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Programmed cell death protein-1 (PD1), PD1 ligand 1 (PD-L1), and human leukocyte antigen (HLA) class I molecule play pivotal roles in T cell-induced anti-tumor immunity; however, the clinical impact of these parameters in resected malignant pleural mesothelioma (MPM) cases is unknown. We immunohistochemically evaluated the tumor infiltrated lymphocytes (TILs), PD1/PD-L1 axis, and expression of HLA class I in resected specimens from 58 patients with MPM who underwent extra-pleural pneumonectomy (EPP). Higher infiltration of CD3-TIL, CD8-TIL, and PD1-TIL, loss of HLA class I, and overexpression of PD-L1 by tumor cells (PD-L1 TC) or immune cells (PD-L1 IC) were observed in 34 (58.6%), 27 (46.6%), 41 (70.7%), 45 (77.6%), 29 (50.0%), and 33 (56.4%) of 58 cases, respectively. Interestingly, the CD3-TIL score positively correlated with PD-L1 TC and PD1-TIL scores. HLA class I expression level was inversely correlated with the expression levels of PD-L1 TC and PD-L1 IC. Multivariate analysis showed that age, histology, and node metastasis were independent prognostic factors for 5-year overall survival (OS) and loss of HLA class I coincided with a positive prognosis (p = 0.011). The concomitant lack of infiltrating CD8+ T cells with no loss of HLA class I predicted worse 5-year OS (p = 0.007). Moreover, cluster classifications among multiple immunoparameters showed that categories among CD3/PD-L1 TC/HLA class I (p = 0.043), CD8/PD1/HLA class I (p = 0.032), CD8/PD-L1 TC/HLA class I (p = 0.011), and PD1/PD-L1 TC/HLA class I (p = 0.032) predicted 5-year OS in EPP cases for MPM. These immunoparameters could guide surgical indications for patients with MPM.

Background Coronavirus disease 2019 (COVID-19) remains a threat to vulnerable populations such as long-term care facility (LTCF) residents, who are often older, severely frail, and have multiple comorbidities. Although associations have been investigated between COVID-19 mRNA vaccine immunogenicity, durability, and response to booster vaccination and chronological age, data on the association of clinical factors such as performance status, nutritional status, and underlying comorbidities other than chronological age are limited. Here, we evaluated anti-spike IgG and neutralizing activity against the wild-type virus and Delta and Omicron variants in the sera of LTCFs residents, outpatients, and healthcare workers before the primary vaccination, at 8, 12, and 24 weeks after the primary vaccination, and approximately 3 months after the booster vaccination. This 48-week prospective longitudinal study was registered in the UMIN Clinical Trials Registry (Trial ID: UMIN000043558). Results Of 114 infection-naïve participants (64 LTCF residents, 29 outpatients, and 21 healthcare workers), LTCF residents had substantially lower anti-spike IgG levels and neutralizing activity against the wild-type virus and Delta variant than outpatients and healthcare workers over 24 weeks after the primary vaccination. In LTCF residents, booster vaccination elicited neutralizing activity against the wild-type virus and Delta variants comparable to that of outpatients, whereas neutralizing activity against the Omicron variants was comparable to that of outpatients and healthcare workers. Multiple regression analyses showed that age was negatively correlated with anti-spike IgG levels, neutralizing activity against the wild-type virus, and Delta variant after the primary vaccination. However, multivariate regression analysis revealed that poor performance status and hypoalbuminemia were more strongly associated with a lower humoral immune response than age, number of comorbidities, or sex after primary vaccination. In contrast, booster vaccination counteracted the negative effects of poor performance status and hypoalbuminemia on humoral immune response. Conclusions LTCF residents exhibited suboptimal immune responses following primary vaccination. Although older age is significantly associated with a lower humoral immune response, poor performance status and hypoalbuminemia are more strongly associated with a lower humoral immune response after primary vaccination. Thus, booster vaccination is beneficial for older adults, especially those with a poor performance status and hypoalbuminemia.

Patients with neuromuscular diseases usually have progressive neuromuscular scoliosis (NMS), requiring invasive surgery. Some patients present with severe scoliosis at the time of consultation and are difficult to treat. Posterior spinal fusion (PSF) surgery combined with anterior release and pre or intraoperative traction would be effective for severe spinal deformity, but invasive. This study aimed to evaluate the outcomes of PSF only surgery for patients with severe NMS with Cobb angle >100 °. Thirty NMS patients (13 boys and 17 girls; mean age 13.8 years) who underwent PSF only surgery for scoliosis with Cobb angle >100 ° were included. We reviewed the lower instrumented vertebra (LIV), duration of surgery, blood loss, perioperative complications, preoperative clinical findings, and radiographic findings including Cobb angle and pelvic obliquity (PO) in the sitting position pre and postoperatively. The correction rate and correction loss of the Cobb angle and PO were also calculated. The mean duration of surgery was 338 min, intraoperative blood loss was 1,440 mL, preoperative %VC was 34.1%, FEV1.0 (%) was 91.5%, and EF was 66.1%. There were eight cases of perioperative complications. The Cobb angle and PO correction rates were 48.5% and 42.0%, respectively. We divided the patients into two groups: the L5 group, in which the LIV was L5, and the pelvis group, in which the LIV was the pelvis. The duration of surgery and PO correction rate in the pelvis group was significantly higher than that of the L5 group. Patients with severe NMS demonstrated severe preoperative restrictive ventilatory impairments. PSF only surgery for severe NMS showed satisfactory outcomes, although highly invasive. Instrumentation and fusion to the pelvis for severe scoliosis in patients with NMS showed good PO correction and low correction loss of Cobb angle and PO, but a longer duration of surgery.

Correction surgeries for spinal malalignment showed good clinical outcomes; however, there were concerns including increased invasiveness, complications, and impact on medico-economics. Ideally, an early intervention is needed. To better understand the patho-mechanism and natural course of spinal alignment, the effect of factors such as muscle mass and strength on spinal sagittal imbalance were determined in a multicenter cross-sectional study.After excluding metal implant recipients, 1823 of 2551 patients (mean age: 69.2 ± 13.8 years; men 768, women 1055) were enrolled. Age, sex, past medical history (Charlson comorbidity index), body mass index (BMI), grip strength (GS), and trunk muscle mass (TM) were reviewed. Spinal sagittal imbalance was determined by the SRS-Schwab classification. Multiple comparison analysis among four groups (Normal, Mild, Moderate, Severe) and multinomial logistic regression analysis were performed.On multiple comparison analysis, with progressing spinal malalignment, age in both sexes tended to be higher; further, TM in women and GS in both sexes tended to be low. On multinomial logistic regression analysis, age and BMI were positively associated with spinal sagittal malalignment in Mild, Moderate, and Severe groups. TM in Moderate and Severe groups and GS in the Moderate group were negatively associated with spinal sagittal malalignment.Aging, obesity, low TM, and low GS are potential risk factors for spinal sagittal malalignment. Especially, low TM and low GS are potentially associated with more progressed spinal sagittal malalignment. Thus, early intervention for muscles, such as exercise therapy, is needed, while the spinal sagittal alignment is normal or mildly affected.

