Application and usefulness of a new eight-wire basket catheter for endoscopic extraction of small common bile duct stones: A retrospective multicenter study.

Objectives: Distally located small common bile duct stones are often difficult to treat or grasp endoscopically. Therefore, multiple devices, such as baskets or balloon catheters, are frequently used in such cases. However, it is desirable to use a single device for stone extraction from the perspective of cost-effectiveness. In this multicenter study, we evaluated the efficacy of a new eight-wire basket catheter for extracting small (≤10 mm) common bile duct stones. Methods: We retrospectively analyzed the records of 144 patients who underwent stone extraction using the eight-wire basket catheter for common bile duct stones ≤10 mm. The success rate of complete stone extraction and the risk factors for the difficulty in stone extraction with the eight-wire catheter alone were mainly evaluated. Results: The success rate of stone extraction with the eight-wire catheter alone was 86.1%. The final rate of complete stone extraction was 98.0%. The mean of the maximum diameter of the common bile duct and the largest stone dimension were 10.5 ± 3.5, and 5.1 ± 2.1 mm, respectively. Common bile duct diameter ≥12 mm and stone diameter ≥6 mm were identified as independent risk factors for the difficulty in stone extraction with the eight-wire catheter alone. Conclusions: The success rate of the new eight-wire basket for small common bile duct stone extraction was acceptable. The device is beneficial and could be used from the start for the extraction of small stones < 6 mm.

Clip Formation in the Complementarity Determining Region of Bevacizumab Lowers Monomer Stability and Affinity for Both FcRn and FcγR: A Comprehensive Characterization of the Clipped Variant Including its Higher Order Structure.

The presence of monoclonal antibody (mAb) fragments in pharmaceutical mAb products is a critical quality attribute and should be controlled for safety. Several mAb fragments derived from clip formation in the complementarity determining regions (CDRs), as well as from cleavage in the hinge region, have been reported. However, the properties of CDR-clipped variants are not fully understood because of difficulties in separating them from intact mAbs under non-denaturing conditions due to similarities in size. We have established a method for separating CDR-clipped variants under non-denaturing conditions using an appropriate size exclusion chromatography column.1 In this report, we provide a comprehensive characterization of a CDR-clipped variant from bevacizumab. The variant exhibited a lower pI, a higher tendency to form dimers, and a lower affinity for both neonatal Fc receptor (FcRn) and Fcγ receptor (FcγR). The effects of clip formation in CDR H3 on the higher order structure were analyzed by hydrogen/deuterium exchange mass spectrometry, and the observed changes in the structures of the VH, CH2, and VL domains were in agreement with the lowered affinity for antigen, FcRn, and FcγR. These findings suggest that clip formation in the CDR may affect the efficacy, safety, and pharmacokinetics of pharmaceutical mAbs.

Nanoneedle-Electrode Devices for In Vivo Recording of Extracellular Action Potentials.

Microscale needle-like electrode technologies offer in vivo extracellular recording with a high spatiotemporal resolution. Further miniaturization of needles to nanoscale minimizes tissue injuries; however, a reduced electrode area increases electrical impedance that degrades the quality of neuronal signal recording. We overcome this limitation by fabricating a 300 nm tip diameter and 200 µm long needle electrode where the amplitude gain with a high-impedance electrode (>15 MΩ, 1 kHz) was improved from 0.54 (-5.4 dB) to 0.89 (-1.0 dB) by stacking it on an amplifier module of source follower. The nanoelectrode provided the recording of both local field potential (<300 Hz) and action potential (>500 Hz) in the mouse cortex, in contrast to the electrode without the amplifier. These results suggest that microelectrodes can be further minimized by the proposed amplifier configuration for low-invasive recording and electrophysiological studies in submicron areas in tissues, such as dendrites and axons.

Development of a comprehensive approach for performance evaluation of a quantitative multi-attribute method as a quality control method.

