Development of an indirect ELISA for the serologic detection of bovine viral diarrhea virus based on E2 antigen sub-genotypes 1b, 1e, and 1d.

Bovine viral diarrhea virus (BVDV) causes ongoing economic losses to cattle industries, directly through reduced herd performance or indirectly through control program costs. ELISA assays, one of the most widely used techniques due to their ease of implementation, have been a valuable tool for mass surveillance and detection of BVDV. In this study, we developed a new indirect ELISA (rE2-ELISA) for serologic detection of BVDV. The assay considers three recombinant E2 protein subtypes as antigens, allowing serologic diagnosis of BVDV-1b (high prevalence worldwide), BVDV-1d and 1e (high prevalence in southern Chile) sub-genotypes. Recombinant E2 (rE2) proteins were successfully expressed in stably transfected CHO cells. Conditions for rE2 ELISAs were established after determining appropriate concentrations of antigen, blocking agent, secondary antibody, and serum dilutions to achieve maximum discrimination between positive and negative serum samples. The developed rE2-ELISA showed a sensitivity of 92.86% and a specificity of 98.33%. Clinical testing of 180 serum samples from herds in southern Chile showed high accuracy (kappa > 0.8) compared to the commercial BVDV Total Ab kit (IDEXX), with 95.37% positive and 87.5% negative predictive value. In addition, the rE2 ELISA has shown the capability to detect anti-BVDV antibodies from naturally infected animals with sub-genotypes 1b, 1e, or undetermined. These results indicate that the developed indirect ELISA could serve as a valid, and efficient alternative for identifying BVDV-infected animals, thus contributing to the success of disease control and eradication programs.

Impact of fluazuron on oocyte maturation: May the antiparasitic affect bovine reproduction?

Fluazuron is a novel veterinary pour-on antitick formulation which can be applied simultaneously with bovine reproduction management strategies. Considering the economic importance of the livestock industry in many countries, it is important to know whether antiparasitics such as fluazuron may cause embryonic loss. The aim of this study was to evaluate the toxicological effect of fluazuron on bovine oocytes during in vitro maturation. The best fluazuron concentrations were determined in a preliminary experiment on Chinese hamster ovary (CHO)-K1 cells and further used to compare fluazuron toxicity in both study models. Results of the annexin V and alkaline single cell gel electrophoresis assays demonstrated that fluazuron caused cytotoxicity and genotoxicity in bovine cumulus cells at all the concentrations tested (50, 75 and 100 µg fluazuron/mL). The evaluation of cortical granules and mitochondria distribution showed that cytoplasmic maturation was not affected by fluazuron treatment. However, a decrease in metaphase II + polar body, degenerate oocytes as well as disorganized chromatin in polar body were observed at all concentrations tested. Whereas the fertilization process was not altered by 50 µg/mL fluazuron, the embryo development rate decreased significantly. No significant differences were observed in any of the oxidative stress parameters assessed. This study contributes to a better understanding of fluazuron in bovines, suggesting that the antiparasitic may affect bovine reproduction and might cause embryo loss.

Differential effects of follicle-stimulating hormone glycoforms on the transcriptome profile of cultured rat granulosa cells as disclosed by RNA-seq.