Aromatic carbonyl thiourea PI-28 has been focused on as a lead compound for the developing radicle elongation inhibitors in germinating Orobanche minor dry seeds. In this study, we have synthesized PI-28 and its derivatives using commercially available phenols, 2-chloroacetamide, and aryl isothiocyanates. In this method, 2-aryloxyacetanilides, which are also attractive as bioactive compounds, were obtained as byproducts. These compounds were formed due to the loss of isothiocyanate moiety from the formed aromatic carbonyl thioureas.

Introduction Spinal muscular atrophy (SMA) is defined as a neuromuscular disorder induced by progressive weakness of the skeletal muscle and is usually accompanied by progressive spinal deformity including scoliosis. The newly developed Nusinersen, which is the first approved drug worldwide for SMA, requires accurate intrathecal injection, which is sometimes difficult in patients with severe spinal deformity. Technical note For an accurate intrathecal approach in patients who have spinal fusion surgery to treat neuromuscular scoliosis, we have combined an L3 laminectomy with spinal correction and fusion surgery. Here, we review four cases of SMA in patients who underwent the additional L3 laminectomy during surgery to treat spinal scoliosis. A successful intrathecal approach was made using fluoroscopic guidance in all four patients, who were then administered with Nusinersen. Conclusions Our findings show that additional lumbar laminectomy during surgery for spinal scoliosis has effectively allowed for intrathecal injection of Nusinersen.

Clinical case report. To report the rare case with post-operative chylous retroperitoneum after corrective surgery for adult spinal deformity. We present a case of a 73-year-old woman with Parkinson’s disease. She sustained a severe split fracture subluxation of the L3 vertebra with AO Spine Thoracolumbar classification type CN2M2, resulting in severe kyphoscoliosis in global alignment. She underwent a two-stage 720-degree anteroposterior-combined corrective surgery with anterior vertebral column resection of L3 and posterior fusion from T4 to the pelvis. On post-operative day 1, milky fluid in the drainage tube was noted, which was diagnosed as post-operative chylous retroperitoneum. Oral intake was discontinued immediately and peripheral parenteral nutrition was started. A low-fat, high-protein diet was started on post-operative day 4, and drainage was removed on day 6. A low-fat diet was continued until 3 months post-operatively, with dietary counselling by a nutritionist. The chylous retroperitoneum resolved without recurrence at the final follow-up evaluation at 3 years. Surgeons should recognize this rare complication, which might be induced by direct damage to the lymphatic flow during an operative maneuver anterior to the lumbar vertebral body and indirect damage due to shearing force during correction of a subluxated vertebra, especially in cases with a severe deformity.

Animal studies suggest that pain-related-molecule upregulation in degenerated intervertebral discs (IVDs) potentially leads to low back pain (LBP). We hypothesized that IVD mechanical stress and axial loading contribute to discogenic LBP’s pathomechanism. This study aimed to elucidate the relationships among the clinical findings, radiographical findings, and pain-related-molecule expression in human degenerated IVDs. We harvested degenerated-IVD samples from 35 patients during spinal interbody fusion surgery. Pain-related molecules including tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-6, calcitonin gene-related peptide (CGRP), microsomal prostaglandin E synthase-1 (mPGES1), and nerve growth factor (NGF) were determined. We also recorded preoperative clinical findings including body mass index (BMI), Oswestry Disability Index (ODI), and radiographical findings including the vacuum phenomenon (VP) and spinal instability. Furthermore, we compared pain-related-molecule expression between the VP (−) and (+) groups. BMI was significantly correlated with the ODI, CGRP, and mPGES-1 levels. In the VP (+) group, mPGES-1 levels were significantly higher than in the VP (−) group. Additionally, CGRP and mPGES-1 were significantly correlated. Axial loading and mechanical stress correlated with CGRP and mPGES-1 expression and not with inflammatory cytokine or NGF expression. Therefore, axial loading and mechanical stress upregulate CGRP and mPGES-1 in human degenerated IVDs, potentially leading to chronic discogenic LBP.

In the current study, multivariate analyses were performed to determine factors associated with low back pain (LBP) in patients with osteoporosis. Aging, high bone turnover, obesity, low trunk muscle mass, spinal global sagittal malalignment, and a high number of previous vertebral fractures were potential independent risk factors of pain-related disorders, gait disturbance, or ADL deficit due to LBP. PURPOSE Patients with osteoporosis often experience low back pain (LBP) even in the absence of acute fractures. This study identifies factors that may affect questionnaires about LBP. METHODS The data of 491 patients with osteoporosis were retrospectively reviewed. Data included patient age, sex, body mass index (BMI), bone mineral density of the lumbar spine, tartrate-resistant acid phosphatase 5b level (TRACP5b), trunk muscle mass, sagittal vertical axis (SVA), previous vertebral fractures, secondary osteoporosis, controlling nutritional status score, pain-related disorders and gait disturbance scores from the Japanese Orthopedic Association Back Pain Evaluation questionnaire (JOABPEQ), and Oswestry disability index (ODI) scores for activities of daily living (ADL) deficit. Patients with scores of 100 for each subsection of the JOABPEQ, or an ODI scores

Therapeutic monoclonal antibodies (mAbs) are mostly of the IgG class and constitute highly efficacious biopharmaceuticals for a wide range of clinical indications. Full-length IgG mAbs are large proteins that are subject to multiple posttranslational modifications (PTMs) during biosynthesis, purification, or storage, resulting in micro-heterogeneity. The production of recombinant mAbs in nonhuman cell lines may result in loss of structural fidelity and the generation of variants having altered stability, biological activities, and/or immunogenic potential. Additionally, even fully human therapeutic mAbs are of unique specificity, by design, and, consequently, of unique structure; therefore, structural elements may be recognized as non-self by individuals within an outbred human population to provoke an anti-therapeutic/anti-drug antibody (ATA/ADA) response. Consequently, regulatory authorities require that the structure of a potential mAb drug product is comprehensively characterized employing state-of-the-art orthogonal analytical technologies; the PTM profile may define a set of critical quality attributes (CQAs) for the drug product that must be maintained, employing quality by design parameters, throughout the lifetime of the drug. Glycosylation of IgG-Fc, at Asn297 on each heavy chain, is an established CQA since its presence and fine structure can have a profound impact on efficacy and safety. The glycoform profile of serum-derived IgG is highly heterogeneous while mAbs produced in mammalian cells in vitro is less heterogeneous and can be "orchestrated" depending on the cell line employed and the culture conditions adopted. Thus, the gross structure and PTM profile of a given mAb, established for the drug substance gaining regulatory approval, have to be maintained for the lifespan of the drug. This review outlines our current understanding of common PTMs detected in mAbs and endogenous IgG and the relationship between a variant's structural attribute and its impact on clinical performance.

