Establishment of a novel cell line, CHO-MK, derived from Chinese hamster ovary tissues for biologics manufacturing.

Chinese hamster ovary (CHO) cells are widely used as a host for producing recombinant therapeutic proteins due to advantages such as human-like post-translational modification, correct protein folding, higher productivity, and a proven track record in biopharmaceutical development. Much effort has been made to improve the process of recombinant protein production, in terms of its yield and productivity, using conventional CHO cell lines. However, to the best of our knowledge, no attempts have been made to acquire new CHO cell lines from Chinese hamster ovary. In this study, we established and characterized a novel CHO cell line, named CHO-MK, derived from freshly isolated Chinese hamster ovary tissues. Some immortalized cell lines were established via sub-culture derived from primary culture, one of which was selected for further development toward a unique expression system design. After adapting serum-free and suspension culture conditions, the resulting cell line exhibited a considerably shorter doubling time (approximately 10 h) than conventional CHO cell lines (approximately 20 h). Model monoclonal antibody (IgG1)-producing cells were generated, and the IgG1 concentration of fed-batch culture reached approximately 5 g/L on day 8 in a 200-L bioreactor. The cell bank of CHO-MK cells was prepared as a new host and assessed for contamination by adventitious agents, with the results indicating that it was free from any such contaminants, including infectious viruses. Taking these findings together, this study showed the potential of CHO-MK cells with a shorter doubling time/process time and enhanced productivity in biologics manufacturing.

Development of a stable antibody production system utilizing an Hspa5 promoter in CHO cells.

Chinese hamster ovary (CHO) cells are widely used for manufacturing antibody drugs. We attempted to clone a novel high-expression promoter for producing monoclonal antibodies (mAbs) based on transcriptome analysis to enhance the transcriptional abundance of mAb genes. The efficacy of conventional promoters such as CMV and hEF1α decrease in the latter phase of fed-batch cell culture. To overcome this, we screened genes whose expression was maintained or increased throughout the culture period. Since CHO cells have diverse genetic expression depending on the selected clone and culture medium, transcriptome analysis was performed on multiple clones and culture media anticipated to be used in mAb manufacturing. We thus acquired the Hspa5 promoter as a novel high-expression promoter, which uniquely enables mAb productivity per cell to improve late in the culture period. Productivity also improved for various IgG subclasses under Hspa5 promoter control, indicating this promoter's potential universal value for mAb production. Finally, it was suggested that mAb production with this promoter is correlated with the transcription levels of endoplasmic reticulum stress-related genes. Therefore, mAb production utilizing the Hspa5 promoter might be a new method for maintaining protein homeostasis and achieving stable expression of introduced mAb genes during fed-batch culture.

Novel cell line development strategy for monoclonal antibody manufacturing using translational enhancing technology.

Chinese hamster ovary (CHO) cells are widely used for constructing expression systems to produce therapeutic proteins. However, the establishment of high-producer clones remains a laborious and time-consuming process, despite various progresses having been made in cell line development. We previously developed a new strategy for screening high monoclonal antibody (mAb)-producing cells using flow cytometry (FCM). We also reported that p180 and SF3b4 play key roles in active translation on the endoplasmic reticulum, and that the productivity of secreted alkaline phosphatase was enhanced by the overexpression of p180 and SF3b4. Here, we attempted to apply the translational enhancing technology to high mAb-producing cells obtained after high-producer cell sorting. A high mAb-producing CHO clone, L003, which showed an mAb production level of >3 g/L in fed-batch culture, was established from a high mAb-producing cell pool fractionated by FCM. Clones generated by the overexpression of p180 and SF3b4 in L003 cells were evaluated by fed-batch culture. The specific productivity of clones overexpressing these two factors was ∼3.1-fold higher than that of parental L003 cells in the early phase of the culture period. Furthermore, the final mAb concentration was increased to 9.5 g/L during 17 days of fed-batch culture after optimizing the medium and culture process. These results indicate that the overexpression of p180 and SF3b4 would be promising for establishing high-producer cell lines applicable to industrial production.

Postoperative clinical outcomes of unicompartmental knee arthroplasty in patients with isolated medial compartmental osteoarthritis following medial meniscus posterior root tear.