It has been documented that variations in glycosylation on glycoprotein hormones, confer distinctly different biological features to the corresponding glycoforms when multiple in vitro biochemical readings are analyzed. We here applied next generation RNA sequencing to explore changes in the transcriptome of rat granulosa cells exposed for 0, 6, and 12 h to 100 ng/ml of four highly purified follicle-stimulating hormone (FSH) glycoforms, each exhibiting different glycosylation patterns: a. human pituitary FSH18/21 (hypo-glycosylated); b. human pituitary FSH24 (fully glycosylated); c. Equine FSH (eqFSH) (hypo-glycosylated); and d. Chinese-hamster ovary cell-derived human recombinant FSH (recFSH) (fully-glycosylated). Total RNA from triplicate incubations was prepared from FSH glycoform-exposed cultured granulosa cells obtained from DES-pretreated immature female rats, and RNA libraries were sequenced in a HighSeq 2500 sequencer (2 x 125 bp paired-end format, 10-15 x 106 reads/sample). The computational workflow focused on investigating differences among the four FSH glycoforms at three levels: gene expression, enriched biological processes, and perturbed pathways. Among the top 200 differentially expressed genes, only 4 (0.6%) were shared by all 4 glycoforms at 6 h, whereas 118 genes (40%) were shared at 12 h. Follicle-stimulating hormone glycocoforms stimulated different patterns of exclusive and associated up regulated biological processes in a glycoform and time-dependent fashion with more shared biological processes after 12 h of exposure and fewer treatment-specific ones, except for recFSH, which exhibited stronger responses with more specifically associated processes at this time. Similar results were found for down-regulated processes, with a greater number of processes at 6 h or 12 h, depending on the particular glycoform. In general, there were fewer downregulated than upregulated processes at both 6 h and 12 h, with FSH18/21 exhibiting the largest number of down-regulated associated processes at 6 h while eqFSH exhibited the greatest number at 12 h. Signaling cascades, largely linked to cAMP-PKA, MAPK, and PI3/AKT pathways were detected as differentially activated by the glycoforms, with each glycoform exhibiting its own molecular signature. These data extend previous observations demonstrating glycosylation-dependent distinctly different regulation of gene expression and intracellular signaling pathways triggered by FSH in granulosa cells. The results also suggest the importance of individual FSH glycoform glycosylation for the conformation of the ligand-receptor complex and induced signalling pathways.

Development of a biotechnology process for the production of a novel hyperglycosylated long-acting recombinant bovine follicle-stimulating hormone.

Follicle-stimulating hormone (FSH) is an important protein used for bovine ovarian hyperstimulation in multiple ovulation and embryo transfer technology (MOET). Several attempts to produce bovine FSH (bFSH) in recombinant systems have been reported, nonetheless, up to date, the most commonly used products are partially purified preparations derived from porcine or ovine (pFSH or oFSH) pituitaries. Here we describe the development of a biotechnology process to produce a novel, hyperglycosylated, long-acting recombinant bFSH (LA-rbFSH) by fusing copies of a highly O-glycosylated peptide. LA-rbFSH and a nonmodified version (rbFSH) were produced in suspension CHO cell cultures and purified by IMAC with high purity levels (>99%). LA-rbFSH presented a higher glycosylation degree and sialic acid content than rbFSH. It also demonstrated a notable improvement in pharmacokinetic properties after administration to rats, including a higher concentration in plasma and a significant (seven-fold) reduction in apparent clearance (CLapp). In addition, the in vivo specific bioactivity of LA-rbFSH in rats was 2.4-fold higher compared to rbFSH. These results postulate this new molecule as an attractive substitute for commercially available porcine pituitary-derived products.

Development and characterization of a novel variant of long-acting bovine follicle-stimulating hormone (brscFSH).

Assisted reproduction is a key aspect of modern animal breeding, providing valuable assistance in improving breeding programs. In this field, the administration of exogenous hormones, such as follicle-stimulating hormone (FSH), plays a crucial role in the induction of multiple ovulations. However, commercial FSH used in veterinary practice has been derived primarily from pituitary glands, obtained mostly from pigs for nearly four decades. Although these hormones have contributed significantly to the advancement of assisted reproductive techniques, they have certain limitations that warrant further improvements. These limitations include contamination with luteinizing hormone (LH), the potential risk of pathogen contamination, the potential to trigger an immune response in non-pig species, and the short half-life in circulation, requiring the implementation of complex 8-dose superovulation schedules. Our research team has developed and characterized a new variant of bovine follicle-stimulating hormone (bscrFSH) to address these limitations. The new hormone is produced recombinantly in CHO cell cultures, with a specific productivity of about 30 pg/cell/day. The bscrFSH can be purified to a high purity of 97 % using a single step of immobilized metal affinity chromatography (IMAC). N-glycan analysis of bscrFSH showed that approximately 74 % of the glycans corresponded to charged structures, including mono-, di-, tri-, and tetra-sialylated glycans. Superovulation trials conducted in cattle revealed that bscrFSH, administered at a total dose of about 0.5 µg per kg of body weight, using a decrescent schedule of 4 doses with 24-h intervals, resulted in an average yield of 8-12 transferable embryos per animal. Further research is required; however, the preliminary findings indicate that bscrFSH, currently packaged under the provisional brand name of Cebitropin B, holds potential as a commercial product for assisted reproduction in ruminants.

