Surfactant protein D inhibits activation of non-small cell lung cancer-associated mutant EGFR and affects clinical outcomes of patients.

Tyrosine kinase inhibitor (TKI)-sensitive and TKI-resistant mutations of epidermal growth factor receptor (EGFR) are associated with lung adenocarcinoma. EGFR mutants were previously shown to exhibit ligand-independent activation. We have previously demonstrated that pulmonary surfactant protein D (SP-D, SFTPD) suppressed wild-type EGFR signaling by blocking ligand binding to EGFR. We herein demonstrate that SFTPD downregulates ligand-independent signaling in cells harboring EGFR mutations such as TKI-sensitive exon 19 deletion (Ex19del) and L858R mutation as well as TKI-resistant T790M mutation, subsequently suppressing cellular growth and motility. Lectin blotting and ligand blotting in lung cancer cell lines suggested that EGFR mutants express oligomannose-type N-glycans and interact with SFTPD directly. Cross-linking assay indicated that SFTPD inhibits ligand-independent dimerization of EGFR mutants. We also demonstrated that SFTPD reduced dimerization-independent phosphorylation of Ex19del and T790M EGFR mutants using point mutations that disrupted the asymmetric dimer interface. It was confirmed that SFTPD augmented the viability-suppressing effects of EGFR-TKIs. Furthermore, retrospective analysis of 121 patients with lung adenocarcinoma to examine associations between serum SFTPD levels and clinical outcome indicated that in TKI-treated patients with lung cancer harboring EGFR mutations, including Ex19del or L858R, high serum SFTPD levels correlated with a lower number of distant metastases and prolonged overall survival and progression-free survival. These findings suggest that SFTPD downregulates both TKI-sensitive and -resistant EGFR mutant signaling, and SFTPD level is correlated with clinical outcome. These findings illustrate the use of serum SFTPD level as a potential marker to estimate the efficacy of EGFR-TKIs.

Surfactant protein D suppresses lung cancer progression by downregulation of epidermal growth factor signaling.

Surfactant protein D (SP-D) is a member of the collectin family that has an important role in maintaining pulmonary homeostasis. In this study, we demonstrated that SP-D inhibited the proliferation, migration and invasion of A549 human lung adenocarcinoma cells. We found that SP-D suppressed epidermal growth factor (EGF) signaling in A549 cells, H441 human lung adenocarcinoma cells and human EGF receptor (EGFR) stable expression CHO-K1 cells. A binding study using (125)I-EGF demonstrated that SP-D downregulated the binding of EGF to EGFR. A ligand blot indicated that SP-D bound to EGFR, and a lectin blot suggested that EGFR in A549 cells had both high-mannose type and complex type N-glycans. We purified the recombinant extracellular domain of EGFR (soluble EGFR=soluble EGFR (sEGFR)), and demonstrated that SP-D directly bound to sEGFR in a Ca(2+)-dependent manner. The binding of SP-D to sEGFR was suppressed by EDTA, mannose or N-glycopeptidase F treatment. Mass spectrometric analysis indicated that N-glycans in domain III of EGFR were of a high-mannose type. These data suggest that SP-D reduces EGF binding to EGFR through the interaction between the carbohydrate recognition domain of SP-D and N-glycans of EGFR, and downregulates EGF signaling. Our finding suggests the novel type of regulation system of EGF signaling involving lectin-to-carbohydrate interaction and downregulation of ligand binding.

Phasic synaptic incorporation of GluR2-lacking AMPA receptors at gonadotropin-releasing hormone neurons is involved in the generation of the luteinizing hormone surge in female rats.

Reproductive success depends on a robust and appropriately timed preovulatory luteinizing hormone (LH) surge, which is induced by the activation of gonadotropin-releasing hormone (GnRH) neurons in response to positive feedback from increasing estrogen levels. Here we document an increase in postsynaptic GluR2-lacking Ca2+ -permeable AMPA-type glutamate receptors (CP-AMPARs) at synapses on GnRH neurons on the day of proestrus in rats, coincident with the increase in estrogen levels. Functional blockade of CP-AMPARs depressed the synaptic responses only on the day of proestrus and concomitantly attenuated the LH surge. Thus, the phasic synaptic incorporation of postsynaptic CP-AMPARs on GnRH neurons is involved in the generation of the LH surge.

