The molecular weight and concentration of dextran sulfate affect cell growth and antibody production in CHO cell cultures

To investigate the effect of dextran sulfate (DS), a widely used anti‐aggregation agent, on cell growth and monoclonal antibody (mAb) production including the quality attributes, DS with the three different MWs (4,000 Da, 15,000 Da, and 40,000 Da) at various concentrations (up to 1 g/L) was added to suspension cultures of two different recombinant CHO (rCHO) cell lines producing mAb, SM‐0.025 and CS13‐1.00. For both cell lines, the addition of DS, regardless of the MW and concentration of DS used, improved cell growth and viability in the decline phase of growth. However, it increased mAb production only in the CS13‐1.00 cells. Among the three different MWs, 40,000 Da DS was most effective in attenuating cell aggregation during the cultures of CS13‐1.00 cells, and showed the highest maximum mAb concentration. For SM‐0.025 cells, it significantly decreased specific mAb productivity, particularly at a high concentration of DS. Overall, DS addition did not negatively affect the quality attributes of mAbs (aggregation, charge variation, and glycosylation), though its efficacy on mAb quality depended on the MW and concentration of DS and cell lines. For both cell lines, the addition of DS did not affect N‐glycosylation of mAbs and decreased basic charge variants in mAbs. For CS13‐1.00 cells, the mAb monomer increased with the addition of 40,000 Da DS at 0.3–1.0 g/L. Taken together, to maximize the beneficial effect of DS addition on mAb production, the optimal MW and concentration of DS should be determined for each specific rCHO cell line. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1113–1122, 2016     

Additive Effect between IL-13 Polymorphism and Cesarean Section Delivery/Prenatal Antibiotics Use on Atopic Dermatitis: A Birth Cohort Study (COCOA)

Background

Although cesarean delivery and prenatal exposure to antibiotics are likely to affect the gut microbiome in infancy, their effect on the development of atopic dermatitis (AD) in infancy is unclear. The influence of individual genotypes on these relationships is also unclear. To evaluate with a prospective birth cohort study whether cesarean section, prenatal exposure to antibiotics, and susceptible genotypes act additively to promote the development of AD in infancy.

Methods

The Cohort for Childhood of Asthma and Allergic Diseases (COCOA) was selected from the general Korean population. A pediatric allergist assessed 412 infants for the presence of AD at 1 year of age. Their cord blood DNA was subjected to interleukin (IL)-13 (rs20541) and cluster-of-differentiation (CD)14 (rs2569190) genotype analysis.

Results

The combination of cesarean delivery and prenatal exposure to antibiotics associated significantly and positively with AD (adjusted odds ratio, 5.70; 95% CI, 1.19–27.3). The association between cesarean delivery and AD was significantly modified by parental history of allergic diseases or risk-associated IL-13 (rs20541) and CD14 (rs2569190) genotypes. There was a trend of interaction between IL-13 (rs20541) and delivery mode with respect to the subsequent risk of AD. (P for interaction = 0.039) Infants who were exposed prenatally to antibiotics and were born by cesarean delivery had a lower total microbiota diversity in stool samples at 6 months of age than the control group. As the number of these risk factors increased, the AD risk rose (trend p<0.05).

Conclusion

Cesarean delivery and prenatal antibiotic exposure may affect the gut microbiota, which may in turn influence the risk of AD in infants. These relationships may be shaped by the genetic predisposition.     

Moxibustion Treatment for Knee Osteoarthritis: A Multi-Centre,Non-Blinded,Randomised Controlled Trial on the Effectiveness and Safety of the Moxibustion Treatment versus Usual Care in Knee Osteoarthritis Patients

Introduction

This study tested the effectiveness of moxibustion on pain and function in chronic knee osteoarthritis (KOA) and evaluated safety.

