Fibronectin plays a major role in hypoxia-induced lenvatinib resistance in hepatocellular carcinoma PLC/PRF/5 cells.

Resistance to lenvatinib mesylate (LEN), a systemic chemotherapy that can be administered orally, has been a major issue for treatment of hepatocellular carcinoma (HCC). Although HCC is the tumor that most exhibits intratumoral hypoxia, which has been shown to be involved in the development of treatment resistance, there are no reports of LEN resistance in HCC treatment under hypoxia. The purpose of our study was to elucidate the mechanism of treatment resistance to LEN under hypoxia using HCC cell lines. We confirmed LEN resistance under hypoxic conditions in HCC cell lines. There was a significant increase in the IC50 value of PLC/PRF/5 cells from 13.0±0.8 µM in normoxia to 21.3±1.1 µM in hypoxia, but in HepG2 cells, the increase was not significant. To elucidate the LEN resistance mechanism of PLC/PRF/5 cells under hypoxia, we performed microarray analysis and extracted genes that are thought to be related to this mechanism. Furthermore, in-silico analysis confirmed significant changes in the extracellular matrix, and among them, FN1 encoding fibronectin was determined as the hub of the gene cluster. The expression of fibronectin in PLC/PRF/5 cells examined with immunofluorescence staining was significantly elevated in and outside of cells under hypoxia, and tended to decrease when cells were exposed to LEN under normoxia. Furthermore, the fibronectin concentration in the culture solution of PLC/PRF/5 cells examined by ELISA was 2.3 times higher under hypoxia than under normoxia under LEN(-) conditions, and 1.6 times higher under hypoxia than under normoxia under LEN(+) conditions. It is assumed that in PLC/PRF/5 cells, fibronectin is probably suppressed as an indirect effect of LEN under normoxia, but transcription factors such as HIF-1α are induced under hypoxia, thus enhancing the production of fibronectin and attenuating the effect of LEN, resulting in drug resistance. This behavior of fibronectin with LEN exposure under hypoxia is probably specific to PLC/PRF/5 cells. Further studies should verify the combined effective inhibition of fibronectin and the MAPK pathway as a promising therapeutic strategy to enhance the value of LEN in HCC treatment.

The expression of miR-125b-5p is increased in the serum of patients with chronic hepatitis B infection and inhibits the detection of hepatitis B virus surface antigen.

MicroRNAs were first discovered as small endogenous RNA molecules and some viruses have been reported to interact with host miRNAs. By investigating miRNA expression in serum derived from HBV-infected patients, we have clarified the relationship between miRNA expression and chronic HBV infection. Additionally, we demonstrate the use of miRNAs as both novel biomarkers and new therapies against HBV. We included the sera of 20 patients with chronic HBV infection, sera of 20 patients with HCV infection and sera of 10 healthy controls in this study. The miRNA libraries were sequenced using a 32-mer single end sequence. The validation study of circulating miRNA in serum was conducted by qRT-PCR. The HBV genomic regions of genotype B and genotype C that were speculated to be targeted by miRNA were constructed using complementary oligonucleotides in the vectors. Reporter assays were performed 48 h after transfection. The expression levels of 21 miRNAs were found to be differentially expressed in the three groups. 10 miRNAs (hsa-miR-100-5p, miR-125b-5p, miR-193b-3p, miR-194-3p, miR-30a-3p, miR-30c-2-3p, miR-3591-5p, miR-4709-3p, miR-574-3p and miR-99a-5p) were found to be upregulated in CH-B by deep sequence analysis. The computer analysis showed that two regions of HBsAg are potential targets of miR-125b-5p and miR-30c-2-3p and that these miRNAs may downregulate the expression of HBV-S. The HBV genotype C segment speculated to be targeted by hsa-miR-125b-5p significantly decreased the expression of the reporter. This study indicated that expression of miR-125b-5p was related to the etiology of chronic hepatitis B infection and regulated the expression of HBsAg.

Vicia villosa B4 lectin inhibits nucleotide pyrophosphatase activity toward UDP-GalNAc specifically.