Background Recently, Oblique lumbar interbody fusion (OLIF) is commonly indicated to correct the sagittal and coronal alignment in adult spinal deformity (ASD). Endplate fracture during surgery is a major complication of OLIF, but the detailed location of fracture in vertebral endplate in ASD has not yet been determined. We sought to determine the incidence and location of endplate fracture and subsidence of the OLIF cage in ASD surgery, and its association with fusion status and alignment. Methods We analyzed 75 levels in 27 patients were analyzed using multiplanar CT to detect the endplate fracture immediately after surgery and subsidence at 1 year postoperatively. The prevalence was compared between anterior and posterior, approach and non-approach sides, and concave and convex side. Their association with fusion status, local and global alignment, and complication was also investigated. Results Endplate fracture was observed in 64 levels (85.3%) in all 27 patients, and the incidence was significantly higher in the posterior area compared with the anterior area (85.3 vs. 68.0%, p=0.02) of affected vertebra in the sagittal plane. In the coronal plane, there was no significant difference in incidence between left (approach) and right (non-approach) sides (77.3 and 81.3%, respectively), or concave and convex sides (69.4 and 79.6%) of wedged vertebra. By contrast, cage subsidence at 1 year postoperatively was noted in 14/75 levels (18.7%), but was not associated with endplate fracture. Fusion status, local and global alignment, and complications were not associated with endplate fracture or subsidence. Conclusion Endplate fracture during OLIF procedure in ASD cases is barely avoidable, possibly induced by the corrective maneuver with ideal rod counter and cantilever force, but is less associated with subsequent cage subsidence, fusion status, and sustainment of corrected alignment in long fusion surgery performed even for elderly patients.

Background: Recently, Oblique lumbar interbody fusion (OLIF) is commonly indicated to correct the sagittal and coronal alignment in adult spinal deformity (ASD). Endplate fracture during surgery is one of major complication of OLIF, but the detailed location of fracture in vertebral endplate has not yet evaluated in ASD. We conducted this study to evaluate the incidence and location of endplate fracture and subsidence of the OLIF cage, and its impact on fusion status and alignment in ASD surgery.Methods: 75 levels in 27 patients were analyzed using multiplanar CT to detect the endplate fracture immediately after surgery and subsidence at postoperative 1 year. The prevalence was compared between anterior and posterior, approach side and non-approach side, and concave and convex side. Their impact on fusion status, local and global alignment, and complication was also investigated.Results: Endplate fracture was observed in 64 levels (85.3%) in all 27 patients, and the incidence was significantly higher in posterior area compared with anterior area (85.3% vs. 68.0%, p=0.02) of affected vertebra in sagittal plane. On the coronal plane, there was no significant difference in incidence between left (approach) side and right (non-approach) side (77.3% and 81.3%, respectively), or concave and convex side (69.4% and 79.6%) of wedged vertebra. On the other hand, cage subsidence at postoperative one year was noted in 18.7% (14/75 levels), which was not associated with endplate fracture. Fusion status, local and global alignment, or complications were not associated with endplate fracture and subsidence.Conclusion: Endplate fracture during OLIF procedure in ASD cases is hardly avoidable, possibly induced by corrective maneuver with ideal rod counter and cantilever force, but impacts less on subsequent cage subsidence, fusion status, and sustainment of corrected alignment in long fusion surgery performed even for elderly patients.

Introduction: Respiratory failure from acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF) is associated with high mortality. Direct hemoperfusion with polymyxin B-immobilized fiber column (PMX-DHP) has been reported to have beneficial effects on patients with AE-IPF. Whether patient characteristics influence the extent of this benefit remains unclear. Methods: We retrospectively examined the records of 30 patients with AE-IPF who underwent PMX-DHP. The favorable factors of survival were determined using Cox proportional hazards analyses. Results: The 1- and 12-month survival rates after PMX-DHP were 70.0% and 50.0%, respectively. The multivariate analysis revealed that low modified Gender-Age-Physiology (GAP) index (≤8 points) (hazard ratio [HR] 0.317, p = 0.015) and PMX-DHP received within 48 h of steroid pulse (HR 0.289, p = 0.012) were favorable factors. Notably, even in the patients with high modified GAP index (>8 points), that is, more advanced IPF, those who received PMX-DHP within 48 h of steroid pulse had a better prognosis than those who did after 48 h of the steroid pulse (p = 0.032). Conclusions: Early PMX-DHP initiation in patients with AE-IPF, specifically within 48 h after the steroid pulse therapy, may improve prognosis regardless of the severity of chronic phase of IPF before AE-IPF.

Background and Aim: The effect of corticosteroids on dyspnea in patients with advanced cancer is expected to be associated with patient prognosis, though details about this are unclear. Thus, we aimed to investigate the relationship between prognostic indicators, actual survival, and the effect of corticosteroid administration on dyspnea in patients with advanced cancer.Method: We predicted the prognosis using the PaP score (palliative prognostic score) at the time of corticosteroid administration in 56 consecutive patients with cancer who started corticosteroid treatment for dyspnea at a single palliative care unit, and compared it with the actual survival. In addition, we investigated the relationship between the effect of corticosteroids on dyspnea, the prognostic score before administration, the actual survival, and other background factors.Results: The effect of corticosteroids strongly correlated with the actual survival, but not the prognostic score before corticosteroid administration, although it was correlated with prognostic score and actual survival. In addition, the effect of corticosteroids on dyspnea was less associated with other factors.Conclusion: The corticosteroid effect strongly associated with actual survival, but not the prognostic score. It was suggested that the effect of corticosteroids in patients could be predicted by using a more accurate prognosis prediction in the future.

