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Callyspongiolide is a marine macrolide known to induce caspaseindependent cancer cell death. While its toxic effects have been known, the mechanism leading to cell death is yet to be identified. We report that Callyspongiolide R form at C-21 (cally2R) causes mitochondrial dysfunction by inhibiting mitochondrial complex I or II, leading to a disruption of mitochondrial membrane potential and a deprivation of cellular energy. Subsequently, we observed, using electron microscopy, a drastic formation of autophagosome and mitophagy. Supporting these data, LC3, an autophagosome marker, was shown to co-localize with LAMP2, a lysosomal protein, showing autolysosome formation. RNA sequencing results indicated the induction of hypoxia and blocking of EGF-dependent pathways, which could be caused by induction of autophagy. Furthermore, mTOR and AKT pathways preventing autophagy were repressed while AMPK was upregulated, supporting autophagosome progress. Finally, the combination of cally2R with known anti-cancer drugs, such as gefitinib, sorafenib, and rapamycin, led to synergistic cell death, implicating potential therapeutic applications of callyspongiolide for future treatments. [BMB Reports 2021; 54(4): 227-232].

A Quality by Design (QbD) concept was applied to characterize a cell culture process for production of the recombinant Factor VIII (rFVIII). We characterized the production bioreactor process and defined the design space by applying risk assessment to determine potential critical process parameters (CPPs) impacting critical quality attributes (CQAs). Characterization studies were subsequently performed using a qualified scaled-down model (SDM) and a multi-factorial design of experiment (DOE) approach to determine both the individual and combined impacts of the potential CPPs on CQAs. Among the operating parameters characterized, production temperature, production pH and a shift in the timing of production affected rFVIII activity and tyrosine sulfation level. Finally, we identified CPPs and established a design space for the cell culture process to identify appropriate conditions for routine manufacturing.

Conditioned media from various sources comprise numerous growth factors and cytokines and are known to promote the regeneration of damaged tissues. Among these, natural killer cell conditioned medium (NK‑CdM) has been shown to stimulate collagen synthesis and the migration of fibroblasts during the wound healing process. With a long‑term aim of developing a treatment for skin photoaging, the ability of NK‑CdM to prevent ultraviolet‑B (UV‑B) damage was assessed in neonatal human dermal fibroblasts (NHDFs) and an in vitro reconstructed skin model. The factors present in NK‑CdM were profiled using an antibody array analysis. Protein and mRNA levels in UV‑B exposed NHDFs treated with NK‑CdM were measured by western blotting and quantitative reverse transcription‑PCR, respectively. The total antioxidant capacity of NK‑CdM was determined to assess its ability to suppress reactive oxygen species. The anti‑photoaging effect of NK‑CdM was also assessed in a 3D reconstituted human full skin model. NK‑CdM induced proliferation of UV‑B‑treated NHDFs, increased procollagen expression, and decreased matrix metalloproteinase (MMP)‑1 expression. NK‑CdM also exhibited a potent antioxidant activity as measured by the total antioxidant capacity. NK‑CdM inhibited UV‑B‑induced collagen degradation by inactivating MAPK signaling. NK‑CdM also elicited potential anti‑wrinkle effects by inhibiting the UV‑B‑induced increase in MMP‑1 expression levels in a 3D reconstituted human full skin model. Taken together, the suppression of both UV‑B‑induced MMP‑1 expression and JNK activation by NK‑CdM suggests NK‑CdM as a possible candidate anti‑skin aging agent.