The multi-attribute method has been recognized as an elegant quantification tool for post-translational modifications (PTMs) of therapeutic proteins, since it can evaluate several attributes spontaneously and site-specifically. Here, the abundance of PTMs calculated by three different types of formula were compared and there was little difference among the results. For the method evaluation, two different kinds of peptides were used as internal standards (ISs) and one of the IS was used as the "standard peak" to define the signal strength of MS. They are also used for system suitability testing to verify whether the condition or sensitivity of mass spectrometry are high enough to evaluate the minor components by confirming the recovery rate of one IS to the another. This system is beneficial that since we have defined the limit of quantification as a certain ratio to IS, consistent MS intensity is applied as the threshold across all detected peaks.

In vitro synthesis of mucin-type O-glycans using saccharide primers comprising GalNAc-Ser and GalNAc-Thr residues.

Mucin-type O-glycosylation of serine or threonine residue in proteins is known to be one of the major post-translational modifications. In this study, two novel alkyl glycosides, Nα-lauryl-O-(2-acetamido-2-deoxy-α-d-galactopyranosyl)-l-serineamide (GalNAc-Ser-C12) and Nα-lauryl-O-(2-acetamido-2-deoxy-α-d-galactopyranosyl)-l-threonineamide (GalNAc-Thr-C12) were synthesized as saccharide primers to prime mucin-type O-glycan biosynthesis in cells. Upon incubating human gastric cancer MKN45 cells with the saccharide primers, 22 glycosylated products were obtained, and their structures were analyzed using liquid chromatography-mass spectrometry and enzyme digestion. The amounts of glycosylated products were dependent on the amino acid residues in the saccharide primers. For example, in vitro synthesis of T antigen (Galß1-3GalNAc), fucosyl-T (Fucα1-2Galß1-3GalNAc), and sialyl-T (NeuAcα2-3Galß1-3GalNAc) preferred a serine residue, whereas sialyl-Tn (NeuAcα2-6GalNAc) preferred a threonine residue. Furthermore, the glycosylated products derived from GalNAc-Ser/Thr-C12 and Gal-GalNAc-Ser/Thr-C12 using cell-free synthesis showed the same amino acid selectivity as those in the cell experiments. These results indicate that glycosyltransferases involved in the biosynthesis of mucin-type O-glycans distinguish amino acid residues conjugated to GalNAc. The saccharide primers developed in this study might be useful for comparing mucin-type oligosaccharides in cells and constructing oligosaccharide libraries to study cell function.

Chemical Probing of Thymine in the TGG/CGG Triad to Explore the Deamination of 5-Methylcytosine in the CGG Repeat.

The methylation of cytosine in the full mutation of the expanded CGG repeat and subsequent deamination to thymine could be a measure of repeat instability. We report the synthesis of NCD-Bpy, which binds to the TGG/CGG site in the repeat hairpin. NCD-Bpy forces the thymine in the TGG/CGG site to flip out from the π-stack, recruits osmium tetroxide in the vicinity of the flipped-out T, and oxidizes the T. The piperidine-induced cleavage band successfully determined the position of the T in the expanded CGG repeat.

A Comparison of the Oligosaccharide Structures of Antithrombin Derived from Plasma and Recombinant Using POTELLIGENT® Technology.

Human antithrombin (AT) has two isoforms of which the predominant α-form is glycosylated on all four possible glycosylation sites and the lower abundant ß-isoform lacks the oligosaccharide on Asn135. The main oligosaccharide structure of human AT consists of biantennary complex-type oligosaccharides lacking a core fucose. Generally, Chinese hamster ovary (CHO) cells produce recombinant human AT (rhAT) with core-fucosylated oligosaccharides. However, rhAT lacking core-fucose oligosaccharides can be produced by POTELLIGENT® technology, which uses FUT8 knockout CHO cells in production. The rhAT has more variable glycan structures, such as tetra-antennary complex type, high-mannose type, and mannose 6-phosphate species as minor components compared to plasma-derived human AT (phAT). In addition, the site-specific glycan profile was different between two ATs. We evaluated the effect of these properties on efficacy and safety based on a comparison of rhAT made by that technology with phAT in terms of their respective oligosaccharide structures, site-specific oligosaccharide profiles, and the ratio of α- and ß-forms. Although some structural differences were found between the rhAT and phAT, we concluded that these differences have no significant effect on the efficacy and safety of rhAT.