The complex diantennary-type oligosaccharides at Asn297 residues of the IgG heavy chains have a profound impact on the safety and efficacy of therapeutic IgG monoclonal antibodies (mAbs). Fc glycosylation of a mAb is an established critical quality attribute (CQA), and its oligosaccharide profile is required to be thoroughly characterized by state-of-the-art analytical methods. The Fc oligosaccharides are highly heterogeneous, and the differentially glycosylated species (glycoforms) of IgG express unique biological activities. Glycoengineering is a promising approach for the production of selected mAb glycoforms with improved effector functions, and non- and low-fucosylated mAbs exhibiting enhanced antibody-dependent cellular cytotoxicity activity have been approved or are under clinical evaluation for treatment of cancers, autoimmune/chronic inflammatory diseases, and infection. Recently, the chemoenzymatic glycoengineering method that allows for the transfer of structurally defined oligosaccharides to Asn-linked GlcNAc residues with glycosynthase has been developed for remodeling of IgG-Fc oligosaccharides with high efficiency and flexibility. Additionally, various glycoengineering methods have been developed that utilize the Fc oligosaccharides of IgG as reaction handles to conjugate cytotoxic agents by "click chemistry", providing new routes to the design of antibody-drug conjugates (ADCs) with tightly controlled drug-antibody ratios (DARs) and homogeneity. This review focuses on current understanding of the biological relevance of individual IgG glycoforms and advances in the development of next-generation antibody therapeutics with improved efficacy and safety through glycoengineering.

Therapeutic monoclonal antibodies (mAbs) are mostly of the IgG class and constitute highly efficacious biopharmaceuticals for a wide range of clinical indications. Full-length IgG mAbs are large proteins that are subject to multiple posttranslational modifications (PTMs) during biosynthesis, purification, or storage, resulting in micro-heterogeneity. The production of recombinant mAbs in nonhuman cell lines may result in loss of structural fidelity and the generation of variants having altered stability, biological activities, and/or immunogenic potential. Additionally, even fully human therapeutic mAbs are of unique specificity, by design, and, consequently, of unique structure; therefore, structural elements may be recognized as non-self by individuals within an outbred human population to provoke an anti-therapeutic/anti-drug antibody (ATA/ADA) response. Consequently, regulatory authorities require that the structure of a potential mAb drug product is comprehensively characterized employing state-of-the-art orthogonal analytical technologies; the PTM profile may define a set of critical quality attributes (CQAs) for the drug product that must be maintained, employing quality by design parameters, throughout the lifetime of the drug. Glycosylation of IgG-Fc, at Asn297 on each heavy chain, is an established CQA since its presence and fine structure can have a profound impact on efficacy and safety. The glycoform profile of serum-derived IgG is highly heterogeneous while mAbs produced in mammalian cells in vitro is less heterogeneous and can be “orchestrated” depending on the cell line employed and the culture conditions adopted. Thus, the gross structure and PTM profile of a given mAb, established for the drug substance gaining regulatory approval, have to be maintained for the lifespan of the drug. This review outlines our current understanding of common PTMs detected in mAbs and endogenous IgG and the relationship between a variant’s structural attribute and its impact on clinical performance.

Summary The cause of pleural effusion remains uncertain in approximately 15% of patients despite exhaustive evaluation. As recently described immunoglobulin (Ig)G4-related disease is a fibroinflammatory disorder that can affect various organs, including the lungs, we investigate whether idiopathic pleural effusion includes IgG4-associated etiology. Between 2000 and 2012, we collected 830 pleural fluid samples and reviewed 35 patients with pleural effusions undiagnosed after pleural biopsy at Yamaguchi-Ube Medical Center. Importantly, IgG4 immunostaining revealed infiltration of IgG4-positive plasma cells in the pleura of 12 patients (34%, IgG4+ group). The median effusion IgG4 level was 41 mg/dl in the IgG4+ group and 27 mg/dl in the IgG4− group (P

BACKGROUND: Intrapulmonary percussive ventilation (IPV) is used for airway clearance and delivery of aerosol medications, including bronchodilators. Despite the common use of IPV for drug delivery, few data are available regarding optimization of inhalation therapy with IPV. In this study, we investigated the influence of IPV setting parameters and lung mechanics on drug delivery via IPV alone. METHODS: An IPV device was connected to a lung model via a trachea model and a flow analyzer. Albuterol nebulized from the IPV device was collected onto a filter attached between the trachea and lung models, and was quantitated by spectrophotometry (230 nm). RESULTS: Albuterol delivery to the lung model was increased up to 2.1-fold, with decreasing percussion frequency. Decreasing percussion frequency concomitantly increased the tidal volume, and albuterol delivery was correlated with tidal volume (r = 0.91, P CONCLUSIONS: Albuterol delivery and tidal volume with IPV can be improved by maintaining low levels of percussion frequency and increasing operational pressure. When increasing operational pressure, the peak inspiratory pressure and airway resistance levels need to be carefully monitored for safe inhalation therapy with IPV.

Malignant pleural mesothelioma (MPM) is an aggressive disease that is resistant to conventional therapies. Cell lines are useful models for studying the biological characteristics of tumors; therefore, the establishment of MPM cell lines is valuable for exploring novel therapeutic strategies for MPM. In the present study, 4 MPM cell lines (YUMC8, YUMC44, YUMC63, and YUMC64) were established, which consisted of 2 epithelioid and 2 sarcomatoid mesothelioma histological subtypes, from Japanese patients with MPM. The DNA methylation status, mutations, copy number gains, protein expression of representative genes, and the sensitivity to several drugs were examined in these 4 cell lines. Methylation of P16 was demonstrated in 3/4 cell lines, in which the protein expression of p16 was lost. Methylation of RASSF1A was observed in 3/4 cell lines. Copy number gains of EGFR, HER2 or MET were not detected in the 4 cell lines. Mutations in various genes, including EGFR, KRAS, HER2, BRAF, and PIK3CA, which are frequently detected in non-small cell lung cancer, were not detected in the 4 cell lines. microRNA-34b/c is a direct transcriptional target of p53 and is often silenced in MPM by promoter methylation. In the present study, miR-34b/c was heavily methylated in 2/4 established MPM cell lines. For cell adhesion molecules, E-cadherin expression was detected in the 2 epithelioid MPM cell lines, whereas N-cadherin expression was detected in all 4 established cell lines by western blotting. Vimentin was strongly expressed in the 2 sarcomatoid MPM cell lines. None of the established MPM cell lines demonstrated significant responses to the drugs tested, including NVP-AUY922, 17-DMAG, Trichostatin A, and Vorinostat. Although novel molecular findings were not observed in the current characterization of these MPM cell lines, these lines will be useful for future extensive analyses of the biological behavior of MPM and the development of novel therapeutic strategies.