Background context Laminoplasty aims to decompress the spinal cord and stabilize the cervical spine in patients with multilevel cervical lesions. Not every patient with cervical compressive myelopathy is a good candidate for laminoplasty. Most studies recommend that neutral or kyphotic alignments are contraindications for laminoplasty. However, cervical sagittal alignment does not have a strong and consistent effect on the clinical outcomes of laminoplasty. Moreover, many reports on the effect of cervical sagittal alignment did not designate the ideal definition of alignment and used different definitions of lordosis. Purpose To identify the effect of preoperative cervical alignment according to two different definitions after midline splitting double-door laminoplasty. Study design Retrospective cohort study. Patient sample From August 2008 to September 2010, 58 patients were diagnosed with cervical myelopathy and treated with midline splitting double-door laminoplasty. Outcome measures The clinical results were assessed with the modified Japanese Orthopedic Association (JOA) score, neck disability index (NDI), and visual analog scale (VAS) and were compared to analyze the rate of change between preoperative and postoperative values. Postoperative radiological results at the final follow-up examinations were compared between groups to obtain the change in range of motion and sagittal alignment. Method The effect of cervical alignment on JOA, NDI, and VAS scales and also on change of alignment and change of range of motion (ROM) at the final follow-up examinations was analyzed statistically between two groups according to two different definitions such as Toyama classification and Cobb angle. Results No difference was found between the two groups according to Toyama classification in terms of the postoperative improvement rate of the modified JOA score (p=.086), decreasing rate of the VAS (p=.940) or NDI (p=.211), postoperatively. Additionally, no difference was found for the decreasing rate of ROM (p=.427) or sagittal alignment (p=.864) based on the radiological evaluation results. Also, there was no difference between two groups according to Cobb angle in terms of the modified JOA score (p=.743), VAS (p=.548), or NDI (p=.32), postoperatively. Additionally, no difference was found for the ROM (p=1.000) or sagittal alignment (p=.440) based on the radiological evaluation results. Conclusions Despite nonlordosis cervical sagittal alignment, double-door laminoplasty would be effective for patients with cervical myelopathy because of cervical spondylotic myelopathy or ossification of the posterior longitudinal ligament. Furthermore, sagittal alignment is not the absolute and sole factor that surgeons should consider when determining the optimal treatment strategy.

A new 3rd generation recombinant factor VIII (rFVIII), GreenGene F (WHO INN: beroctocog alfa), which is a highly homogenous B-domain deleted FVIII protein comprising of two peptides as heavy chain (A1 and A2 domain) and light chain (A3, C1, and C2 domain) at 80 and 90 kDa, was developed from its predecessor product GreenGene (2nd generation product previously approved by Korea FDA after clinical studies in South Korea) by process improvements of i) addition of Solvent/Detergent treatment for virus inactivation, ii) nanofiltration (20 nm pore size) for viral removal and iii) alterations to an albumin-free formulation to minimize the risk of viral contamination. An assessment of comparability between the two products was made to see if process improvements for safer product manufacturing affected the rFVIII structural and functional characteristics. Physicochemical and physiological characteristics were observed, in vivo efficacy following a single intravenous administration to FVIII knock-out mice and toxicity by various GLP in vivo tests were evaluated. All results showed equivalence, proving that no changes in protein characteristics of rFVIII occurred from process changes in formulation, viral inactivation, and viral removal which minimize the risk of pathogen transmission to enhance safety.

We describe a case of foreign body synovitis caused by delayed intra-articular migration of the outer sheath 5 months after anterior cruciate ligament (ACL) reconstruction with a quadrupled tibialis allograft tendon using the IntraFix device for tibial fixation. The postoperative course was unremarkable. At 5 months after surgery, the patient experienced a sudden catching sensation and a slight pain without any obvious twisting or trauma. At 6 months after surgery, extension deficit was 20°. At arthroscopy, intra-articular migration of the outer sheath from the tibial tunnel and reactive synovitis were observed. The outer sheath in the joint and the inner screw in the tibial tunnel were removed successfully. The ACL graft was well incorporated under good tension. Patient was able to return to her previous level of all daily activities with no further episodes of swelling. To our knowledge, described here is the only case of foreign body synovitis due to intra-articular migration of the unbroken sheath.

Background: The natural course of sarcoidosis is variable, but no single parameter has been generally accepted as a good marker for disease activity. Adhesion molecules are required for the migration of inflammatory cells; thus, they may be markers of activity in sarcoidosis. Methods: In 16 patients with active sarcoidosis and 11 with inactive disease (10 were male, 17 were female; mean age [6 SD], 39.6 6 11.0 years; mean follow-up, 21 6 16 months), the expression of adhesion molecules on cells obtained with BAL (measured by flow cytometry) and the level of soluble intercellular adhesion molecule 1 (sICAM-1) in the serum and BAL fluid (BALF) were measured at the time of diagnosis and during the follow-up. The changes in serum sICAM-1 level and ICAM-1 expression on cells obtained with BAL were compared with the clinical course of the disease. Results: In patients with active disease, the ICAM-1 on alveolar macrophage (AM) (relative linear median fluorescence intensity [RMFI], 3.21 6 1.55) and sICAM-1 levels in serum (575 6 221 ng/mL) and BALF (47.3 6 19.3 ng/mL) were higher than those for patients with inactive disease (RMFI, 1.67 6 0.66; p 5 0.0034; serum, 263 6 98.5 ng/mL; p 5 0.0001; BALF, 27.5 6 19.0 ng/mL; p 5 0.0209). In the patients with active disease, ICAM-1 on AM and serum sICAM-1 decreased (RMFI, 1.51 6 0.84; 284 6 118 ng/mL, respectively) after steroid therapy, but no significant change was noted in patients with inactive disease. We also found that the initial ICAM-1 on AM and serum sICAM-1 had a significant correlation with the degree of improvement in pulmonary function tests after the therapy. The disease relapsed in four patients after the discontinuation of steroids, and the serum sICAM-1 level was elevated again at the time of relapse. Conclusion: Our data suggest that the serum sICAM-1 level and the ICAM-1 expression on AM may be good markers of disease activity and also a predictor of outcome in sarcoidosis. (CHEST 1999; 115:1059 ‐1065)