In vivo morphometric analysis of inflammatory condylar changes in rat temporomandibular joint.

OBJECTIVE: Injection of complete Freund's adjuvant (CFA) into the temporomandibular joint (TMJ) causes acute swelling around the joint and subsequent morphological alterations in the condyle. We aimed to evaluate changes in the three-dimensional architecture of the condyle induced with CFA. MATERIALS AND METHODS: The CFA was injected into the unilateral TMJ of rats and morphological changes in the condyle were assessed repeatedly for 14 days by in vivo micro-CT. RESULTS: Osseous abnormalities of condyle were first observed at 3-5 days after CFA injection on the tomographic images, and the condylar deformation became more obvious thereafter. Among 12 condyles examined at 14 days postinjection, osteophytosis was observed in all of the specimens and bone erosion coexisted in five condyles. None of the saline-treated condyles showed architectural changes. Significant changes were detected in the mesiolateral and rostrocaudal widths of the CFA-treated condyles at 10-14 days postinjection (P < 0.01). The extent of both condylar bone formation and resorption was greater in the CFA-injected TMJs than in saline-injected TMJs (P < 0.05). CONCLUSION: These results indicate that CFA causes dynamic morphological changes in the condyle and that our experimental approach will provide new insights into the subacute inflammatory processes in the TMJ.

JTT-130, a novel intestine-specific inhibitor of microsomal triglyceride transfer protein, ameliorates impaired glucose and lipid metabolism in Zucker diabetic fatty rats.

AIM: Microsomal triglyceride transfer protein (MTP) takes part in the mobilization of triglyceride-rich lipoproteins from enterocytes and hepatocytes. We investigated the effects of JTT-130, a novel intestine-specific MTP inhibitor, on impaired glucose and lipid metabolism in Zucker diabetic fatty (ZDF) rats. METHODS: Male ZDF rats were fed a regular powdered diet with or without JTT-130 as a food admixture (0.01-0.02%) for 6 weeks. Food intake, body weight, blood biochemical parameters, fecal lipid contents, hepatic lipid contents, tissue mRNA levels and glucose utilization in adipose tissues were assessed. An intraperitoneal glucose tolerance test (IPGTT) and histological analysis of the pancreas were performed. RESULTS: JTT-130 treatment decreased food intake, glycated hemoglobin, plasma levels of glucose, triglycerides and total cholesterol, hepatic levels of triglycerides and cholesterol and hepatic mRNA levels of glucose-6-phosphatase, phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase. JTT-130 treatment increased fecal levels of free fatty acids and cholesterol, plasma levels of glucagon-like peptide-1 and peptide YY, mRNA levels of glucose transporter 4 (GLUT4) and lipoprotein lipase in adipose tissues and GLUT4 in muscle and glucose utilization in adipose tissues. Plasma insulin decreased after 2 weeks and increased after 4 weeks of JTT-130 treatment. Plasma glucose in the JTT-130-treated rats was lower with higher plasma insulin than in the control rats during the IPGTT. The islets of the JTT-130-treated rats were larger and contained more insulin than those of the control rats. CONCLUSIONS: JTT-130 ameliorates impaired glucose and lipid metabolism in the ZDF rats thereby suggesting that JTT-130 could be useful for prevention and treatment of type 2 diabetes.

JTT-130, a novel intestine-specific inhibitor of microsomal triglyceride transfer protein, suppresses high fat diet-induced obesity and glucose intolerance in Sprague-Dawley rats.