BACKGROUND Donor lymphocyte infusion (DLI) is used for treatment of hematologic malignancy relapse or mixed chimerism after allogeneic hematopoietic stem cell transplantation. Although graft-versus-host disease is well recognized as one of the adverse effects of DLI, there are limited reports on noninfectious pulmonary complications (NIPCs) after DLI. CASE REPORT A 55-year-old woman with acute myeloid leukemia received DLI for conversion from recipient predominant to complete donor chimerism on Day +193 after allogeneic HSCT. Eight weeks later, she complained of dyspnea with fever; chest computed tomography revealed diffuse, bilateral, ground glass opacity and reticular appearance. She was diagnosed as having NIPC based on serum and bronchoalveolar lavage fluid (BALF) findings. She was successfully treated with prednisolone (PSL) and completely recovered. DISCUSSION We analyzed the cell profile from the BALF and 27 cytokines and chemokines in the serum using the Bio-Plex platform. The cells consisted of recipient predominant macrophages and T cells. The serum cytokine and chemokine profile showed significant elevation of interleukin (IL)−1β, IL-6, IL-8, tumor necrosis factor-α, macrophage inflammatory protein (MIP)−1α, and MIP-1β, which declined with the improvement of symptoms after initiation of PSL treatment. CONCLUSION Inflammatory effectors by recipient cells, rather than allogeneic responses by donor cells, played an important role in the pathogenesis of NIPCs after DLI in the present case.

Objective: Gavage administration of antidepressant drugs is required for some depressed patients with dysphagia. In the recent years, a simple suspension method has drawn increasing attention as a method that prevents changes in the stability and safety of various drugs. However, only 59 of the 354 orally administered antidepressant drugs (16.7%) approved in Japan by April 2013 have been examined with this method. In this study, we investigated whether 44 oral antidepressant drugs, which have not previously been tested for effi cacy through gavage, could be adapted to the simple suspension method. Materials and Methods: Adaptability of antidepressants to the simple suspension method was assessed by incubating at room temperature a drug in 20 mL of warm water (at 55°C) in a 30 mL-syringe for 5 and 10 min. The ability of the decayed drugs to pass through the tubing was also examined. Results: Most of the 44 oral antidepressant drugs tested could be adapted to the simple suspension method. Unexpectedly, fl uvoxamine maleate tablets, milnacipran hydrochloride tablets, and Cymbalta® capsules required 10 min or longer to decay. Conclusion: We were able to qualitatively assess all 44 oral antidepressant drugs. These results provide useful information for administration of oral antidepressant drugs to depressed patients using a simple suspension method. Key words: Difficulty swallowing, gavage administration, generic drugs, oral antidepressant drugs, simple suspension method.

Acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF) is characterized by severe worsening dyspnea of unknown etiology and high mortality without effective treatment. Recently, direct hemoperfusion with polymyxin B (PMX)-immobilized fiber cartridge (PMX-DHP) has been reported to improve pulmonary oxygenation and survival in patients with AE-IPF although its mechanism of action remains unknown. To gain insights into the pathobiology of AE-IPF through the beneficial effects of PMX-DHP, we analyzed the profile of cytokines adsorbed onto PMX-fibers used in 9 AE-IPF patients. In addition, the sera of these AE-IPF patients collected immediately before and after PMX-DHP, 9 stable IPF patients and 8 healthy individuals were also analyzed. The serum levels of cytokines including IL-9, IL-12, IL-17, PDGF and VEGF were significantly decreased immediately after PMX-DHP (P

Background: Acute exacerbations of idiopathic pulmonary fibrosis (AE-IPF) are fatal episodes of acute respiratory worsening of unknown etiology. Previous studies on acute respiratory distress syndrome have shown that direct hemoperfusion with polymyxin B-immobilized fiber column (PMX-DHP) beneficially impact respiratory status. Aimes: To investigate the prognosis and survival outcome of patients with AE-IPF who underwent PMX-DHP. Methods: We examined records of 50 patients with AE-IPF treated in our hospital. All patients received corticosteroid pulse therapy. We compared the disease outcome between 27 patients who underwent PMX-DHP (PMX group) and 23 patients who did not (non-PMX group). Independent predictors of survival were determined with Cox proportional hazards analyses. Results: A multivariate analysis of all patients revealed that PMX-DHP therapy was a significant predictor of survival (HR=0.442, p=0.019). Throughout the median observational period of 42.0 days (range, 1-1656 days) 38 patients (76.0%) died. Overall survival was significantly longer in the PMX group than the non-PMX group: median survival time, 192 days in the PMX vs. 29 days in the non-PMX (p=0.040, log rank test). The 12-month survival rate was also significantly better in the PMX group than the non-PMX group (41.7% vs. 9.8%). In a subanalysis of the PMX group, time from AE-IPF onset to PMX-DHP was a significant predictor of survival (HR=1.080, p=0.049). Conclusions: PMX-DHP improved the prognosis of AE-IPF. Time from AE-IPF onset to PMX-DHP may be informative for outcome prediction.

Expression of the transcription factor FOXP3 characterizes regulatory T cells (Tregs) that engage in the maintenance of immunological self-tolerance and immune homeostasis. Intra-tumoral accumulation of Tregs is associated with unfavorable prognosis in several kinds of cancers. Recently, expression of FOXP3 and its association with prognosis have also been shown in some cancer cells in clinical studies. For non-small cell lung cancer (NSCLC), however, prognostic significance of tumor FOXP3 expression and its relationship with Tregs remain unknown. FOXP3 expression in cancer cells and tumor-infiltrating lymphocytes was examined by immunohistochemical staining of surgical specimens from 87 patients with NSCLC. Prognostic values of the tumor-infiltrating Treg count and tumor FOXP3 expression status were evaluated retrospectively. FOXP3-positive cancer cells were observed in 27 of 87 (31.0%) patients. There was no significant relationship between Treg count and tumor FOXP3 status. Increased Treg counts were associated with worse overall and relapse-free survival whereas the influence of tumor FOXP3 status on survival was not significant. However, when FOXP3-positive cancer cells were present, the relationship between Treg accumulation and worse prognosis was attenuated. In contrast, patients without tumor FOXP3 expression and high Treg count had significantly worse overall and relapse-free survival (hazard ratio: 3.118 and 3.325, p=0.028 and 0.024, respectively) than other groups. These results suggest that tumor FOXP3 expression has a better prognostic potential in NSCLC and that in combination with tumor-infiltrating Treg count the absence of tumor FOXP3 allows the selection of high-risk patients.