Plant seed lectins play a defense role against plant-eating animals. Here, GalNAc-specific Vicia villosa B4 lectin was found to inhibit hydrolysis of UDP-GalNAc by animal nucleotide pyrophosphatases, which are suggested to regulate local levels of nucleotide sugars in cells. Inhibition was marked at low concentrations of UDP-GalNAc, and was reversed largely by the addition of GalNAc to the reaction mixture. In contrast, lectin inhibited enzymatic hydrolysis of other nucleotide sugars, such as UDP-Gal and UDP-GlcNAc, only to a small extent, and GalNAc did not affect such an inhibition. The binding constant of the lectin for UDP-GalNAc was as high as 2.8 x 10(5) M-1 at 4 degrees C, whereas that for GalNAcalpha-1-phosphate was 1.3 x 10(5) M-1. These findings indicate that lectin inhibition of pyrophosphatase activity toward low concentrations of UDP-GalNAc arises mainly from competition between lectin and enzyme molecules for UDP-GalNAc. This type of inhibition was also observed to a lesser extent with GalNAc-specific Wistaria floribunda lectin, but not apparently with GalNAc-specific soybean or Dolichos biflorus lectin. Thus, V. villosa B4 lectin shows unique binding specificity for UDP-GalNAc and has the capacity to modulate UDP-GalNAc metabolism in animal cells.

Purification and characterization of N-acetyl-D-galactosamine-binding lectin from Falcata japonica.

An N-acetyl-D-galactosamine-binding lectin from Falcata japonica seeds was purified by affinity column chromatography of N-acetyl-D-galactosamine coupled to epoxy-activated Sepharose 6B. A 1000-fold purification of lectin was obtained from the crude extracts. The purified lectin agglutinated blood group A red cells, but neither blood group B nor O red cells. Polyacrylamide gel electrophoresis of the lectin showed one diffuse band. Molecular weights of 125,000 and 117,000 were estimated by gel filtration and ultracentrifugal analysis, respectively. SDS-polyacrylamide gel electrophoresis of the lectin also showed a single band which has a molecular weight of 34,000. Therefore, the lectin molecule was estimated to be a tetramer composed of four identical non-covalently bound subunits. F. japonica lectin was a glycoprotein containing 5% total carbohydrate, and the amino acid composition was characterized by a high content of aspartic acid, serine and glycine, a low content of methionine and the absence of cysteine.

Identification and purification of a novel phospholipid/ganglioside-binding protein in rabbit serum.

We have isolated a novel phospholipid/ganglioside-binding protein from rabbit sera or platelet-free plasma. Using an affinity chromatography of a commercial gel (Sephacryl S-series gel, Pharmacia) column and a preparative polyacrylamide gel electrophoresis, the protein can be easily purified. The protein agglutinates human red cells irrespective of the ABO blood types, and its hemagglutination reaction is specifically inhibited by some phospholipids (phosphatidylserine and phosphatidylglycerol) and ganglioside (N-acetylneuraminyl-galactosylglucosyl ceramide, GM3). The hemagglutination and its inhibition reactions are independent on any divalent cations (Ca2+, Mg2+, Mn2+, Ni2+). The protein seems to be assembled as multimers of disulfide-bonded molecular of 86 kDa and 59 kDa subunits.

Inflamed site-specific gene delivery using bone marrow-derived CD11b+CD18+ vehicle cells in mice.

We report a novel technique that may allow site-specific gene delivery into inflamed tissues. Bone marrow cells from DBA/2 mice were incubated for 7 days in L-929 cell-conditioned medium containing elements that favor the development of mononuclear cells, such as colony-stimulating factors. Flow cytometric analysis revealed that 99.1 +/- 0.9% of the subcloned cells were positive for CD11b and CD18, both of which are ligands of the intercellular adhesion molecule 1 (ICAM-1). These vehicle cells were labeled with a fluorescent lipophilic probe and returned intravenously to the DBA/2 mice. The mice then received, for 1 week, intraperitoneal injections of either lipopolysaccharide (LPS) to enhance ICAM-1 expression in the glomerulus, or saline as a control. In the LPS-treated mice, labeled vehicle cells were detected within the glomerulus cross-section (gcs) 24 hr after the first injection (0.73 +/- 0.10/gcs). The number of labeled vehicle cells within the glomerulus gradually increased for 1 week (1.47 +/- 0.19/gcs) and decreased after discontinuation of the LPS injections. However, in the saline-treated control group, only a negligible number of vehicle cells could be detected in the glomerulus (0.05 +/- 0.03/gcs). A second administration of LPS 4 weeks after injection of the vehicle cells was also able to promote accumulation in the glomerulus. Furthermore, immunohistochemical analysis revealed that the kinetics of the vehicle cell recruitment into the glomerulus corresponded to the level of ICAM-1 expression. On the assumption that the LPS-induced ICAM-1 expression may regulate the site and timing of the delivery of vehicle cells into the glomerulus, vehicle cells were transduced with human glucocerebrosidase (GC) gene, using an adenovirus vector, and reintroduced into the mice. The basal expression of GC gene in the isolated glomeruli of vehicle cell-treated mice rose by 1.7-fold compared with endogenous activity, whereas the GC activity was enhanced 3.2-fold by LPS treatment. Polymerase chain reaction designed to detect human GC-specific sequence revealed that isolated glomeruli of vehicle cell-treated mice contained exclusively the vehicle cell-oriented GC. This indicates that vehicle cells can be used to carry a certain gene to a specific inflamed site. Injection of vehicle cells, with or without LPS, had small effect on urinary protein excretion or serum creatinine levels. These findings suggest that our novel method allows site-specific gene delivery into inflamed glomeruli through interaction of adhesion molecules.