Characterization of Sodium Channel Peptides Obtained from the Venom of the Scorpion Centruroides bonito.

Five peptides were isolated from the venom of the Mexican scorpion Centruroides bonito by chromatographic procedures (molecular weight sieving, ion exchange columns, and HPLC) and were denoted Cbo1 to Cbo5. The first four peptides contain 66 amino acid residues and the last one contains 65 amino acids, stabilized by four disulfide bonds, with a molecular weight spanning from about 7.5 to 7.8 kDa. Four of them are toxic to mice, and their function on human Na+ channels expressed in HEK and CHO cells was verified. One of them (Cbo5) did not show any physiological effects. The ones toxic to mice showed that they are modifiers of the gating mechanism of the channels and belong to the beta type scorpion toxin (ß-ScTx), affecting mainly the Nav1.6 channels. A phylogenetic tree analysis of their sequences confirmed the high degree of amino acid similarities with other known bona fide ß-ScTx. The envenomation caused by this venom in mice is treated by using commercially horse antivenom available in Mexico. The potential neutralization of the toxic components was evaluated by means of surface plasmon resonance using four antibody fragments (10FG2, HV, LR, and 11F) which have been developed by our group. These antitoxins are antibody fragments of single-chain antibody type, expressed in E. coli and capable of recognizing Cbo1 to Cbo4 toxins to various degrees.

In silico identification of a biarylamine acting as agonist at human ß3 adrenoceptors and exerting BRL37344-like effects on mouse metabolism.

Human ß3-adrenoceptor (ß3AR) agonists were considered potential agents for the treatment of metabolic disorders. However, compounds tested as ß3AR ligands have shown marked differences in pharmacological profile in rodent and human species, although these compounds remain attractive as they were successfully repurposed for the therapy of urinary incontinence. In this work, some biarylamine compounds were designed and tested in silico as potential ß3AR agonists on 3-D models of mouse or human ß3ARs. Based on the theoretical results, we identified, synthesized and tested a biarylamine compound (polibegron). In CHO-K1 cells expressing the human ß3AR, polibegron and the ß3AR agonist BRL 37344 were partial agonists for stimulating cAMP accumulation (50 and 57% of the response to isoproterenol, respectively). The potency of polibegron was 1.71- and 4.5-fold higher than that of isoproterenol and BRL37344, respectively. These results indicate that polibegron acts as a potent, but partial, agonist at human ß3ARs. In C57BL/6N mice with obesity induced by a high-fat diet, similar effects of the equimolar intraperitoneal administration of polibegron and BRL37344 were observed on weight, visceral fat and plasma levels of glucose, cholesterol and triglycerides. Similarities and differences between species related to ligand-receptor interactions can be useful for drug designing.

Development of IFNß-1a versions with reduced immunogenicity and full in vitro biological activity for the treatment of multiple sclerosis.

IFNß (recombinant interferon Beta) has been widely used for the treatment of Multiple sclerosis for the last four decades. Despite the human origin of the IFNß sequence, IFNß is immunogenic, and unwanted immune responses in IFNß-treated patients may compromise its efficacy and safety in the clinic. In this study, we applied the DeFT (De-immunization of Functional Therapeutics) approach to producing functional, de-immunized versions of IFNß-1a. Two de-immunized versions of IFNß-1a were produced in CHO cells and designated as IFNß-1a(VAR1) and IFNß-1a(VAR2). First, the secondary and tertiary protein structures were analyzed by circular dichroism spectroscopy. Then, the variants were also tested for functionality. While IFNß-1a(VAR2) showed similar in vitro antiviral activity to the original protein, IFNß-1a(VAR1) exhibited 40% more biological potency. Finally, in vivo assays using HLA-DR transgenic mice revealed that the de-immunized variants showed a markedly reduced immunogenicity when compared to the originator.