症例は61歳, 男性. 胃がんの診断で胃全摘術を施行されたのち, リンパ節転移と骨転移で再発し, 両側尿管閉塞による慢性腎不全を併発したため緩和ケア病棟へ紹介されて転院となった. 遷延する尿路感染症に対して抗生剤点滴を行い改善がみられたが, その後, 食事摂取量が減少し, 血尿が出現し, さらに口腔内にも出血が認められるようになった. PT, APTTは測定不能と延長していたが, 血小板, フィブリノーゲン, FDPは正常でDICは否定された. 血清のPIVKA-IIが著明高値, ビタミンKは低値であり, ビタミンKの投与にてPT, APTT, 出血症状の改善を認めた. 本症例では, 食欲低下と抗生剤投与によりビタミンK欠乏が起こり, その結果, 出血症状が出現したと考えられる. 進行がん患者を対象とする緩和ケア領域では, 経口摂取の低下や抗生剤使用などのためビタミンKの欠乏が生じやすく, 出血症状がビタミンK欠乏症と終末期DICとの鑑別が必要な場合もあり, 注意が必要と考えられた.

A conserved structural feature of human IgG molecules is the presence of an oligosaccharide moiety within the Fc region at Asn297. In addition, 15-20% of normal polyclonal IgG molecules bear N-linked oligosaccharides in the variable (V) regions of the light (L) and/or heavy (H) chains. Electrospray ionization mass spectrometry (ESI-MS) has been applied to the glycan analysis of two IgG1 myeloma proteins (Wid and Cri) after mild reduction and acidification. Heterogeneous ion peaks were observed for both the H and L chains of Wid in contrast to Cri whose L chain peak was homogeneous. Site-specific deglycosylation of the H and L chains of IgG Wid was achieved under native conditions with peptide-N-glycosidase F and endoglycosidase F2, respectively. The Fc glycoforms differed between the two proteins in that Cri-Fc bears diantennary complex-type glycans that are fully core-fucosylated and partially sialylated while Wid-Fc glycans are non-fucosylated, partially galactosylated and non-sialylated. In contrast to the Fc glycans, the L chain glycans of Wid were shown to be fucosylated, fully galactosylated and sialylated, indicating that the glycosylation machinery of the Wid-producing myeloma cells is intact. Thus, combination of the two endoglycosidases can provide a simple means of glycan analysis of both Fab and Fc by ESI-MS, which may contribute to the development of therapeutic IgG with customized glycan profiles.

The potential of optical circuit packaging technology is discussed. Special attention is paid to introduction of "optical wiring" at the printed wiring board level (i.e., in the "last 1 meter area") to overcome the bandwidth limitations of electrical copper-based wiring. The suitability of optical surface mount technology (O-SMT) as a possible solution is reviewed. It is shown that the key to the utility of O-SMT is high efficiency and alignment-free coupling between optical wiring and optical devices. O-SMT requires a method to change the beam direction from the horizontal to the vertical and vice versa in order to couple optical wiring in an OE-board and OE-devices mounted on the board. A novel method using an "optical pin" is proposed and investigated. Furthermore, an optical coupling method using a self-written waveguide called "optical solder" is reviewed. Several applications of self-written waveguides using a green laser and a photo-mask are demonstrated.

Glycosylation of the IgG-Fc is essential for optimal binding and activation of Fcγ receptors and the C1q component of complement. However, it has been reported that the effector functions are down-regulated when the Fc glycans terminate in sialic acid residues and that sialylated IgG mediates anti-inflammatory effects of intravenous immunoglobulin (IVIG). Although recombinant IgG is hypo-sialylated, Fc sialylation is shown to be markedly increased when a mouse/human chimeric IgG3 Phe243Ala (F243A) variant is expressed in Chinese hamster ovary (CHO)-K1 cells. Here we investigate whether sialylation is increased in IgG1 F243A when expressed in CHO-K1, mouse myeloma J558L and human embryonic kidney (HEK) 293. Although the sialylation level was 2-5% for IgG1 wild type (WT), it was increased to 31%, 10% and 33% for the variant from CHO-K1, J558L and HEK293 cells, respectively. Interestingly, an increased addition of bisecting GlcNAc and α(1-3)-galactose residues to the Fc glycan was observed for HEK293-derived and J558L-derived IgG1 F243A, respectively. Fucosylation of HEK293-derived IgG1 F243A was maintained despite increased bisecting GlcNAc content. Although sialic acid and bisecting GlcNAc residues are reported to have an opposing effect on antibody-dependent cellular cytotoxicity (ADCC), IgG1 F243A showed 7 times lower ADCC activities than IgG1 WT, irrespective of bisecting GlcNAc residue. Thus, highly sialylated, human cell-derived IgG1 F243A with lowered ADCC activity may be of interest for the development of therapeutic antibodies with anti-inflammatory properties as an alternative to IVIG.

BACKGROUND Donor lymphocyte infusion (DLI) is used for treatment of hematologic malignancy relapse or mixed chimerism after allogeneic hematopoietic stem cell transplantation. Although graft‐versus‐host disease is well recognized as one of the adverse effects of DLI, there are limited reports on noninfectious pulmonary complications (NIPCs) after DLI. CASE REPORT A 55‐year‐old woman with acute myeloid leukemia received DLI for conversion from recipient predominant to complete donor chimerism on Day +193 after allogeneic HSCT. Eight weeks later, she complained of dyspnea with fever; chest computed tomography revealed diffuse, bilateral, ground glass opacity and reticular appearance. She was diagnosed as having NIPC based on serum and bronchoalveolar lavage fluid (BALF) findings. She was successfully treated with prednisolone (PSL) and completely recovered. DISCUSSION We analyzed the cell profile from the BALF and 27 cytokines and chemokines in the serum using the Bio‐Plex platform. The cells consisted of recipient predominant macrophages and T cells. The serum cytokine and chemokine profile showed significant elevation of interleukin (IL)−1β, IL‐6, IL‐8, tumor necrosis factor‐α, macrophage inflammatory protein (MIP)−1α, and MIP‐1β, which declined with the improvement of symptoms after initiation of PSL treatment. CONCLUSION Inflammatory effectors by recipient cells, rather than allogeneic responses by donor cells, played an important role in the pathogenesis of NIPCs after DLI in the present case.

The influence of sodium butyrate on the production and glycosylation of recombinant mouse/human chimeric antibody by transfected CHO-K1 cells was investigated. We selected cells expressing 'wild-type' antibody with a human IgG3 heavy chain and a mutant of this molecule in which Phe 243 is replaced by Ala. These proteins have previously been shown to exhibit very different glycoform profiles with the mutant IgG being comprised of glycoforms having a high galactose and sialic acid content. Cell culture with 0-5 mM butyrate was shown to effect a 2-4-fold increase in antibody production whilst the induction of apoptosis was observed in a dose-dependent manner. The optimal butyrate concentration was observed to be 2 mM. The glycoform profile of each antibody produced in the presence of butyrate was analyzed by HPAEC-PAD and shown to be unchanged, relative to that produced in the absence of butyrate. Biological activity was evaluated by the ability of the antibodies to trigger superoxide generation, through Fc gamma RI, and shown to be independent of production in the presence or absence of butyrate. A similar increase in production was observed for a high antibody-producing cell line when expanded in a hollow fibre bioreactor under low-serum conditions (1%). These results demonstrated that butyrate is of value for increasing the productivity of CHO-K1 for recombinant IgG and does not compromise either glycosylation or biological activity.