BACKGROUND: Cartilage degradation progresses rapidly following medial meniscus posterior root tear (MMPRT). Unicompartmental knee arthroplasty (UKA) has been performed for medial compartmental osteoarthritis following MMPRT. We evaluated the clinical and radiographic outcomes of UKA for medial compartmental osteoarthritis after an untreated MMPRT. METHODS: Twenty-one patients who underwent UKA for isolated medial compartment osteoarthritis following MMPRT were retrospectively investigated. Clinical outcomes were assessed using the Knee Injury and Osteoarthritis Outcome Score and knee range of motion. The posterior tibial slope and tibial component inclination were evaluated using plain radiographs. RESULTS: The mean follow-up periods were 25.5 ± 13.8 months. Clinical outcomes improved significantly postoperatively. The mean postoperative knee extension angle was -1.1° ± 2.1°, and the knee flexion angle was 134.3° ± 4.9°. The posterior tibial slope angle decreased from 9.0° ± 2.0° preoperatively to 5.4° ± 1.8° postoperatively, and postoperative tibial component inclination at the final follow-up was 2.9° ± 1.1° varus. No aseptic loosening or deep infections were observed. CONCLUSION: UKA significantly improved clinical outcomes and could be a viable surgical option for treating isolated medial compartmental osteoarthritis accompanied by untreated MMPRT.

Tibial Tunnel Positioning Using the Posterolateral (PL) Divergence Guide in Anterior Cruciate Ligament Reconstruction.

The aim of this study was to evaluate tunnel coalition and inter-tunnel distance by comparing the tibial tunnel position in double-bundle anterior cruciate ligament (ACL) reconstruction performed with a conventional guide versus a posterolateral (PL) divergence (PLD) guide. Subjects were 43 patients (ACL tip aimer: 20 knees; PLD guide: 23 knees) who underwent double-bundle ACL reconstruction between September 2014 and December 2017. In all cases, the tibial tunnel position, tunnel edge distance and tunnel angles were evaluated based on CT images. Clinical outcome was evaluated using the Lachman test, pivot-shift test, and Lysholm score. Tibial tunnel positions were similar between the conventional and PLD guide groups, while tibial tunnel edge distance was significantly less in the conventional group. Tunnel coalition was observed in 5 knees in the conventional and no knees in the PLD guide group. Distance between two tibial tunnel centers was 9.1 mm for the tip aimer, and 10.5 mm for the PLD guide. Creation of the PL tunnel tended to involve insertion from a more medial aspect for the PLD guide group than the conventional guide group. No differences in clinical outcomes were noted. The PLD guide can be used to create anatomically-positioned PL tunnels, and reduce the probability of occurrence of tunnel coalition.

Combining pullout suture and retrograde screw fixation for anterior cruciate ligament tibial eminence avulsion fractures: A case report.

This report describes a novel arthroscopic technique for the treatment of anterior cruciate ligament (ACL) tibial eminence avulsion fractures. A 16-year-old boy who was diagnosed with a left ACL tibial eminence avulsion fracture was treated by arthroscopic fixation. Two bone tunnels were created from the anterior tibial cortex into the fracture bed, and a strong suture passed through the ACL just above its insertion was pulled out through them for reduction and fixation. A retrograde cannulated screw fixation was added for stronger fixation. Weight-bearing and range of motion exercises were started immediately after surgery. Radiographically, bone union was obtained 6 months postoperatively. During second-look arthroscopy (24 months postoperatively), there was no loss of reduction and no subsequent meniscal or cartilage injuries. At that point, the Lysholm score was 95, and the International Knee Documentation Committee score was 96.

Outcomes and Prognosis of Neurological Decompression and Stabilization for Spinal Metastasis: Is Assessment with the Spinal Instability Neoplastic Score Useful for Predicting Surgical Results?