AIM: Microsomal triglyceride transfer protein (MTP) takes part in the mobilization and secretion of triglyceride-rich lipoproteins from enterocytes and hepatocytes. We investigated the effects of JTT-130, a novel intestine-specific MTP inhibitor, on high fat diet-induced obesity and glucose intolerance. METHODS: Male Sprague-Dawley rats were fed a 3.1% fat diet or a 35% fat diet with or without JTT-130 as a food admixture (0.029%). Food intake, body weight, abdominal fat, hepatic triglyceride, faecal free fatty acids and plasma levels of glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) were assessed. Plasma levels of glucose and insulin were measured during intraperitoneal glucose tolerance tests. In addition, indirect calorimetry was performed on rats fed with a 35% fat diet. RESULTS: JTT-130 treatment decreased body weights, abdominal fat and hepatic triglyceride with suppression of food intake and elevation of faecal free fatty acids and plasma GLP-1 and PYY levels in rats fed with the 35% fat diet, whereas no significant effects on these parameters except for increased faecal free fatty acids were observed in rats fed with the 3.1% fat diet. JTT-130 treatment decreased plasma levels of glucose and insulin during intraperitoneal glucose tolerance tests on rats fed with the 35% fat diet, but not on rats fed with the 3.1% fat diet. JTT-130-treated rats showed increased O(2) consumption and CO(2) production on a 35% fat diet. CONCLUSIONS: JTT-130 suppresses high fat diet-induced obesity and glucose intolerance with suppression of food intake and fat absorption and could be useful for prevention and treatment of obesity and obesity-related insulin resistance.

Japanese-style intensive medical care improves prognosis for acute liver failure and the perioperative management of liver transplantation.

The Japanese style of intensive medical care for acute liver failure has yielded high survival rates. The care system comprises artificial liver support (ALS) together with treatment for the underlying disease. Plasma exchange in combination with high-volume hemodiafiltration using an high performance membrane has become the standard ALS system. It is safe, efficiently removing more low and middle molecular weight toxic substances than other methods because of the large volumes of buffer (more than 200 L per session), resulting in recovery from coma in patients with severe fulminant hepatitis, a status comparable with the ahepatic state. This ALS is therefore an effective tool to sustain patients with fulminant hepatitis in a favorable condition until liver function recovers or liver transplantation becomes available. The accompanying treatment for underlying disease serves to limit the liver destruction that hampers regeneration. The treatment has remarkably improved the prognosis for patients with subacute types of fulminant hepatitis, which generally carry a less favorable prognosis than the acute type. This treatment system thus provides more time for physicians to assess the indications for liver transplantation as well as giving the patient a greater chance of undergoing transplantation.

Increasing antiviral activity of surfactant protein d trimers by introducing residues from bovine serum collectins: dissociation of mannan-binding and antiviral activity.

Collectins contribute to host defence through interactions with glycoconjugates on pathogen surfaces. We have prepared recombinant trimeric neck and carbohydrate recognition domains (NCRD) of collectins, and we now show that the NCRD of bovine conglutinin and CL-46 (like that of CL-43) have greater intrinsic antiviral activity for influenza A virus (IAV) than the human SP-D NCRD (hSP-D-NCRD). The three serum collectins differ from SP-D by having insertions adjacent to amino acid 325 and substitution of hydrophobic residues for arginine 343. We previously showed that a three amino acid (RAK) insertion, as found in CL-43, increases antiviral activity and mannan-binding activity of the hSP-D-NCRD, while the substitution of valine at 343, as in conglutinin, more strongly increased these activities. Mannan-binding activity of collectins has been considered to predict for ability to bind to high mannose glycans on viruses or other pathogens. We now show, however, that combined mutants containing the RAK insertion and R343V or R343I substitutions have greatly increased mannan-binding ability, but lower IAV binding or inhibiting activity than mutants containing R343V or R343I substitutions only. These findings indicate differences in the recognition of glycan structures of mannan and IAV by the NCRD and emphasize the importance of the flanking sequences in determining the differing interactions of human SP-D and bovine serum collectins with mannose-rich glycoconjugates on IAV and other pathogens. Of interest, we show conservation of some monoclonal antibody-binding epitopes between bovine collectin NCRD and hSP-D, suggesting shared structural motifs.

Effect of alendronate on bone metabolic indices and bone mineral density in patients treated with high-dose glucocorticoid: a prospective study.