Antibodies are multifunctional molecules that following the formation of antibody antigen complexes, may activate mechanisms to effect the clearance and destruction of the antigen (pathogen). The IgG molecule is comprised of three globular protein moieties (2Fab+Fc) linked through a flexible hinge region. While the Fabs bind antigens, the Fc triggers effector mechanisms through interactions with specific ligands, e.g. cellular receptors (FcgammaR), and the C1 component of complement. Glycosylation of IgG-Fc has been shown to be essential for efficient activation of FcgammaR and C1. We report the generation of a series of truncated glycoforms of IgG-Fc, and the analysis of the contribution of the residual oligosaccharide to IgG-Fc function and thermal stability. Differential scanning microcalorimetry has been used to compare the stabilities of the homogeneous glycoforms of IgG1-Fc. The results show that all truncated oligosaccharides confer a degree of functional activity, and thermodynamic stability to the IgG1-Fc, in comparison with deglycosylated IgG1-Fc. The same truncated glycoforms of an intact IgG1 anti-MHC Class II antibody are shown to exhibit differential functional activity for FcgammaRI and C1 ligands, relative to deglycosylated IgG1. The minimal glycoform investigated had a trisaccharide attached to each heavy chain and can be expected to influence protein structure primarily in the proximity of the N-terminal region of the C(H)2 domain, implicated as a binding site for multiple effector ligands. These data provide a thermodynamic rationale for the modulation of antibody effector functions by different glycoforms.

The heavy and light chains of IgG monoclonal antibodies (mAbs) can be shown to be heterogeneous, with respect to isoelectric points, when analyzed by two-dimensional electrophoresis (2-DE). The molecular basis for this charge heterogeneity has not been clearly defined but it has been suggested that it could be due, in part, to differences in glycosylation. To investigate this possibility we have compared the 2-DE pattern of glycosylated and aglycosylated forms of the mouse IgG1 mAb (1B7-11), produced in vitro in the presence and absence of tunicamycin. Charge heterogeneity was shown not to be a consequence of glycosylation status. Intracellular and secreted IgG mAbs were also analyzed to investigate the time course of change in charge properties of the heavy and light chains. The charge heterogeneity was found to be generated intracellularly, and alterations in charge properties could be induced during incubation under physiological conditions. Semilogarithmic plots of the density of the principal heavy and light chain spots against incubation time showed linear relationships, suggesting that the charge shifts result from a first-order reaction. The semilogarithmic plot for the light chain correlated well with the time after IgG synthesis. These results suggest that the charge heterogeneity of an IgG mAb is due to intra- and extracellular modifications of the polypeptide chains which reflect "aging" of antibody molecules.

We proposed and demonstrated a method for reconstructing waveguide portions that were removed when dicing 45/spl deg/ mirrors onto other waveguides on the same substrate. The polymeric waveguide core divided by dicing at not optical input or output positions was repaired using a self-written waveguide formed with green laser irradiation. The optical coupling loss at the cut point was obtained to be 0.4 dB. We obtained optical output with low excess loss from the repaired waveguide.

The optical solder effect of the self-written waveguide (SWW) using Rhodamine 6G dye-dispersed ultraviolet (UV) curable resin by a green laser is demonstrated. This enables formation of SWW from the end of polyimide waveguide having strong UV absorption. High efficient coupling between an optical fiber and a vertical-cavity surface-emitting laser is successfully achieved

The immune response to different doses of a hapten of fluorescein isothiocyanate (FITC) in BALB/c mice was analyzed by two-dimensional affinity electrophoresis (2-D AEP). The mice were immunized with different doses (0.5 microgram, 5 micrograms, 50 micrograms, 500 micrograms and 2.5 mg) of FITC-conjugated bovine serum albumin (BSA). The antibodies to FITC bovine serum albumin (BSA) were separated into a large number of IgG spots due to differences in their isoelectric points (pI) and binding affinity to FITC ligand immobilized in the gel. The IgG spots, showing identical affinity to the ligand but different pI, have been considered as an IgG family. The affinity and quantity of IgG families changed with the increase in FITC-BSA dosage. With a low dose (5 micrograms) most of the families showed high affinity (Kd1 microMKd50 microM) and low affinity (Kd50 microM) antibodies were produced. The increase of FITC-BSA up to 500 micrograms markedly increased the quantity of IgG spots showing a variety of affinity to FITC. However, 2.5 mg FITC-BSA did not increase the quantity and heterogeneity of IgG spots significantly. The changes in the heterogeneity and quantity of anti-FITC antibodies and the subclass switch were observed over the course of immunization. The heterogeneity and the quantity of IgG1, IgG2b and IgG3 antibodies increased markedly during the first and the second immunization, whereas an increase in the heterogeneity and the quantity of IgG2a antibody was observed in the third immunization. This suggests that the subclass switch to IgG1, IgG2b and IgG3 and the somatic mutation of IgG1, IgG2b and IgG3 occur during the first and the second immunization, but the subclass switch to IgG2a and the somatic mutation of IgG2a seem to occur later than that of the other IgG subclasses.

Background: Lung cancer is one of the refractory malignancies and the leading cause of cancer-related death worldwide. Although prognosis of lung cancer was improved by the anticancer drugs and molecular targeted therapies, the results are not sufficient. Cellular senescence is irreversible cell cycle arrest. The drug-dependent induction of cellular senescence in neoplastic cells is considered an important tumor suppressive mechanism. 5-aza-2’-deoxycytidine (5-aza-dC) causes cellular senescence in solid tumors. Hypermethylation of CpG islands is well known as a major inactivation mechanism of tumor suppressor genes such as E-cadherin. Loss of E-cadherin confers a poor prognosis in lung cancer patients and is associated with in vitro resistance to epidermal growth factor receptor inhibitors. This study was designed to assess the relationship of E-cadherin expression cellular senescence induced by 5-aza-dC in non-small cell lung cancer (NSCLC) cell lines. Materials and Methods: We investigated two NSCLC cell lines; H1299 and A549 reported they have hypermethylation in promoter lesion of E-cadherin. There are two clinically approved DNA methyltransferase inhibitors (DNMTi), 5-aza-dC and 5-azacytidine (5-aza-CR). We also used Sodium Butyrate (NaB). We treated them with each drug for 120 hours. Flow cytometry analysis, immunofluorescence staining for E-cadherin and senescence associated beta-galactosidase assays and immunoblotting for senescence related proteins assessed NSCLC cell outgrowth and morphology. Results: Both cell lines with 5-aza-dC or NaB treatment changed morphology and increased size but decreased cell numbers. 5-aza-CR causes apoptosis in both cell lines. In A549, E-cadherin expression increased after 5-aza-dC treatment. There is no E-cadherin expression in H1299 with or without treatment. Both cell lines with 5-aza-dC were positive for Senescence-associated beta-galactosidase staining, H3K9me3 and gamma-H2AX. Conclusion: Taking together these findings strongly suggest the ability of 5-aza-dC to induce cellular senescence in NSCLC cells. The cellular senescence induced by a 5-aza-dC approach has to be considered a therapeutic option for NSCLC and an important phenomenon to further investigate in the next future. Citation Format: Masashi Furukawa, Yuka Mimura, Hiroyuki Tao, Yusuke Mimura, Kazunori Okabe. 5-aza-2’-Deoxycytidine induces cellular senescence in non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2842.