STUDY DESIGN: Retrospective study. PURPOSE: To evaluate the efficacy of the Spinal Instability Neoplastic Score (SINS) in predicting surgical outcomes and survival. Patients were categorized into two groups according to the SINS, and their surgical outcomes and survival following decompression and stabilization were assessed. OVERVIEW OF LITERATURE: Palliative surgery in patients with a life expectancy ≥3 months may effectively improve their overall condition in the long term. Currently, the effectiveness of the SINS for predicting surgical results and survival remains controversial. METHODS: This study included 44 patients who underwent decompression and stabilization for spinal metastases at Yokosuka Kyosai Hospital between 2008 and 2017. The patients were divided into two groups: stable (SINS ≤12) and unstable (SINS ≥13). Changes in the Frankel score and Eastern Cooperative Oncology Group Performance Status (ECOG-PS) were compared between the two groups, and patient survival was evaluated according to the SINS, Tokuhashi score, and Katagiri score. RESULTS: The stable group (SINS range, 7-12) included 24 patients while the unstable group (SINS range, 13-16) included 20 patients. The Frankel score significantly improved from 2.8 to 3.6 in the stable group (p <0.001) and from 2.7 to 3.9 in the unstable group (p <0.001). The ECOG-PS significantly improved from 3.2 to 2.1 in the stable group (p <0.001) and from 3.0 to 1.8 in the unstable group (p <0.001). There was a statistically significant difference in median survival between the two groups. CONCLUSIONS: All patients treated with palliative surgery showed favorable outcomes, as indicated by improved the Frankel score and ECOG-PS following surgery. However, median survival was significantly better in the stable group. The results of this study indicate that the SINS is appropriate for surgical decision making and may be used to predict survival.

The surgical outcome of decompression alone versus decompression with limited fusion for degenerative lumbar scoliosis.

OBJECTIVE The aim of this study was to assess the usefulness of radiological parameters for surgical decision-making in patients with degenerative lumbar scoliosis (DLS) by comparing the clinical and radiological results after decompression or decompression and fusion surgery. METHODS The authors prospectively planned surgical treatment for 298 patients with degenerative lumbar disease between September 2005 and March 2013. The surgical method used at their institution to address intervertebral instability is precisely defined based on radiological parameters. Among 64 patients with a Cobb angle ranging from 10° to 25°, 57 patients who underwent follow-up for more than 2 years postoperatively were evaluated. These patients were divided into 2 groups: those in the decompression group underwent decompression alone (n = 25), and those in the fusion group underwent decompression and short segmental fusion (n = 32). Surgical outcomes were reviewed, including preoperative and postoperative Cobb angles, lumbar lordosis based on radiological parameters, and Japanese Orthopaedic Association (JOA) scores. RESULTS The JOA scores of the decompression group and fusion group improved from 5.9 ± 1.6 to 10.0 ± 2.8 and from 7.2 ± 2.0 to 11.3 ± 2.8, respectively, which was not significantly different between the groups. At the final follow-up, the postoperative Cobb angle in the decompression group changed from 14° ± 2.9° to 14.3° ± 6.4° and remained stable, while the Cobb angle in the fusion group decreased from 14.8° ± 4.0° to 10.0° ± 8.5° after surgery. CONCLUSIONS The patients in both groups demonstrated improved JOA scores and preserved Cobb angles after surgery. The improvement in JOA scores and preservation of Cobb angles in both groups show that the evaluation of spinal instability using radiological parameters is appropriate for surgical decision-making.

Identification of regulatory motifs in the CHO genome for stable monoclonal antibody production.

Chinese hamster ovary (CHO) cell lines are widely used for therapeutic protein production. When a transgene is integrated into the genome of a CHO cell, the expression level is highly dependent on the site of integration because of positional effects such as gene silencing. To overcome negative positional effects and establish stable CHO cell lines with high productivity, several regulatory DNA elements are used in vector construction. Previously, we established the CHO DR1000L-4N cell line, a stable and high copy number Dhfr gene-amplified cell line. It was hypothesized that the chromosomal location of the exogenous gene-amplified region in the CHO DR1000L-4N genome contains regulatory motifs for stable protein production. Therefore, we isolated DNA regulatory motifs from the CHO DR1000L-4N cell line and determined whether these motifs act as an insulator. Our results suggest that stable expression of a transgene can be promoted by the CHO genome sequence, and it would be a powerful tool for therapeutic protein manufacturing.

Features of human autologous hamstring graft elongation after pre-tensioning in anterior cruciate ligament reconstruction.