Methods

A multi-centre, non-blinded, parallel-group, randomised controlled trial compared moxibustion with usual care (UC) in KOA. 212 South Korean patients aged 40–70 were recruited from 2011–12, stratified by mild (Kellgren/Lawrence scale grades 0/1) and moderate-severe KOA (grades 2/3/4), and randomly allocated to moxibustion or UC for four weeks. Moxibustion involved burning mugwort devices over acupuncture and Ashi points in affected knee(s). UC was allowed. Korean Western Ontario and McMaster Universities Questionnaire (K-WOMAC), Short Form 36 Health Survey (SF-36v2), Beck Depression Inventory (BDI), physical performance test, pain numeric rating scale (NRS) and adverse events were evaluated at 5 and 13 weeks. K-WOMAC global score at 5 weeks was the primary outcome.

Results

102 patients (73 mild, 29 moderate-severe) were allocated to moxibustion, 110 (77 mild, 33 moderate-severe) to UC. K-WOMAC global score (moxibustion 25.42+/−SD 19.26, UC 33.60+/−17.91, p<0.01, effect size  = 0.0477), NRS (moxibustion 44.77+/−22.73, UC 56.23+/−17.71, p<0.01, effect size  = 0.0073) and timed-stand test (moxibustion 24.79+/−9.76, UC 25.24+/−8.84, p = 0.0486, effect size  = 0.0021) were improved by moxibustion at 5 weeks. The primary outcome improved for mild but not moderate-severe KOA. At 13 weeks, moxibustion significantly improved the K-WOMAC global score and NRS. Moxibustion improved SF-36 physical component summary (p = 0.0299), bodily pain (p = 0.0003), physical functioning (p = 0.0025) and social functioning (p = 0.0418) at 5 weeks, with no difference in mental component summary at 5 and 13 weeks. BDI showed no difference (p = 0.34) at 5 weeks. After 1158 moxibustion treatments, 121 adverse events included first (n = 6) and second degree (n = 113) burns, pruritus and fatigue (n = 2).

Conclusions

Moxibustion may improve pain, function and quality of life in KOA patients, but adverse events are common. Limitations included no sham control or blinding.

Trial Registration

Clinical Research Information Service (CRIS) KCT0000130     

Gene-specific patterns of coregulator requirements by estrogen receptor-α in breast cancer cells

Progesterone receptor (PgR) controls the menstrual cycle, pregnancy, embryonic development, and homeostasis, and it plays important roles in breast cancer development and progression. However, the requirement of coregulators for estrogen-induced expression of the PgR gene has not been fully explored. Here we used RNA interference to demonstrate dramatic differences in requirements of 10 different coregulators for estrogen-regulated expression of six different genes, including PgR and the well-studied TFF1 (or pS2) gene in MCF-7 breast cancer cells. Full estrogen-induced expression of TFF1 required all ten coregulators, but PgR induction required only four of the 10 coregulators. Chromatin immunoprecipitation studies demonstrated several mechanisms responsible for the differential coregulator requirements. Actin-binding coregulator Flightless-I, required for TFF1 expression and recruited to that gene by estrogen receptor-α (ERα), is not required for PgR expression and not recruited to that gene. Protein acetyltransferase tat-interactive protein 60 and ATP-dependent chromatin remodeler Brahma Related Gene 1 are recruited to both genes but are required only for TFF1 expression. Histone methyltransferase G9a is recruited to both genes and required for estrogen-induced expression of TFF1 but negatively regulates estrogen-induced expression of PgR. In contrast, histone methyltransferase myeloid/lymphoid or mixed-lineage leukemia 1 (MLL1), pioneer factor Forkhead box A1, and p160 coregulator steroid receptor coactivator-3 are required for expression of and are recruited to both genes. Depletion of MLL1 decreased ERα binding to the PgR and TFF1 genes. In contrast, depletion of G9a enhanced ERα binding to the PgR gene but had no effect on ERα binding to the TFF1 gene. These studies suggest that differential promoter architecture is responsible for promoter-specific mechanisms of gene regulation.     