Prophylaxis of antibody-induced acute glomerulonephritis with genetically modified bone marrow-derived vehicle cells.

Glomerulonephritis is an inflammatory disease of the renal glomerulus, which often progresses either slowly or rapidly, ending in renal death despite the availability of various antiinflammatory drugs. Gene therapy may be a promising method of suppressing the progression of glomerulonephritis through the blockage of key inflammatory molecule(s). However, the difficulty of local gene delivery into the glomerulus has made the clinical use of gene therapy difficult. As a solution to this issue, we applied a novel ex vivo technique that may allow site-specific gene delivery into the inflamed site and thus suppress local inflammation in the glomerulus, and examined the feasibility of this system as a prophylaxis of glomerulonephritis. The gene encoding the antiinflammatory cytokine interleukin 1 receptor antagonist (IL-1ra) was delivered into animal models of inflamed glomeruli evoked by anti-glomerular basement membrane antibody; this animal model is an analog of the human Goodpasture syndrome. Vehicle cells did indeed accumulate in the glomeruli on the induction of nephritis and were confirmed to secrete recombinant IL-1ra. Renal functions as well as morphology were preserved by this intervention for up to 14 days after IL-1ra introduction. These data demonstrate the possible application of gene therapy for acute glomerulonephritis. A gene encoding an antiinflammatory molecule, IL-1 receptor antagonist, was delivered into inflamed glomeruli, using a technique that may allow site-specific gene delivery into inflamed tissues. The progression of experimental acute glomerulonephritis was effectively suppressed by this intervention for at least 14 days after gene introduction. This success may strengthen the rationale for gene therapy in the treatment of inflammatory diseases such as glomerulonephritis.

High gain efficiency amplifier based on an erbium doped aluminosilicate holey fiber.

We demonstrate an optical amplifier based on an erbium doped holey fiber (EDHF) with a small core. Owing to the high NA, which is readily achievable using holey fiber technology, and the tight physical confinement of the erbium ions, we show that it is possible to achieve an internal gain efficiency of >8.5dB/mW using an aluminosilicate based glass within the core. The dependence of the gain and noise figure performance with respect to fiber length and wavelength are experimentally characterized.

Identification and characterization of UDP-GalNAc: NeuAc alpha 2-3Gal beta 1-4Glc(NAc) beta 1-4(GalNAc to Gal)N-acetylgalactosaminyltransferase in human blood plasma.

Normal human plasma was found to contain beta 1-4N-acetylgalactosaminyltransferase catalyzing the transfer of N-acetylgalactosamine from UDP-GalNAc to 3'-sialyl-lactose, NeuAc alpha 2-3Gal beta 1-4Glc. The transferred N-acetylgalactosaminyl residue was cleaved from the desialylated reaction product by the beta-N-acetylhexosaminidase from jack beans. Methylation and hydrolysis of the desialylated reaction product yielded only 2,3,6-tri-O-methylgalactose and 2,3,6-tri-O-methylglucose as neutral sugars, indicating that the N-acetylgalactosaminyl residue was introduced at position C-4 of the galactosyl residue of 3'-sialyllactose. The enzyme required Mn2+ ions for its activity and showed a pH optimum between 6.5 and 8.5. By using a wide variety of oligosaccharides and glycoconjugates, the acceptor specificity of the beta 1-4N-acetylgalactosaminyltransferase was investigated. No detectable amount of N-acetylgalactosamine was transferred to either 6'-sialyllactose or lactose. The enzyme did not act on ganglioside GM3, NeuAc alpha 2-3Gal beta 1-4Glc-ceramide, suggesting that the hydrophobic ceramide portion of GM3 interferes with the enzyme reaction. On the other hand, glycoproteins carrying terminal NeuAc alpha 2-3Gal beta 1-4GlcNAc structures on their N-linked oligosaccharide chains, e.g. Tamm-Horsfall glycoprotein, were efficient acceptors.

Human serum contains N-acetyllactosamine: beta 1-3 N-acetylglucosaminyltransferase activity.

