with non-AD neurological diseases and in controls. High CSF-tau levels were found irrespective of age at onset, apolipoprotein E genotype, clinical stage, and ethnic group. Western blots of AD CSF proteins revealed two to three immunoreactive bands with apparent molecular mass between 50 and 65 kDa, which is consistent with phosphorylated CSF-tau. These results suggest that CSF-tau reflects progressive accumulation of tau due to the progressive death of neurons in the AD brain. Assay of CSF-tau may prove to be a reliable diagnostic test for AD.

Role of Leukotriene-degrading enzymes in pulmonary response to antigen infusion in sensitized guinea pigs in vivo.

To determine the role of leukotriene (LT)-degrading enzymes in allergic reactions, we studied the effects of inhibitors of gamma-glutamyl transpeptidase (gamma-GTP) and dipeptidases on increases in pulmonary insufflation pressure (PIP) and vascular permeability induced by ovalbumin (OA) antigen in guinea pigs sensitized to OA antigen in vivo. Vascular permeability was assessed by the amount of extravasated Evans blue dye from the trachea, main bronchi, and segmental bronchi. An intravenous (i.v.) administration of OA antigen (200 micrograms/kg) caused increases in PIP and extravasated Evans blue dye, and OA antigen-induced effects were potentiated by gamma-GTP inhibitor L-serine borate (3 x 10(-5) M/kg, i.v.) (P < 0.05) and an inhibitor of dipeptidases, L-cysteine (3 x 10(-5) M/kg, i.v.) (P < 0.01). OA antigen-induced increases in PIP and Evans blue dye extravasation were in part inhibited by LT-receptor antagonist ONO-1078 (10(-4) M/kg, i.v.). Guinea-pig tracheal tissues contained gamma-GTP and microsomal dipeptidase activities. Histochemical and immunohistochemical studies indicate that gamma-GTP-like activity existed in the epithelium and smooth muscle, and an activity of microsomal dipeptidase was observed in the endothelial cells of microvessels and epithelium. These results suggest that LT-degrading enzymes have an important role in regulating allergic reaction in the airway in vivo.

Regulatory function of delta/YY-1 on the locus control region-like sequence of mouse glycophorin gene in erythroleukemia cells.

The far upstream region (-1.2-0.9 kilobase pairs) of the mouse glycophorin gene contains the locus control region (LCR)-like region, which acts as an erythroid-specific enhancer dependent on chromosomal integration in murine erythroleukemia (MEL) cells. In the present study, we demonstrated that this region binds six nuclear factors. The binding of GATA-1 to corresponding sites did not show any change before or after induction with dimethyl sulfoxide, but the binding of Spi-1/PU.l and an unidentified factor called glycophorin regulatory element binding factor (GRBF) showed a change during induction. While binding activity of Spi-l/PU.l dropped soon after induction, the GRBF activity increased after induction when expression of the glycophorin gene began. After identification of the consensus binding site of GRBF, we cloned cDNA for that factor by Southwestern method, and it was identified as a previously reported transcription factor, delta, a murine form of YY-l which is a versatile transcription factor. Mutation analysis in the delta/YY-1 binding site within the LCR-like region indicated that delta/YY-1 acts as a regulatory protein in combination with the E-box-binding protein that binds to the neighboring sequence.