Expanding chemical space of DNA-binding molecules with three base-binding units.

A new molecule NC3-3 designed to expand chemical space of parent molecule NCD by adding the third base-binding unit was reported. NC3-3 bound to the G-G mismatch in the 5'-CGG-3'/5'-CGG-3' motif but not to that in 5'-GGC-3'/5'-GGC-3'. This binding selectivity is similar to that reported for NCD. Fluorimetric screening of NCD and NC3-3 to dsDNA library containing yGw/xGz motifs showed that NC3-3 still kept the sequence selectivity as we observed for NCD-binding. The third naphthyridine heterocycle in NC3-3 affected the mode of the binding, but a little effect on the sequence selectivity.

Rhinosinusitis and disseminated cutaneous infection caused by Mycobacterium chelonae in an immunocompromised patient.

Mycobacterium chelonae frequently involves the skin, and the disseminated form can be observed in immunocompromised patients. In contrast, rhinosinusitis caused by the bacterium is a rare manifestation, which occurs independently of immune status. We report here a rare case of M. chelonae infection presenting as both disseminated cutaneous infection and rhinosinusitis in an immunocompromised patient. He had received systemic corticosteroids for 11 months due to cryptogenic organizing pneumonia. Before admission, he sustained injuries to his left arm and hand; those injuries succumbed to an infection that would subsequently spread to his other limbs, face, and even nasal cavities. This valuable case suggests that disseminated cutaneous infection by M. chelonae could spread to other organs.

[Investigation of lung cancer patients with cardiovascular disease].

BACKGROUND: According to the aging in Japan, surgery of lung cancer with cardiovascular disease is increasing. Among them, the merger of the heart and large blood vessel disease would be expected to be directly linked to the perioperative management. MATERIALS AND METHODS: We conducted a retrospective analysis of 1,005 patients who had undergone surgical resection of primary lung adenocarcinoma between January 1997 and June 2014 at Hokkaido University Hospital. Among them, 81 patients had more than one cardiovascular disease. RESULTS: The median age was 71 years old. Men are 62 cases(76%). 64 cases (79%)had smoking history. Ischemic heart disease( IHD) was 29 cases(33%), followed by atrial fibrillation(Af), arrhythmia other than Af, valve disease and macrovascular disease. Anticoagulant or antiplatelet therapy were preoperatively done in 41% of IHD cases, 39% of Af cases. Thirty four% of IHD, 50% of vascular disease received the treatment other than medication. Video-assisted thoracic surgery was performed in 67 cases(83%). Lobectomy, sublobectomy were performed for 59 cases(73%), 19 cases(23%), respectively. Postoperative complications were occurred in 32 cases(40%). Two patients(2.5%)died in hospital without discharge. Univariate analysis revealed tumor size, pStage, lymph node dissection, operation time or blood loss were correlated with postoperative complication. CONCLUSIONS: For the patients with cardiovascular disease, careful evaluation of surgical indication, perioperative management is required.

[Fiberoptic-guided tracheal intubation through the i-gel supraglottic airway].

BACKGROUND: The i-gel is a rescue device for ventilation or tracheal intubation in patients with a difficult airway. The aim of this study was to evaluate the safety and reliability of fiberoptic-guided intubation through the i-gel in anesthetized patients with no history of difficult intubation undergoing elective surgery. METHODS: Patients were enrolled in the study with prior informed consent. After insertion of the i-gel, the larynx was observed by bronchoscopy, and the bronchoscopic view through the i-gel was graded. Tracheal intubation was performed under fiberoptic guidance, and the i-gel was removed. The outcome was evaluated using the success rate of initial intubation as the primary variable, and complications were evaluated as a secondary variable. RESULTS: The first attempt at intubation was successful in all 52 patients evaluated, and there was no problem with i-gel removal. No arterial oxygen desaturation was noted throughout the induction of anesthesia, and no serious complication was observed. CONCLUSIONS: Fiberoptic-guided intubation could be performed safely through the i-gel. The i-gel is considered to be potentially useful as an alternative conduit for fiberoptic-guided intubation.