Human serum was shown to contain N-acetyllactosamine: N-acetylglucosaminyltransferase activity. The reaction product was hydrolyzed by beta-N-acetylglucosaminidase and released [14C]N-acetylglucosamine, indicating that the N-acetylglucosaminyl residue was beta-linked to N-acetyllactosamine. Methylation and hydrolysis of the reaction product yielded 2,4,6-trimethyl[3H]galactose, indicating that the N-acetylglucosaminyl residue was introduced at position C-3 of the terminal galactose of N-acetyllactosamine. In our experiments, 2,3,4-trimethyl[3H]galactose was not detected. Substrate competition studies between N-acetyllactosamine and lactose showed that this enzyme also catalyzed the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to lactose. Since the Km value for N-acetyllactosamine, which was 7.0 mM, was approximately a fourth of that for lactose (29.8 mM), N-acetyllactosamine was more effective than lactose as an acceptor.

Clinical course of acute middle cerebral artery occlusion.

Knowledge of the natural course of stroke patients has become increasingly important since new therapeutic methods have been proposed for patients with cerebral infarction in the acute stage. In order to clarify the acute stage of this disease, 188 patients admitted within 24 hours after onset of middle cerebral artery (MCA) occlusion were followed for 2 months, and data relating to mortality and changes in disturbances of consciousness and motor function were investigated. It was shown that the prognosis for MCA occlusion cases is poor, and about 80% of these patients are unable to return to their previous lifestyle. The level of consciousness in the acute stage is a good index for estimating the patients' quality and time of survival, and motor function in the acute stage is a good indicator of functional recovery. Thus, when evaluating the effectiveness of a new therapy for cerebral infarction, rapid improvement in the acute stage before and after treatment should be carefully noted.

Systemic lupus erythematosus complicated by disseminated intravascular coagulation: the role of serum soluble cell surface markers.

We describe a 31-year-old Japanese female patient with systemic lupus erythematosus (SLE), who developed disseminated intravascular coagulation (DIC), fever, erythema on the hands, and aphthous stomatitis despite the absence of circulating anticoagulant. Since no other cause for DIC besides SLE could be demonstrated, she was treated with prednisolone and anticoagulants, which rapidly corrected the DIC as well as the other manifestations of SLE. During the episode of DIC, elevated serum anti-DNA antibody titers and decreased serum complement concentrations were not observed. In contrast, the serum concentration of soluble CD8 (sCD8) paralleled SLE disease activity. In addition, the concentration of plasma thrombomodulin was also increased. These observations suggest that the serum concentration of sCD8 is related to the clinical aspects of SLE, and that vasculitis might contribute to the development of SLE-associated DIC.

Clinical and immunomodulatory effects of fun-boi, an herbal medicine, on collagen-induced arthritis in vivo.

OBJECTIVE: Crude preparations of Fun-boi (Stephania tetrandra), a traditional antirheumatic herb, have been reported to have immunomodulatory effects on both cell-mediated and humoral immunity in vitro, but little is known about the mode of action in vivo. The objective of this study was therefore to evaluate the efficacy of Fun-boi against arthritis and its effect on the immune system. METHODS: Mice were divided into the following 3 groups of 7 mice each: 1) a normal group, not treated to cause collagen-induced arthritis (CIA), received water orally; 2) a control group with CIA received water orally; and 3) the Fun-boi group with CIA, received Fun-boi (3 mg/g body weight/day) orally. We analyzed the arthritis score, the serum anti-type II collagen (CII) antibody level, and the percentage of the following lymphocyte subsets from lymphoid organs: B220, CD3/CD4, CD3/CD8 and CD40L/CD4 lymphocytes from blood or lymph nodes; and CD4-CD8-, CD4+CD8+, CD4+CD8- and CD4-CD8+ from the thymus. RESULTS: Fun-boi therapy markedly reduced the severity of arthritis (p < 0.001) and tended to reduce the serum anti-CII antibody level (p = 0.06). Whereas CII immunization of DBA/1J mice caused a significant redistribution of CD3/CD8 lymphocytes from blood or lymph nodes, Fun-boi therapy caused significant normalization of the same types of lymphocyte subsets from lymph nodes, but did not affect the CD4 or CD4/CD40L lymphocyte subsets. CONCLUSION: These results demonstrate that Fun-boi therapy exerts therapeutic effects in CIA mice, possibly by causing immunomodulatory effects at specific sites.

Radiological manifestations of oesophageal involvement in acanthosis nigricans.

Radiologic features of two cases of acanthosis nigricans with oesophageal involvement are reported. The first case demonstrated diffuse, granular shadows throughout the oesophagus which were difficult to differentiate from oesophageal moniliasis. Another case showed many discrete and tiny elevations resembling pseudopolyposis of the colon. Emphasis is placed on radiological differential diagnosis from moniliasis and leukoplakia of the oesophagus.