A formulation with stabilizers replacing albumin was developed for lyophilization of recombinant factor VIII (FVIII), GreenGene F (WHO INN: beroctocog alfa), to achieve stability and eliminate safety issues of blood-derived albumin. L-Arginine (hydrophilic amino acid, positively charged side chain), L-glutamic acid (hydrophilic amino acid, negatively charged side chain), and L-isoleucine (hydrophobic amino acid, nonpolar) were selected as stabilizers, and the mixture of the three amino acids were optimized. The mixture had results comparative with albumin and other commonly used stabilizers showing good preservation of recombinant FVIII during lyophilization, robust stability with consistently high recovery of FVIIII, very low aggregate formation, and good storage stability without alterations in protein characteristics. In vivo test results showed that the efficacy was maintained and had no signs of toxicity. The study demonstrated that the three amino acid mixture acts as a good stabilizer for lyophilization of recombinant FVIII and as a safe excipient. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012

Background The novel bioreactor system from PBS Biotech is the first with a pneumatic mixing device powered solely by gas buoyancy, eliminating the need for an external mechanical agitator. The patented design of the AirWheel mixing device promotes not only high tangential fluid flow around the wheel but also efficient radial and axial flows. The leverage effect of the Air-Wheel mixing device also allows for lower gassing requirement (v/v) with increasing working volume, making the power utilization from the gas buoyancy more efficient with scale. A series of physical tests were performed to demonstrate that PBS systems can promote uniform, homogenous mixing over a wide range of working volumes and can offer a low-shear environment for cell culture. A series of biological tests were then performed at various evaluation sites to confirm the physical test results.

Cervical artificial disc replacement is increasingly becoming popular among spine surgeons. Cervical disc arthroplasty aims to afford spinal stability and then balance this with flexibility. One of the fundamental benefits from performing cervical arthroplasty instead of fusion is preservation of motion in both the functional spinal unit (FSU) and the overall cervical spine. Eventually, preservation of segmental motion is believed to prevent the development of adjacent segment degeneration. But to justify its use, disc replacement prosthesis must demonstrate actual motion in vivo and preserve range of motion (ROM) after surgery as long as it allows. Without preservation of motion, disc prosthesis becomes just a functional arthrodesis equivalent.The purpose of this study was to analyze the possible factors affecting cervical spine ROM after single-level cervical disc arthroplasty.This is a retrospective radiological study of patients with symptomatic single-level cervical disc disorder who received the cervical disc prosthesis (Bryan Cervical Disc Prosthesis; Medtronic Sofamor Danek, Memphis, TN, USA).Procedure was performed in 39 patients.The outcome measures were statistical correlation of possible factors and ROM.We investigated possible factors that could affect cervical ROM after surgical intervention using cervical disc replacement. For this, we focused on two main components, namely, patient factors and technical factors. First, we examined patient factors, such as age, sex, preoperative FSU ROM, and preoperative overall cervical spine (whole cervical spine) ROM. Second, we then investigated technical factors, such as the amount of bone resection, disc insertion angle, and disc insertion depth. Then, our study searched if there was any statistical correlation between these factors and the postoperative cervical ROM.Significant correlation was found between the postoperative overall cervical spine ROM and preoperative overall cervical spine ROM (p.0001, R(2)=0.9062). Postoperative FSU ROM is closely correlated to both the preoperative FSU ROM (p.0001) and the disc insertion angle (p=.0097). However, no significant correlation was noted between age, sex, disc insertion angle, and disc insertion depth.Significant correlation was found between the postoperative overall cervical spine ROM and preoperative overall cervical spine ROM. Postoperative FSU ROM is closely correlated to both the preoperative FSU ROM and the disc insertion angle. Careful preoperative evaluation of the patient's radiographs and meticulous surgical technique during the surgical procedure could aid in achieving the goals and benefits of cervical disc arthroplasty.