Application of microchip electrophoresis sodium dodecyl sulfate for the evaluation of change of degradation species of therapeutic antibodies in stability testing.

We evaluated the performance of a commercial microchip electrophoresis instrument (LabChip(®) GXII) for the evaluation of change of degradation species of therapeutic antibodies in stability testing. This system requires a sample volume of only 5 µg, and indicates fine resolution of size variant species such as light chain, heavy chain, non-glycosylated heavy chain and various degradation species. Precision and accuracy were high; the intermediate precision of 18 determinations was only 2.1% or less as RSD and recoveries ranged from 97.8 to 103.0% for major species as heavy chain, light chain and intact molecule of a therapeutic antibody. The applicability of this method was demonstrated by applying the method for the analysis of heat-degraded products of three pharmaceutical antibodies. Though some fragment peaks commonly appeared and increased according to temperature regardless of the source of preparations, one of them indicated specific peaks implying the cleavage of the peptide chain of the heavy chain. We also compared the performance of the method with those using conventional capillary-based SDS electrophoresis. Although the absolute purity values expressed as peak area % were different for the two methods, probably due to the difference in the detection methods, similar quality profiles were obtained within 40 s by microchip-based SDS electrophoresis. In addition, the degradation manner of three marketed antibodies depending on temperature was almost the same for the two methods. At the first stage in the development of manufacturing antibody pharmaceuticals, various factors including cell selection, cell cultivation, and formulation development should be evaluated using limited sample amounts. The stability testing using microchip-based electrophoresis seems suitable for these purposes.

Specific detection of N-glycolylneuraminic acid and Galα1-3Gal epitopes of therapeutic antibodies by partial-filling capillary electrophoresis.

The oligosaccharide structure is very important in biopharmaceuticals because of its effects on protein function, including efficacy and half-life. N-glycolylneuraminic acid (Neu5Gc) and Galα1-3Gal (α-Gal) residues are known to show immunogenicity in humans. It is now understood that murine cell lines, such as NS0 or SP2, which are typically used for biopharmaceutical manufacture, produce proteins containing Neu5Gc and α-Gal residues. The expression of these specific residues is affected by the cell line and culture conditions. Therefore, monitoring and controlling the levels of these epitopes are important for the quality control of biopharmaceuticals. To detect the two epitopes on a therapeutic antibody produced by NS0 cells, we applied partial-filling capillary electrophoresis using anti-Neu5Gc antibody and α-galactosidase. In the anti-Neu5Gc antibody filling method, one minor glycan peak with Neu5Gc residues at the nonreducing end disappeared specifically from the electropherogram. In the α-galactosidase filling method, some minor peaks with α1,3-linked Gal residues disappeared. However, in a therapeutic antibody from Chinese hamster ovary cells, no peaks disappeared with the two methods. These results show this method can be used to specifically detect and quantify the two epitopes on biopharmaceuticals with high sensitivity.

Partial filling affinity capillary electrophoresis using large-volume sample stacking with an electroosmotic flow pump for sensitive profiling of glycoprotein-derived oligosaccharides.