A TEM study of long-period mica polytypes: determination of the stacking sequence of oxybiotite by means of atomic resolution images and Periodic Intensity Distribution (PID).

In this study it is shown that the stacking sequences in long-period mica polytypes (including a rare example of a mixed-rotation polytype, with a 36-layer long periodicity) can be unambiguously determined using atomic resolution images recorded down two zone axes separated by 30 degrees (e.g. [100] and [31;0]) at the same area. Uniformity of these stacking sequences in a large area is further confirmed by the correspondence between computed and observed Periodic Intensity Distributions (PIDs) in electron diffraction patterns. The appearance of long-period polytypes containing layers with different orientation parity may be explained by the coalescence of two small crystals during crystal growth.

Radioimmunodetection of cancer of gastrointestinal tract and liver metastasis with I-131 anti-CEA and I-131 anti-CA19-9 monoclonal antibody cocktail (IMACIS-1).

We evaluated the intravenous infusion of a cocktail of I-131 anti-CEA and anti-CA19-9 monoclonal antibody F(ab')2 (IMACIS-1) in patients with gastrointestinal neoplasm and liver metastases in order to assess its efficacy in detecting the presence of cancer. Seven patients with primary or recurrent gastrointestinal cancer in whom liver metastases were also detected were studied. Accumulation of radioactivity in the primary tumor was seen in only one patient. Visualization of the liver metastases was achieved in all patients. Thus detection of liver metastasis was better than in primary or recurrent tumors. While tumor visualization was most often seen in the 3 day image, optimal visualization of the tumor was seen at 5-7 days. There was no correlation between the serum concentration of CEA or CA19-9 and the visualization of tumors. Serum kinetics of I-131 IMACIS-1 showed biexponential components with a 1st phase T1/2 of 5.0 hours and 2nd phase T1/2 of 34.7 hours. The mean whole body (I-131) half-life determined from the whole-body scans was 1.95 days. The mean urinary excretion of I-131 in 7 days was 85%. This value agreed closely with total radioactivity retention detected by scanning. This series of studies demonstrated the potential utility of a cocktail of antibodies consisting of an anti-CEA and an anti-CA19-9 monoclonal F(ab')2.

Multiple gastric diverticulosis: report of a case.

A case of multiple gastric diverticula with pyloric stenosis is reported here. More than 30 diverticula were located throughout the stomach except in the pyloric antrum. The diverticula were divided into two types according to shape: shallow depression and mushroom-like outpouching. This case has the greatest number of gastric diverticula ever reported.

Secondary esophageal carcinoma: report of two cases showing intraluminal tumor.

Two cases of secondary esophageal carcinoma showing intraluminal tumor are reported here. The one from pancreatic carcinoma showed a solitary sessile tumor which was misdiagnosed as primary adenocarcinoma of the esophagus. The other from epipharyngeal cancer revealed small multiple nodules of the mid-esophagus. When an esophageal tumor is noted, the possibility of esophageal metastasis should be included in the differential diagnosis especially when tumors are multiple and are accompanied with central depression or have a tendency of intramural filling defect.

A rare case of desquamative esophagitis occurring during radiotherapy for esophageal carcinoma.

With the recent progress and increase in availability of fiberendoscopy, acute esophagitis is now much more frequently detected than before. Desquamative esophagitis is a rare form of esophagitis classified as acute esophagitis, but it has come to be reported more frequently in recent years. However, since desquamation seemed to have occurred in relation with the administration of radiotherapy in the present case, this brings to two the total number of cases with radiological involvement, the other case reported by Itai being the first in the world. This is the second report of desquamative esophagitis occurring during radiotherapy for esophageal carcinoma.

A case of the incomplete double aortic arch diagnosed in adulthood by MR imaging.

Double aortic arch is a rare vascular anomaly, particularly in adults. Most of patients were infants or children. In this report we describe a case of the incomplete double aortic arch in a 55 years old woman complaining of esophageal symptoms was found by esophagography and examined by MR imaging. This anomaly had double aortic arch with partial atresia of the left arch. The right common carotid artery (RCCA) and the right subclavian artery (RSA) arose from the large right aortic arch (RAA). The left common carotid artery (LCCA) and subclavian artery (LSA) arose from the left aortic arch (LAA). Aortic diverticulum was detected at the interior site of the descending aorta connected with the left subclavian artery (LSA). The mediastinal organs were surrounded by the vascular ring (RAA, LAA, LSA and aortic diverticulum).