Prognostic factors in primary diffuse large B-cell lymphoma of adrenal gland treated with rituximab- CHOP chemotherapy from the Consortium for Improving Survival of Lymphoma (CISL)

ABSTRACT: BACKGROUND: The objective of this study was to identify prognostic factors for survival in patients with primary diffuse large B-cell lymphoma (DLBCL) of the adrenal gland. METHODS: Thirty one patients diagnosed with primary adrenal DLBCL from 14 Korean institutions and treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone) were analyzed. RESULTS: Complete remission (CR) and overall response rate after R-CHOP chemotherapy were 54.8% and 87.0%. The 2-year estimates of overall survival (OS) and progression-free survival (PFS) were 68.3% and 51.1%. In patients achieving CR, significant prolongations of OS (P = 0.029) and PFS (P = 0.005) were observed. Ann Arbor stage had no influence on OS. There was no significant difference in OS between patients with unilateral involvement of adrenal gland and those with bilateral involvement. When staging was modified to include bilateral adrenal involvement as one extranodal site, early stage (I or II) significantly correlated with longer OS (P = 0.021) and PFS (P <0.001). CONCLUSIONS: Contrary to prior reports, our data suggests that outcomes of primary adrenal DLBCL are encouraging using a regimen of R-CHOP, and that achieving CR after R-CHOP is predictive of survival. Likewise, our modified staging system may have prognostic value.     

Staphylococcal enterotoxin C1-induced pyrogenic cytokine production in human peripheral blood mononuclear cells is mediated by NADPH oxidase and nuclear factor-kappa B

The staphylococcal enterotoxins produced by Staphylococcus aureus are associated with pyrogenic response in humans and primates. This study investigates the role of NADPH oxidase and nuclear factor-kappa B (NF-kappaB) on enterotoxin staphylococcal enterotoxin C1 (SEC1)-induced pyrogenic cytokine production in human peripheral blood mononuclear cells (PBMC). The results indicate that the febrile response to the supernatant fluids of SEC1-stimulated PBMC in rabbits was in parallel with the levels of interleukin-1beta and interleukin-6 in the supernatants. The release of interleukin-1beta and interleukin-6, nuclear translocation of NF-kappaB and its DNA binding activity in the SEC1-stimulated PBMC were time-dependent and were completely eliminated by pyrrolidine dithiocarbamate or SN-50 (NF-kappaB inhibitors). The release of reactive oxygen species in the supernatants and translocation of the NADPH oxidase p47(phox) subunit to the plasma membrane of SEC1-stimulated PBMC were time-dependent. Administration of apocynin (NADPH oxidase inhibitor) attenuated the febrile response to the supernatants in rabbits and decreased the translocation of NADPH oxidase p47(phox) subunit and NF-kappaB activity in the SEC1-stimulated PBMC, and suppressed reactive oxygen species and pyrogenic cytokine production in the supernatants. Taken together, SEC1 may act through an NADPH oxidase mechanism to release reactive oxygen species, which activate NF-kappaB in PBMC to stimulate the synthesis of pyrogenic cytokines that trigger a fever response in rabbits.     

Cocoa procyanidins protect PC12 cells from hydrogen-peroxide-induced apoptosis by inhibiting activation of p38 MAPK and JNK