An online preconcentration technique, large-volume sample stacking with an electroosmotic flow pump (LVSEP) was combined with partial filling affinity capillary electrophoresis (PFACE) to realize highly sensitive analysis of the interaction of glycoprotein-derived oligosaccharides with some plant lectins. Oligosaccharides derivatized with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) were delivered to an entire neutrally-coated capillary and then lectin solution was hydrodynamically introduced from the outlet of the capillary as a short plug. A negative voltage was then applied after immersion of both ends of the capillary in 100 mM Tris-acetate buffer, pH 7.0 containing 0.5% hydroxypropylcellulose as electrophoresis buffers. A low concentration of electrolytes in the sample solution causes a significant flow by electroendosmosis from anode to cathode and the APTS-labeled oligosaccharides move quickly towards the anode and concentrate in the lectin phase. Finally, electroosmotic flow becomes negligible when the capillary is filled with the background electrolyte delivered from the anodic reservoir and APTS-labeled saccharides pass through the lectin plug and are detected at the anodic end. If the APTS-labeled oligosaccharides are recognized by the lectin, the migration profiles should be altered. The sensitivity was enhanced by a factor of ca. 900 compared to typical hydrodynamic injection (3.45 kPa, 10s). By this method, increased residence time of APTS-saccharides in the lectin plug indicates highly efficient interaction with lectins, which differs completely from the results obtained by ordinary lectin PFACE. The run-to-run repeatability (n=18) of the migration time and peak area was high, with relative standard deviations of less than 0.7% and 6.1%, respectively.

Application of partial-filling capillary electrophoresis using lectins and glycosidases for the characterization of oligosaccharides in a therapeutic antibody.

Oligosaccharides in therapeutic recombinant antibodies play important roles in regulation of various biological functions. To monitor the glycosylation profiles of antibody pharmaceuticals in the manufacturing process, a highly sensitive and specific method is required. We extended partial-filling techniques using lectins and exoglycosidases in capillary electrophoresis for the characterization of 8-aminopylene-1,3,6-trisulfonic acid labeled N-linked oligosaccharides derived from the therapeutic antibody rituximab. In the lectin-filling method, Galb1­4GlcNAc-specific Erythrina cristagali agglutinin, a1, 6-linked Fuc-specific Aleuria aurantia lectin and Neu5Aca2­3Gal-specific Maackia amurensis lectin were used. The oligosaccharides migrated through the lectin plug during separation; the changes in separation profiles were observed according to the interaction with the lectins. The glycosidase-filling method allowed rapid digestion as suggested by the electropherograms. Partial-filling CE methods can avoid tedious hands-on procedures such as overnight incubation and optimization reaction condition with lectins and exoglycosidases. Combination of these partial-filling capillary electrophoresis methods makes the characterization of oligosaccharide profiles of therapeutic antibodies easier and faster.

[A case of amyotrophic lateral sclerosis with bilateral vocal cord paralysis necessitating tracheotomy].

Vocal cord movement disorders are increasingly recognized in patients with amyotrophic lateral sclerosis (ALS). We describe a patient with limb-onset ALS who developed vocal cord paralysis. A 74-year-old Japanese male consulted our clinic with a 6-month history of weakness in both arms. His family history was unremarkable. There were fasciculations and mild atrophy of the tongue and both arms. In the legs, muscle strength was almost normal but widespread fasciculations were present. All tendon reflexes were hypoactive and pathological reflexes were absent. Thereafter, he developed weakness of the legs and showed increased eating time. Babinski sign was positive bilaterally at this stage. The forced vital capacity dropped from 90% at the initial evaluation to 62% of the predicted value 14 months later. Two years after disease onset, the patient developed aspiration pneumonia with hoarseness and had difficulty clearing his throat of phlegm. Laryngoscopy demonstrated severe vocal cord paresis on both sides, particularly in the abductor muscles possibly leading to obstruction. Tracheotomy was performed because of the risk that the patient could choke to death. A review of the literature suggests that severe impairment of vocal cord abduction could be a prelude to sudden death in ALS. Follow up by laryngoscopic examination is necessary.

A comparative study of monosaccharide composition analysis as a carbohydrate test for biopharmaceuticals.