PURPOSE: Precise pre-tensioning protocol of the graft has not been determined in anterior cruciate ligament (ACL) reconstruction. The aims of this study are to measure the human autologous graft elongation, and to reveal what portion of the graft elongated greater after pre-tensioning in ACL reconstruction. METHODS: Twenty-four hamstring tendon grafts which were harvested from patients were included. A continuous load of 150 N was applied to the graft twice for 30 seconds each (150 N-1 minute), and the same loading was repeatedly applied (150 N-2 minute). The amount of elongation of the tendon portion (Length T) and the stitched portion (Length S) were measured after each pre-tensioning. RESULTS: Length S gradually increased by 1.57 ± 0.67 mm after the 150 N-1 minute pre-tensioning and by 2.12 ± 0.76 mm after the 150 N-2 minute pre-tensioning, respectively. Length T was not significantly elongated after 150 N-1 min (p = 0.66) and 150 N-2 min (p = 0.59). CONCLUSIONS: Graft elongation of the approximately 2 mm was observed, particularly in the stitched portion. It is necessary for a surgeon to focus on careful removal of slack from each stitch during suturing.

Efficient enrichment of high-producing recombinant Chinese hamster ovary cells for monoclonal antibody by flow cytometry.

To screen a high-producing recombinant Chinese hamster ovary (CHO) cell from transfected cells is generally laborious and time-consuming. We developed an efficient enrichment strategy for high-producing cell screening using flow cytometry (FCM). A stable pool that had possibly shown a huge variety of monoclonal antibody (mAb) expression levels was prepared by transfection of an expression vector for mAb production to a CHO cell. To enrich high-producing cells derived from a stable pool stained with a fluorescent-labeled antibody that binds to mAb presented on the cell surface, we set the cell size and intracellular density gates based on forward scatter (FSC) and side scatter (SSC), and collected the brightest 5% of fluorescein isothiocyanate (FITC)-positive cells from each group by FCM. The final product concentration in a fed-batch culture of cells sorted without FSC and SSC gates was 1.2-1.3-times higher than that of unsorted cells, whereas that of cells gated by FSC and SSC was 3.4-4.7-fold higher than unsorted cells. Surprisingly, the fraction with the highest final product concentration indicated the smallest value of FSC and SSC, and the middle value of fluorescence intensity among all fractionated cells. Our results showed that our new screening strategy by FCM based on FSC and SSC gates could achieve an efficient enrichment of high-producing cells with the smallest value of FSC and SSC.

Effect of mustard gas exposure on incidence of lung cancer: a longitudinal study.

Sulfur mustard, an agent used in chemical warfare, is an alkylating substance with carcinogenic potential. However, the precise long-term carcinogenic effects of mustard gas are unclear. Since 1952, the authors have conducted health surveys of former workers who were employed from 1929 to 1945 in a poisonous gas factory in Okuno-jima, Hiroshima, Japan. This prospective study was undertaken from 1952 to 2005 to examine the incidence of lung cancer among the workers who were exposed to mustard gas (n=480), lewisite (n=55), and/or diphenylcyanarsine (n=178), as well as the incidence among unexposed workers (n=969). The stochastic relation between exposure and lung cancer was explored on the basis of multistage carcinogenesis by using an accelerated hazard model with a transformed age scale. Mustard gas exposure was found to transform the age scale for developing lung cancer. One year of exposure in subjects ≤18 or >18 years old at first exposure shifted the age scale down by 4.9 years and 3.3 years, respectively. On the basis of the long-term follow-up of former workers in the poisonous gas factory, the authors concluded that sulfur mustard decreased the age at which people were at risk of developing lung cancer and that the effect declined with aging.

Rapid and high-sensitivity cell-based assays of protein-protein interactions using split click beetle luciferase complementation: an approach to the study of G-protein-coupled receptors.

To identify biologically relevant compounds in basic biology and drug discovery processes, rapid quantitative methods for elucidating protein-protein interactions have become necessary. We describe a novel optical technique for monitoring protein-protein interactions in living cells based on complementation of split luciferase fragments from click beetle (Brazilian Pyrearinus termitilluminans). A new pair of amino-terminal and carboxy-terminal fragments of the luciferase was identified using semirational library screening, demonstrating achieved markedly higher sensitivity and signal-to-background ratio. The identified fragments were applied to the study of five G-protein coupled receptors (GPCR) that interact with beta-arrestin on the plasma membrane. By generating cell lines stably expressing the GPCRs and beta-arrestin connected with the luciferase fragments, we demonstrated rapid and sensitive screening of potential chemicals that act on GPCRs using a 96-well microtiter plate format. The screening time was reduced to 5-10 min after ligand stimulation. The maximum response became more than 15-fold higher than the background signal. This luciferase complementation method also enabled accurate spatial and temporal analyses of interactions in single living cells using bioluminescence microscopy. These GPCR assays will facilitate developments of high-throughput screening systems in a multiwell plate format. Furthermore, using specific proteins of interest, the novel fragments of luciferase will provide different assay methods for the study of many intracellular signals in living cells and animals.