Chemical characterization of Callingcard Vine (Entada polystachya (L.) DC. var. polystachya) aqueous seed extract and evaluation of its cytotoxic, genotoxic and mutagenic properties.

Callingcard Vine (Entada polystachya (L.) DC. var. polystachya - Fabaceae) is a common plant in coastal thickets from western Mexico through Central America to Colombia and Brazil, especially in Amazon biome. It has been popularly used as a urinary burning reliever and diuretic. However, the plant chemical constituents are poorly understood and Entada spp. genotoxic potential have not been previously investigated. In the present study we determined the chemical composition of the aqueous E. polystachya crude seed extract (EPCSE) and evaluated the cytotoxic, genotoxic and mutagenic properties of EPCSE in Salmonella typhimurium and Chinese hamster fibroblast (V79) cells. Cytotoxic activity was also evaluated in tumor cell lines (HT29, MCF7 and U87) and non-malignant cells (MRC5). The chemical analysis by High Resolution Mass Spectrometry (HRMS) of EPCSE indicated the presence of saponin and chalcone. The results of the MTT and clonal survival assays suggest that EPCSE is cytotoxic to V79 cells. Survival analysis showed higher IC50 in non-tumor compared with tumor cell lines. EPCSE showed induction of DNA strand breaks as revealed by the alkaline comet assay and micronucleus test. Using the modified comet assay, it was possible to detect the induction of oxidative DNA base damage by EPCSE in V79 cells. Consistently, the extract induced increase lipid peroxidation (TBARS), superoxide dismutase (SOD) and catalase (CAT) activities in V79 cells. In addition, EPCSE induced mutations in S. typhimurium TA98 and TA100 strains, confirming a mutagenic potential. Taken together, our results suggest that EPCSE is cytotoxic and genotoxic to V79 cells and mutagenic to S. typhimurium. These properties can be related to the pro-oxidant ability of the extract and induction of DNA lesions. Additionally, EPCSE could inhibit the growth of tumor cells, especially human colorectal adenocarcinoma (HT29) cell line, and can constitute a possible source of antitumor natural agents.

ShlA toxin of Serratia induces P2Y2- and α5ß1-dependent autophagy and bacterial clearance from host cells.

Serratia marcescens is an opportunistic human pathogen involved in antibiotic-resistant hospital acquired infections. Upon contact with the host epithelial cell and prior to internalization, Serratia induces an early autophagic response that is entirely dependent on the ShlA toxin. Once Serratia invades the eukaryotic cell and multiples inside an intracellular vacuole, ShlA expression also promotes an exocytic event that allows bacterial egress from the host cell without compromising its integrity. Several toxins, including ShlA, were shown to induce ATP efflux from eukaryotic cells. Here, we demonstrate that ShlA triggered a nonlytic release of ATP from Chinese hamster ovary (CHO) cells. Enzymatic removal of accumulated extracellular ATP (eATP) or pharmacological blockage of the eATP-P2Y2 purinergic receptor inhibited the ShlA-promoted autophagic response in CHO cells. Despite the intrinsic ecto-ATPase activity of CHO cells, the effective concentration and kinetic profile of eATP was consistent with the established affinity of the P2Y2 receptor and the known kinetics of autophagy induction. Moreover, eATP removal or P2Y2 receptor inhibition also suppressed the ShlA-induced exocytic expulsion of the bacteria from the host cell. Blocking α5ß1 integrin highly inhibited ShlA-dependent autophagy, a result consistent with α5ß1 transactivation by the P2Y2 receptor. In sum, eATP operates as the key signaling molecule that allows the eukaryotic cell to detect the challenge imposed by the contact with the ShlA toxin. Stimulation of P2Y2-dependent pathways evokes the activation of a defensive response to counteract cell damage and promotes the nonlytic clearance of the pathogen from the infected cell.

Toxicological and chemoprevention studies of Brazilian brown propolis from Araucaria sp.