SUMMARY: This prospective study, in the very early phase after initiation of glucocorticoid (GC) treatment, showed that alendronate was effective in suppressing accelerated bone resorption and subsequent decrease in bone mineral density (BMD) at the lumbar spine of patients with high-dose GC treatment. INTRODUCTION: How bisphosphonates affect bone metabolism and BMD of patients with high-dose GC in the early phase, especially within 1 month is unclear. METHODS: We examined the prospective effects of daily 5 mg alendronate on bone metabolism and BMD in 20 patients with high-dose GC (at least 40 mg prednisolone/day) and compared them to 34 high-dose GC-treated patients without alendronate. RESULTS: Serum levels of calcium decreased at day 28 in the alendronate group. Urinary calcium excretion significantly increased after day 7 in both groups. The increase in serum parathyroid hormone (PTH) level at day 7 in the control group was not observed in the alendronate group, but PTH levels increased at day 28 and month 3 in the alendronate group. As for the bone turnover markers, the serum osteocalcin level decreased in both alendronate and control groups, but serum bone-type alkaline phosphatase levels did not show significant changes. Although the urinary type I collagen cross-linked N-telopeptide (NTX) level showed significant increases on days 7 and 28 in the control group; such early increases in urinary NTX were not observed in the alendronate group. Thereafter, the urinary NTX levels fell slowly in the alendronate group significantly. BMD at the lumbar spine significantly decreased from month 1 in the control group, whereas in the alendronate group, BMD at the lumbar spine maintained almost the same level at all time points observed. CONCLUSION: Alendronate was effective in suppressing bone resorption and subsequent BMD decrease at the lumbar spine in patients with high-dose GC treatment.

Diagnostic accuracy of microcomputed tomography for osseous abnormalities in the rat temporomandibular joint condyle.

OBJECTIVES: Our aim was to investigate the diagnostic accuracy of in vivo micro-CT for osseous abnormalities of the rat temporomandibular joint (TMJ) condyle, using macroscopic observations as the "gold standard". METHODS: A 30 TMJ arthritis model was prepared by injecting inflammatory complete Freund's adjuvant (CFA) into one side of the TMJ cavities of rats. The TMJ condyles were then imaged using micro-CT. The samples were macroscopically evaluated for osseous abnormalities, including erosions, osteophytes, flattening and concavity. The micro-CT images were independently assessed for abnormalities using the same criteria. Images in three planes were produced using the micro-XYZ technique with the micro-CT equipment. RESULTS: According to the macroscopic observations, 26 of the 60 rat condyles showed osseous abnormalities. The micro-XYZ images detected abnormalities in 25 of the condyles. The condyle diagnostic accuracy of micro-CT was 0.98, the sensitivity was 0.96 and the specificity was 1.0. CONCLUSIONS: Good diagnostic results were obtained using micro-CT. It is therefore an effective technique for the evaluation of osseous abnormalities in the rat TMJ condyle.

Relationship between patient characteristics, mandibular head morphology and thickness of the roof of the glenoid fossa in symptomatic temporomandibular joints.

OBJECTIVE: The purpose of this clinical study was to investigate the minimum thickness of the roof of the glenoid fossa (RGF) of grossly normal temporomandibular joints (TMJ) and to correlate this with patient gender, age and the morphological classification of the mandibular head. METHODS: The study was performed on 191 TMJs from 109 patients (25 male and 84 female, age range 3-79 years, mean age 28.1 years) who visited Nihon University Dental Hospital, Japan with suspected TMJ disorders. The patients underwent cone beam computed tomography (3DX CT) to enable observation of the morphological features of the mandibular head. The minimum thickness of the RGF was measured using frontal section images acquired by CT. The morphology of the mandibular heads was classified according to the method of Yale and colleagues. Mean linear measurements were used for statistical analyses of patient gender, age and mandibular head morphology. RESULTS: The average minimum thickness of the RGF was 0.79 mm. No significant difference in thickness was found between male and female patients. In addition, no differences were recorded as a result of variation in age or mandibular head morphology. CONCLUSIONS: These results indicate that RGF thickness is not significantly correlated with gender, age, or mandibular head morphology, at least in this cohort of patients.

Reproducibility and accuracy of measuring unerupted teeth using limited cone beam X-ray CT.