Mouse antidextran monoclonal antibodies showed microheterogeneity which was analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Not only the heavy (H) chains but also the light (L) chains were heter ogeneous in terms of isoelectric point (pI). The higher the pI, the more prominent the H chain spots. To demonstrate the cause of the microheterogeneity an IgG1 monoclonal antibody (mAb 35.8.2H) was examined especially for involvement of the sugar moiety in the microheterogeneity. The glycosylated region was determined in the Fc portion from serine 239 to methionine 309 by a glycan detection method using mild periodate oxidation, which confirms that the sugar chain is attached to the conserved glycosylation site of asparagine 297. However, charge heterogeneity of the H chain was not entirely attributed to the Fc because the papain digest of the antibody was separated into two Fc spots, a few Fd spots and two L chain spots by 2-D PAGE. This indicates that factors other than the sugar moiety are responsible for charge heterogeneity of IgG monoclonal antibody. On the other hand, the H chain isoforms of lower pI were shown to be more susceptible to V8 protease by peptide mapping. This result strongly suggests the occurrence of deamidation at glutamine or asparagine residues.

Immune response to a hapten of fluorescein isothiocyanate (FITC) in a single BALB/c strain mouse was analyzed by two-dimensional affinity electrophoresis (2D-AEP). Anti-FITC antibodies were induced by immunization with FITC-conjugated bovine serum albumin. The antibodies were separated into a large number of spots of IgG due to differences in their isoelectric points(pI) and binding affinities to the FITC ligand. These spots consisted of IgG families which were composed of several spots having an identical affinity to the ligand but a different pI. The spots were not clearly detected in the antiserum taken on day 7 after the primary immunization, but on day 21 the spots of IgG were clearly detected, with a high diversity and specificity for the ligand. The size and number of IgG spots were markedly increased by the secondary immunization; however, the third immunization did not increase the size and number of IgG spots. The IgG spots of each family were specifically stained with an antimouse IgG subclass antibody. Furthermore, a monoclonal antibody (FL-D6) was separated by 2D-AEP into a single family which consisted of seven IgG1 spots having an identical affinity to FITC but different pIs. Therefore, each of the IgG families of anti-FITC antibodies in the antiserum can be generated by a single clone of anti-FITC antibody-producing cells. The substitution of dextran T 2000 or lipopolysaccharide for bovine serum albumin as a carrier for FITC induced much smaller amounts of anti-FITC antibodies with a low diversity but high specificity to FITC.

SummaryGlycan recognition leading to cell-cell interactions, signaling, and immune responses is mediated by various glycan-binding proteins (GBPs) showing highly diverse ligand specificities. We describe here a rapid glycan immobilization technique via 4-hydrazinobenzoic acid (HBA)-functionalized beads and its application to high-throughput screening of miniature pig kidney N-glycan-binding proteins by using a mass-spectrometric approach. Without any derivatization steps, the purified pig kidney N-glycans were directly immobilized on to HBA-functionalized beads and subsequently used to identify GBPs from human serum. This screening method showed remarkable performance for identifying potential GBPs closely involved in pig-to-human xenograft rejection mediated by human serum, including antibodies, cytokines, complement components, siglec, and CD antigens. Thus, these results demonstrate that the GBP screening method was firmly established by one-step immobilization of the N-glycans on to microsphere and highly sensitive mass-spectrometric analysis.

A simple and efficient synthesis of N-linked glycoamino acids and glycopeptides from deprotected sugars using the Staudinger reaction.

We have developed "tapered self-written waveguide (SWW)" for coupling of optical components in the board level optical interconnection. Tapered waveguides have a possibility of achieving larger alignment tolerance for optical coupling. The fabrication condition of tapered SWW was studied by adjusting both the optical power and irradiation time of curing resin, and tapered SWW was successfully realized. The optical tolerance vertical to the optical axis twice as compared with straight SWW was obtained.

In the optical interconnection of board level, 45-degree micro mirrors are used to achieve 90-degree optical path change. These mirrors are often fabricated by using a rotating blade and this method has a serious problem of cutting other waveguides in the vicinity. A self-written waveguide ha been successfully applied to repari and connect these waveguides. So, it will be possible to allow optical signals to be input and output at a specific location on the board.

Class II molecules of the major histocompatibility complex (MHC class II molecules) bind peptides derived from protein antigens delivered into endocytic compartments and present these peptides to CD4+ T cells (Cresswell, 1994; Germain, 1994). In the endocytic pathway antigens are unfolded and cleaved into fragments during transport through the increasingly acidic endosomal network, from early endosomes to lysosomes. Newly synthesized MHC class II molecules associated with the invariant chains (Ii) are transported to a late endocytic compartment where the Ii is removed by proteolytic cleavage. MHC class II molecules bind polypeptide antigens by removal of the Ii-derived peptide CLIP (class II invariant chain-derived peptide) through a peptide-exchange process that is catalyzed by MHC-encoded DM molecules (Fig. 1) (Denzin and Cresswell, 1995; Sherman et al., 1995; Sloan et al., 1995; Wolf and Ploegh, 1995). The initial form of antigen that binds to class II molecules may be short peptides 10-20 amino acids in length, generated by acid endopeptidases (Watts, 2001). This pathway could be termed ‘‘cut/trim first, bind later’’ model and became the paradigm for the binding of peptides to MHC class II molecules. However, there is an alternative pathway independent of DM molecules that involves early capture of unfolded, extended sequence byMHC class II molecules (Pinet et al., 1994; Pinet et al., 1995; Sercarz et al., 1993). Subsequent trimming of