The virus neutralizing efficacy of HB-C7A, a human monoclonal antibody raised against the surface antigen of hepatitis B virus (HBsAg), was proved using hepatitis B virus (HBV)-naive chimpanzees. One control chimpanzee which received 100CID(50) of HBV, subtype adw, without HB-C7A antibody became infected by HBV as evidenced by the appearance of HBV DNA on week 10 and subsequent appearance of HBsAg, anti-HBc and anti-HBs in the serum. Two experimental chimpanzees were inoculated intravenously with same dose of HBV as the control chimpanzee, which was previously incubated with 0.1mg and 10mg of HB-C7A antibody prior to inoculation. HBV infection was not observed in the antibody-treated chimpanzees during 12 months of follow-up, exhibiting neither detectable HBsAg nor anti-HBc antibody. This work demonstrates the neutralization of HBV by HB-C7A monoclonal antibody and shows the possibility of prevention of HBV infection using this antibody in liver transplantation and exposure to HBV.

Purpose: Beroctocog alfa is a second generation recombinant factor VIII manufactured by removing the B-domain from factor VIII. This prospective clinical trial was conducted to evaluate the efficacy, safety, and pharmacokinetics of beroctocog alfa in patients of ages ≥12 years previously treated for severe hemophilia A. Materials and Methods: Seventy subjects received beroctocog alfa as an on-demand treatment for acute hemorrhage. Results: The final hemostatic effect was excellent in 35 subjects (50%) and good in 26 subjects (37.1%). The drug showed an overall efficacy rate of 87.1%. The majority of acute hemorrhages was treated by administer ing the study drug once (86.2%) or twice (10.0%), and the mean dose administered per single infusion was 28.55±6.53 IU/kg. Ten subjects underwent 12 surgical procedures, and hemostatic efficacy was excellent in seven cases (58.3%) and good in five cases (41.7%), showing a 100% efficacy rate. A total of 52 of 88 subjects (59.0%) experienced 168 adverse events. There were 18 serious adverse events (10.7%) in 11 subjects, and two (mild dyspnea and facial edema) in one subject were related to the study drug. Only one subject formed a de novo factor VIII inhibitor, for an occurrence rate of 1.4% (one-sided 95% upper confidence limit: 3.85%). The final elimi nation half-life was 13.3 h and 12.6 h at baseline and 6 months after administration, respectively. Conclusion: Our results suggest that beroctocog alfa is safe and effi cacious as either an on-demand treatment for acute hemorrhage or a surgical prophylaxis in patients with hemophilia A.

We describe a case of 43-year-old man who had a pseudoaneurysm of the medial superior genicular artery after arthroscopic partial meniscectomy with standard anterolateral and anteromedial portals. Pseudoaneurysm of the medial superior genicular artery has been reported at the previous superomedial portal site after arthroscopy. Described herein is a unique case that involved the medial superior genicular artery at the previous anteromedial portal site after arthroscopy. The pseudoaneurysm was successfully treated with transcatheter embolization.

Background: The natural course of sarcoidosis is variable from spontaneous remission to significant morbidity or death. So the assessment of disease activity is important but no single parameter was generally accepted as a good marker. Recently several studies suggested that adhesion molecules, especially ICAM-1 can be a marker, but there are some controversies. And only few data are available about the relationship of ICAM-1 with clinical follow-up course. Methods: We measured the expression of adhesion molecules on BAL cells by flow cytometry and the level of soluble ICAM-1(sICAM-1) in serum and BALF at the time of diagnosis in 12 patients with active disease and 7 inactive sarcoidosis(5 male, 14 female, mean age: years, mean follow-up : months). Follow-up clinical course were compared with the changes in serum sICAMA-1 level and the adhesion molecule on BAL cells. Results: In the patients with active disease, the ICAM-1 on AM(RMFI: ) and sICAM-1 level in serum(ng/ml) and BAL fluid(ng/ml) were all higher than those of 7 inactive disease(RMFI: , p=0.0298, serum: ng/ml, p=0.0049, BALF: ng/ml). In the active sarcoidosis, ICAM-1 on AM(RMFI : ) and serum sICAM-1 were decreased after the therapy( ng/ml) but no significant change was noted in inactive disease. Also we found the initial ICAM-1 on AM and serum sICAM-1 had a significant correlation with the degree of improvement in PFT after the therapy. During the follow-up, the disease relapsed in 4 patients after the discontinuation of steroid and the serum sICAM-1 level went-up again at the time of relapse. Conclusion: Our data suggest that the serum sICAM-1 level and the ICAM-1 expression on AM can be a good marker of disease activity and also a predictor of outcome in sarcoidosis.