Oxidative stress induced by reactive oxygen species has been strongly associated with the pathogenesis of neurodegenerative disorders, including Alzheimer's disease. In this study, we investigated the possible protective effects of a cocoa procyanidin fraction (CPF) and procyanidin B2 (epicatechin-(4β-8)-epicatechin) – a major polyphenol in cocoa – against apoptosis of PC12 rat pheochromocytoma (PC12) cells induced by hydrogen peroxide (H₂O₂). CPF (1 and 5 μg/ml) and procyanidin B2 (1 and 5 μM) reduced PC12 cell death caused by H₂O₂, as determined by MTT and trypan blue exclusion assays. CPF and procyanidin B2 attenuated the H₂O₂-induced fragmentation of nucleus and DNA in PC12 cells. Western blot data demonstrated that H₂O₂ induced cleavage of poly(ADP-ribose)polymerase (PARP), downregulated Bcl-X_L and Bcl-2 in PC12 cells. Pretreatment with CPF or procyanidin B2 before H₂O₂ treatment diminished PARP cleavage and increased Bcl-X_L and Bcl-2 expression compared with those only treated with H₂O₂. Activation of caspase-3 by H₂O₂ was inhibited by pretreatment with CPF or procyanidin B2. Furthermore, H₂O₂-induced rapid and significant phosphorylation of c-Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), and both of these effects were attenuated by CPF or procyanidin B2 treatment. These results suggest that the protective effects of CPF and procyanidin B2 against H₂O₂-induced apoptosis involve inhibiting the downregulation of Bcl-X_L and Bcl-2 expression through blocking the activation of JNK and p38 MAPK.     

Improved cryopreservation of bovine preimplantation embryos cultured in chemically defined medium

The aim of this study was to examine the effects of modifications to a standard slow freezing protocol on the viability of in vitro produced bovine embryos. Bovine oocytes were matured, fertilized with frozen-thawed semen, and presumptive zygotes cultured in defined two-step culture media. The standard freezing medium was 1.5M ethylene glycol (EG), 0.1M sucrose, 10% fetal bovine serum (FBS) in Dulbecco's phosphate buffered saline (D-PBS). A preliminary trial showed that in vitro produced embryos cryopreserved in this medium had a survival rate of 74.6% at 24h and 53.5% at 48 h post-thaw. Experiment 1 studied the effects of omitting the sucrose supplement or replacing it with 0.1M xylose. In Experiment 2, the effects of partial (0%, 25% or 50%) or total (100%) replacement of sodium chloride with choline chloride in the cryopreservation medium were examined (the medium with 100% replacement was designated CJ1). The effects of replacing the 10% FBS with 0.4% BSA or 0.4% lipid-rich BSA (Albumax I) in CJ1 was studied in Experiment 3. In Experiment 4, pregnancy/calving rates following the post-thaw transfer of in vitro produced embryos cryopreserved in the standard freezing medium were compared with those of in vitro and in vivo produced embryos cryopreserved in the improved medium (Albumax I in CJ1). Supplementation of the cryopreservation medium with 0.1M sucrose resulted in higher post-thaw survival rates at 24 h (71.3% versus 53.5 and 51.7%; P<0.05), 48 h (51.1% versus 45.3 and 40.2%), and 72 h (34.0% versus 24.4 and 23.0%) than 0.1M xylose or no supplement, respectively, in Experiment 1. Experiment 2 showed that embryos cryopreserved in the standard medium had poorer survival rates at 24 h (72.8% versus 86.5%; P<0.05), 48 h (53.1% versus 66.3%) or 72 h (28.4% versus 44.9%) than those frozen in CJ1. The post-thaw survival rate of embryos frozen in medium supplemented with Albumax I was better than that for the FBS or BSA supplements at 24h (92.0% versus 90.7 and 87.3%), 48 h (87.3% versus 76.9 and 70.9%; P<0.05), and 72 h (70.4% versus 49.1 and 46 4%; P<0.05; Experiment 3). In Experiment 4, in vitro produced embryos cryopreserved in CJ1 medium supplemented with Albumax I resulted in higher pregnancy rates at Day 35 (31.9% versus 22.9%) and Day 60 (24.1% versus 14.3%) of gestation, and calving rates (22.6% versus 10.0%; P<0.05) than similar embryos frozen in the standard medium. However, in vivo produced embryos cryopreserved in Albumax I in CJ1 resulted in higher pregnancy rates at Day 35 (50.7%; P<0.05) and Day 60 (45.1%; P<0.05) of gestation, and calving rate (43.7%; P<0.05). It was concluded that modification of the freezing medium by addition of lipid-rich BSA and replacing sodium chloride with choline chloride improves the post-thaw survival of in vitro produced embryos, and their viability post-transfer.