OBJECTIVE: The purpose of this investigation was to determine the reproducibility among observers and accuracy of the measurement of the tooth crown width of unerupted teeth using limited area cone beam X-ray CT. METHODS: 3DX multi-image micro-CT (3DX, Morita Co., Kyoto, Japan) images of impacted supernumerary teeth in the median maxillary region taken prior to extraction were used for the samples. The width of the tooth on the 3DX image was measured five times by five individual observers. Significant differences in values among the observers in the measurement were determined by one-way analysis of variance for examining reproducibility. The measurement results of the ten samples on 3DX images were compared with the laboratory measurements using a three-dimensional co-ordinate measuring apparatus, using the Wilcoxon signed-rank sum test. RESULTS: There was no significant difference among the observers in the measurement (P>0.05). The measurement results shown on 3DX images were significantly larger than those of the laboratory measurements (P<0.05). The mean difference was +0.088 mm. CONCLUSIONS: 3DX has high reproducibility for measuring the tooth crown width of unerupted teeth. While 3DX measurement values were larger than the laboratory measurements, the difference is clinically insignificant.

Structural analysis of leucine-rich-repeat variants in proteins associated with human diseases.

A number of human diseases have been shown to be associated with mutation in the genes encoding leucine-rich-repeat (LRR)-containing proteins. They include 16 different LRR proteins. Mutations of these proteins are associated with 19 human diseases. The mutations occur frequently within the LRR domains as well as their neighboring domains, including cysteine clusters. Here, based on the sequence analysis of the LRR domains and the known structure of LRR proteins, we describe some features of different sequence variants and discuss their adverse effects. The mutations in the cysteine clusters, which preclude the formation of sulfide bridges or lead to a wrong paring of cysteines in extracellular proteins or extracellular domains, occur with high frequency. In contrast, missense mutations at some specific positions in LRRs are very rare or are not observed at all.

Effects of ageing and smoking on SP-A and SP-D levels in bronchoalveolar lavage fluid.

Surfactant protein (SP)-A and SP-D are collagen-like glycoproteins that are synthesised in the distal pulmonary epithelium. This study examined the effects of ageing and long-term smoking on SP-A and SP-D in the lungs. The possible links to the development of pulmonary emphysema were also investigated. Sequential lavage was performed in young and middle-aged or elderly nonsmokers and asymptomatic current smokers with various smoking histories. Middle-aged or elderly smokers were further categorised according to the presence of emphysema by high-resolution computed tomography. Levels of SP-A and SP-D in bronchial lavage (BL) fluid and in bronchoalveolar lavage (BAL) fluid were quantified by ELISA. Significant decreases in SP-A were seen with age in nonsmokers in BL fluid, but not in BAL fluid. Middle-aged or elderly smokers with emphysema had lower levels of SP-A in both BL and BAL fluids when compared with young subjects, and in BL fluid when compared with middle-aged or elderly smokers without emphysema. SP-D did not change with age alone, however, it was decreased in middle-aged or elderly smokers when compared with similarly aged nonsmokers. In conclusion, surfactant protein-A may decrease with age alone or due to the cumulative effects of long-term smoking and development of emphysema, while surfactant protein-D decreases due to long-term smoking.

DNA sequence and comparative analysis of chimpanzee chromosome 22.

Human-chimpanzee comparative genome research is essential for narrowing down genetic changes involved in the acquisition of unique human features, such as highly developed cognitive functions, bipedalism or the use of complex language. Here, we report the high-quality DNA sequence of 33.3 megabases of chimpanzee chromosome 22. By comparing the whole sequence with the human counterpart, chromosome 21, we found that 1.44% of the chromosome consists of single-base substitutions in addition to nearly 68,000 insertions or deletions. These differences are sufficient to generate changes in most of the proteins. Indeed, 83% of the 231 coding sequences, including functionally important genes, show differences at the amino acid sequence level. Furthermore, we demonstrate different expansion of particular subfamilies of retrotransposons between the lineages, suggesting different impacts of retrotranspositions on human and chimpanzee evolution. The genomic changes after speciation and their biological consequences seem more complex than originally hypothesized.