Background: It is well known that various cytokines and growth factors secreted mainly from alveolar macrophages do the key role in the pathogenesis of IPF. But recently it has been known that structural cells like fibroblast can also release cytokines. So the phenotypic changes in fibroblasts of IPF may do a role in continuous progression of fibrosis. The aim of this study is to find out whether there is a change in the biologic properties of the lung fibroblasts of IPF. Subjects and Method: The study was done on 13 patients with IPF diagnosed by open or thoracoscopic lung biopsy and 7 control patients who underwent resectional surgery for lung cancer. Lung fibroblast cell lines (FB) were established by explant culture technique from the biopsy or resected specimen Result: Basal proliferation of the fibroblast of IPF(IFB) measured by BrdU uptake tended to be highter than control fibroblast(NFB) (0.212 0.107 vs , p=0.0922), also there was no significant difference in proliferation after the stimulation with PDGF or 10% serum. On the contrary, the degree of inhibition in proliferation by PGE2 was significantly lower() in IFB than control(, p=0.0429). The IFB secreted significantly higher amount of MCP-l( pg/ml) spontaneously than NFB( pg/ml) and also after the stimulation with TGF-( ng/ml vs ng/ml, p=0.0012). Similarly IL-8 and IL-6 seretion of IFB was significantly higher than NFB at basal state and with TGF- stimulation. But after the maximal stimulation with IL-1,8, no significant difference in cytokine secretion was found between IFB and NFB. Conclusion : Above data suggest that the fibroblasts of IPF were phenotypically changed and these change may do a role in the pathogenesis of IPF.

Background: Lung cancer is the second most frequent malignancy in man in Korea. Surgery is the best treatment modality for non-small cell lung cancer, but most patients were presented in far advanced stage. So radiation therapy(RT) with or without chemotherapy is the next choice and radiation-induced pneumonitis and pulmonary fibrosis is the major limiting factor for the curative RT. Radiation pneumonitis is manifested with fever, cough and dyspnea, 2~3 months after the termination of radiotherpy. Chest X ray shows infiltration, typically limited to the radiation field, but occasionally bilateral infiltration was reported. Also Gibson et al reported that BAL lymphocytosis was found in both lungs, even though the radiation was confined to one lung. The aim of this study is to investigate the change of adhesion molecules expression on BAL cells and serum soluble ICAM-1(sICAM-1) level after the RT and its relationship to the development of radiation pneumonitis. The second aim is to confirm the bilaterality of change of BAL cell pattern and adhesion molecule expression. Subjects: BAL and the measurement of sICAM level in serum and BALF were done on 29 patients with lung cancer who received RT with curative intention. The BAL was done before the RT in 16 patients and 1~2 month after RT in 18 patients. 5 patients performed BAL before and after RT. Result: Clinically significant radiation pneumonitis developed in 7 patients. After RT, total cell count in BAL was significantly increased from to (p=0.0344) and %lymphocyte was also increased from to (p=0.0001) in all patient group. There was no difference between ipsilateral and contraleteral side to RT, and between the patients with and without radiation-pneumonitis. In whole patient group, the level of sICAM-1 showed no significant change after RT(in serum: , , BALF: , , respectively), but there was a significant difference between the patients with pneumonitis and without pneumonitis (serum: vs , p=0.0253, BALF: vs , p=0.0112). The expression of ICAM-1 on alveolar macrophages (AM) tends to increase after RT (RMFI: from to , p=0.0605), but it was significantly high in patients with pneumonitis () compared to the patients without pneumonitis (, p=0.0002). ICAM-1 expression on lymphocytes and CD 18 (-integrin) expression tended to be high in the patients with pneumonitis but the difference was statiastically not significant. Conclusion: Subclinical alveolitis on the basis of BAL finding developed bilaterally in all patients after RT. But clinically significant pneumonitis occurred in much smaller fraction and the ICAM-1 expression on AM and the sICAM-1 level in serum were good indicator of it.