Primary cervical epidural malignant lymphoma with rheumatoid arthritis: a case report.

We report a case of primary cervical epidural malignant lymphoma with rheumatoid arthritis. Because of the acute progression of paralysis in both legs, surgical decompression and stabilization of the cervical spine were performed. The resected specimen showed proliferation of lymphoblastic cells diagnosed as malignant lymphoma. Four series of chemotherapy were administered after surgery, and the patient recovered from paralysis.

The role of interface framework residues in determining antibody V(H)/V(L) interaction strength and antigen-binding affinity.

While many antibodies with strong antigen-binding affinity have stable variable regions with a strong antibody heavy chain variable region fragment (V(H))/antibody light chain variable region fragment (V(L)) interaction, the anti-lysozyme IgG HyHEL-10 has a fairly strong affinity, yet a very weak V(H)/V(L) interaction strength, in the absence of antigen. To investigate the possible relationship between antigen-binding affinity and V(H)/V(L) interaction strength, a novel phage display system that can switch two display modes was employed. We focused on the two framework region 2 regions of the HyHEL-10 V(H) and V(L), facing each other at the domain interface, and a combinatorial library was made in which each framework region 2 residue was mixed with that of D1.3, which has a far stronger V(H)/V(L) interaction. The phagemid library, encoding V(H) gene 7 and V(L) amber codon gene 9, was used to transform TG-1 (sup+), and the phages displaying functional variable regions were selected. The selected phages were then used to infect a nonsuppressing strain, and the culture supernatant containing V(H)-displaying phages and soluble V(L) fragment was used to evaluate the V(H)/V(L) interaction strength. The results clearly showed the existence of a key framework region 2 residue (H39) that strongly affects V(H)/V(L) interaction strength, and a marked positive correlation between the antigen-binding affinity and the V(H)/V(L) interaction, especially in the presence of a set of particular V(L) residues. The effect of the H39 mutation on the wild-type variable region was also confirmed by a SPR biosensor as a several-fold increase in antigen-binding affinity owing to an increased association rate, while a slight decrease was observed for the single-chain variable region.

Primary lymphoma of the heart: case report and literature review.

Primary cardiac lymphoma (PCL) is a rare and usually fatal neoplasma. A case of PCL in a 78-year-old man who complained of exertional dyspnea and peripheral edema is presented. Echocardiography revealed a mass in the right atrium and a diagnosis of low-grade B-cell lymphoma was obtained with the surgically resected tumor. The lesion appeared to have originated in the right atrium and involved the right ventricle. The patient died of bronchopneumonia 8 months after the initial consultation. The present case and 39 patients with PCL reported between 1995 and 2002 were reviewed. Forty patients showed various and non-specific symptoms such as dyspnea, edema, arrhythmia and pericardial effusion. Primary cardiac lymphoma occurred slightly more often in male patients (M : F = 23:17) and in the elderly in general (mean age, 67 years), with lesions found in the following locations, listed in order of frequency: right atrium, pericardium, right ventricle, left atrium, left ventricle, and other sites. Antemortem diagnosis was obtained in 37 of the 40 patients. Thirty-seven cases were of B-cell lineage and two cases were of T-cell lineage. Complete remission was obtained in only 15 of the 40 patients. Although PCL antemortem diagnoses have been made in the majority of recent cases, the prognosis still remains poor.

Diversity of restitution after deoxycholic acid-induced small intestinal mucosal injury in the rat.