The various monosaccharide composition analysis methods were evaluated as monosaccharide test for glycoprotein-based pharmaceuticals. Neutral and amino sugars were released by hydrolysis with 4-7N trifluoroacetic acid. The monosaccharides were N-acetylated if necessary, and analyzed by high-performance liquid chromatography (HPLC) with fluorometric or UV detection after derivatization with 2-aminopyridine, ethyl 4-aminobenzoate, 2-aminobenzoic acid or 1-phenyl-3-methyl-5-pyrazolone, or high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Sialic acids were released by mild acid hydrolysis or sialidase digestion, and analyzed by HPLC with fluorometric detection after derivatization with 1,2-diamino-4,5-methylenedioxybenzene, or HPAEC-PAD. These methods were verified for resolution, linearity, repeatability, and accuracy using a monosaccharide standard solution, a mixture of epoetin alfa and beta, and alteplase as models. It was confirmed that those methods were useful for ensuring the consistency of glycosylation. It is considered essential that the analytical conditions including desalting, selection of internal standards, release of monosaccharides, and gradient time course should be determined carefully to eliminate interference of sample matrix. Various HPLC-based monosaccharide analysis methods were evaluated as a carbohydrate test for glycoprotein pharmaceuticals by an inter-laboratory study.

The S1P receptor modulator FTY720 prevents the development of experimental colitis in mice.

To evaluate the therapeutic effects of the new synthetic sphingosine-1-phosphate (S1P) receptor modulator, FTY720, we investigated how FTY720 affects the development of dextran sulfate sodium (DSS)-induced colitis and CD4+CD62L+ T cell transfer colitis. BALB/c mice were fed a chow containing 3.5% (wt/wt) DSS to induce colitis. The CD4+CD62L+ T cell transfer colitis was induced by an intraperitoneal injection of CD4+CD62L+ spleen T cells into recipient CB17 SCID mice. The FTY720 was administered by lavage at a dose of 0.3 mg/kg/day. FTY720 was effective in preventing the body weight loss in the DSS-colitis model and the CD4+CD62L+ T cell transfer model. The disease activity index, histological colitis score, and MPO activity were all significantly lower in FTY720-treated mice than in the non-treated mice. Microscopically, mucosal edema, cellular infiltration and epithelial disruption were much more moderate in the FTY720-treated mice than in the non-treated mice. In both colitis models, FTY720 prevented the infiltration of CD4+ T cells into the inflamed colonic lamina propria. In conclusion, the development of DSS-colitis and CD4+CD62L+ T cell transfer colitis were significantly attenuated by FTY720. Since FTY720 is an immunosuppressive product that does not modulate T cell functions, it could be useful in the treatment of IBD patients.

Immunohistochemical study of chymase-positive mast cells in inflammatory bowel disease.

Mast cell-derived chymase promotes inflammatory responses and tissue fibrosis. Although previous studies have reported changes in the number of mucosal mast cells in inflammatory bowel disease (IBD), the behaviour of chymase immunopositive mast cells has not been studied. In this study, we immunohistochemically investigated chymase immunopositive mast cells in the inflamed mucosa of IBD patients. Surgically-obtained or biopsy specimens from 10 patients with ulcerative colitis (UC), 10 patients with Crohn's disease (CD) and 10 normal colorectal tissue specimens were used. The chymase immunopositive cells were identified by immunohistochemical analysis using a monoclonal anti-human chymase antibody. In the normal colonic mucosa, a small number of chymase immunopositive mast cells were detected at the basal sites of the mucosa. There were no immunopositive cells in the submucosa. Chymase immunopositive mast cells were similarly observed in the inactive UC mucosa, but these cells decreased significantly in number in the active UC mucosa. In the inactive CD mucosa, the number of chymase immunopositive mast cells increased significantly (P < 0.05), and this was more clearly observed in the active CD mucosa. Furthermore, in the active CD mucosa, these cells were detected in the submucosa, propria muscularis, and surrounding fatty tissue. These observations suggest a crucial role for chymase immunopositive mast cells in the pathophysiology of CD. Since intestinal fibrotic changes such as stricture formation are a characteristic feature of CD, chymase immunopositive mast cells may act as a stimulus for the process of tissue fibrosis and tissue remodelling in the pathophysiology of CD.