Repair of superficial damage to gastrointestinal mucosa occurs by a process called restitution, with epithelial integrity and continuity reestablished before cell proliferation occurs. The aim of the present study was to investigate the diversity of restitution in rat jejunum exposed to different concentrations of deoxycholic acid (DOC; 1.5-100 mmol/liter). Following a 30-min exposure, the intestine was allowed to recover for 15-330 minutes. DOC caused dose-dependent tissue destruction. Exfoliating epithelial cells were already observed after 5 min of exposure (1.5 mmol/liter), with simple sloughing off and resealing of the tips. Moderately affected epithelium (20 mmol/liter) demonstrated denudation of villous tips and then became covered with goblet cells. Severely affected epithelium (100 mmol/liter) also appeared to be replaced with goblet cells. These data suggest that the reversibility of mucosal damage induced by DOC is due to a variety of processes, which depend on the severity of the mucosal insult.

A general method to select antibody fragments suitable for noncompetitive detection of monovalent antigens.

Previously, immunological detection of a small hapten was only possible in competitive format, which needed a competitor antigen either labeled by a reporter or attached to a carrier protein. Recently, we proposed the open sandwich (OS) immunoassay, a simple immunoassay that can noncompetitively determine monovalent antigen concentration by measuring the antigen-dependent change in a heavy-chain variable region (VH)/light-chain variable region (VL) interaction of an antibody. However, there was a limitation in the assay that the antibody used should have a suitable property such that the VH/VL interaction would become fairly strong along with the addition of antigen. Here, we devised a phage-based "split-Fv system" to rapidly evaluate and select antibody variable region (Fv) fragments that are suitable to OS immunoassay. When three antibodies raised against endocrine disruptor bisphenol A were tested with this system, all were more or less suitable to OS-ELISA. Among them, the best Fv selected was used to construct fusion proteins of VH tethered to an alkaline phosphatase and a tagged VL that can be site-specifically biotinylated to perform direct OS-ELISA. The results showed that the OS-ELISA detects bisphenol A with higher sensitivity than the corresponding competitive assay, also implying that many antibodies to small haptens have suitable properties for OS-ELISA.

Solid-state 13C and 1H spin diffusion NMR analyses of the microfibril structure for bacterial cellulose.

To obtain further information about the cause for the rather large splitting of the C4 resonance line into the downfield (C4D) and upfield (C4U) lines in CP/MAS 13C NMR spectra for native cellulose, 13C and 1H spin diffusion measurements have been conducted by using different types of bacterial cellulose samples. In 13C spin diffusion measurements, the C4D resonance line is selectively inverted by the Dante pi pulse sequence and the 13C spin diffusion is allowed to proceed from the C4D carbons to other carbons including the C4U carbons with use of the 13C4-enriched bacterial cellulose sample. The analysis based on the simple spin diffusion theory for the process experimentally observed reveals that the C4U carbons may be located at distances less than about 1 nm from the C4D carbons. In 1H spin diffusion measurements, poly(vinyl alcohol) (PVA) films in which ribbon assemblies of bacterial cellulose are dispersed are employed and the 1H spin diffusion process is examined from the water-swollen PVA continuous phase to the dispersed ribbon assemblies by the 13C detection through the 1H-13C CP technique. As a result, it is found that the C4D and C4U carbons are almost equally subjected to the 1H spin diffusion from the PVA phase, indicating that the C4U carbons are not localized in some limited area, e.g. in the surfacial region, but are distributed in the whole area in the microfibrils. These experimental results suggest that the C4U carbons may exist as structural defects probably due to conformational irregularity associated with disordered hydrogen bonding of the CH(2)OH groups in the microfibrils.

Structure and enzymatic properties of a stable complex of the human REV1 and REV7 proteins.

With yeast Saccharomyces cerevisiae, results from a variety of genetic and biochemical investigations have demonstrated that the REV genes play a major role in induction of mutations through replication processes that directly copy the damaged DNA template during DNA replication. However, in higher eucaryotes functions of homologues are poorly understood and appear somewhat different from the yeast case. It has been suggested that human REV1 interacts with human REV7, this being specific to higher eucaryotes. Here we show that purified human REV1 and REV7 proteins form a heterodimer in solution, which is stable through intensive purification steps. Results from biochemical analysis of the transferase reactions of the REV1-REV7 complex demonstrated, in contrast to the case of yeast Rev3 whose polymerase activity is stimulated by assembly with yeast Rev7, that human REV7 did not influence the stability, substrate specificity, or kinetic parameters of the transferase reactions of REV1 protein. The possible role of human REV7 is discussed.