Brazilian brown propolis (BBP) is a natural product derived predominantly from the south region of Brazil, where Araucaria forests are dominant. Despite its potential as a source of bioactive compounds with leishmanicidal, anti-inflammatory, nociceptive, and antimicrobial properties, BBP has not been comprehensively studied compared to green propolis. Therefore, this study aimed to determine the safety and chemopreventive potential of BBP. The cytotoxicity attributed to BBP was assessed using two different assays, while the Salmonella/microsome assay was employed to evaluate mutagenicity. The acute toxicity attributed to BBP was determined using a zebrafish model, while the chemopreventive potential was investigated utilizing Chinese hamster lung (V79) cell lines. Data demonstrated that BBP exerted cytotoxic effects at concentrations greater than or equal to 10 µg/ml and did not exhibit mutagenicity in Salmonella typhimurium strains TA98 and TA100. However, at the highest concentration tested (4000 µg/plate), BBP induced a significant increase in revertant colonies in S. typhimurium TA102 strain. The LC50 equivalent to 8.83 mg/L was obtained in the acute toxicity evaluation in zebrafish. BBP also showed antigenotoxic effect by significantly reducing chromosomal damage induced by the mutagen doxorubicin in V79 cell cultures at a concentration of 2.5 µg/ml. Compared to Brazilian green and red propolis, BBP exhibited greater toxicity. On the other hand, at lower concentrations, BBP displayed chemopreventive potential, which may be associated with the antioxidant capacity of the extract. These findings contribute to a better understanding of the biological properties and potential applications of BBP in treating various diseases.

Engineering of Chinese hamster ovary cells for co-overexpressing MYC and XBP1s increased cell proliferation and recombinant EPO production.

Improving the cellular capacity of Chinese hamster ovary (CHO) cells to produce large amounts of therapeutic proteins remains a major challenge for the biopharmaceutical industry. In previous studies, we observed strong correlations between the performance of CHO cells and expression of two transcription factors (TFs), MYC and XBP1s. Here, we have evaluated the effective of overexpression of these two TFs on CHO cell productivity. To address this goal, we generated an EPO-producing cell line (CHOEPO) using a targeted integration approach, and subsequently engineered it to co-overexpress MYC and XBP1s (a cell line referred to as CHOCXEPO). Cells overexpressing MYC and XBP1s increased simultaneously viable cell densities and EPO production, leading to an enhanced overall performance in cultures. These improvements resulted from the individual effect of each TF in the cell behaviour (i.e., MYC-growth and XBP1s-productivity). An evaluation of the CHOCXEPO cells under different environmental conditions (temperature and media glucose concentration) indicated that CHOCXEPO cells increased cell productivity in high glucose concentration. This study showed the potential of combining TF-based cell engineering and process optimisation for increasing CHO cell productivity.

Effects of liquid extract from Plinia cauliflora fruits residues on Chinese hamsters biochemical parameters / Efeitos do extrato líquido de resíduos de frutos de Plinia cauliflora sobre os parâmetros bioquímicos de hamsters chineses

Plinia cauliflora (Mart.) Kausel, popularly known as jabuticaba, is rich in polyphenols. Phenolic compounds exhibit several biological properties, which reflect on biomarkers such as biochemical parameters. In the present study, we evaluated the plasmatic levels of glucose, total cholesterol, HDL-cholesterol, triglycerides, and uric acid of Chinese hamsters fed for 45 days with a regular diet or cholesterol-enriched diet supplemented with a liquid extract obtained from P. cauliflora fruits residues standardized in ellagic acid and total phenolic compounds. The results showed that the concentrated extract obtained from jabuticaba residues increased the glycemia of animals fed with a regular diet and reduced the plasmatic uric acid levels of animals fed with a cholesterol-enriched diet. Since hyperuricemia is considered to be a significant risk factor of metabolic disorders and the principal pathological basis of gout, the liquid extract from P. cauliflora fruits residues would be a promising candidate as a novel hypouricaemic agent for further investigation.

Plinia cauliflora (Mart.) Kausel, popularmente conhecida como jabuticaba, é rica em polifenois. Os compostos fenólicos apresentam diversas propriedades biológicas, que refletem em biomarcadores, como os parâmetros bioquímicos. No presente estudo, avaliamos os níveis plasmáticos de glicose, colesterol total, HDL-colesterol, triglicerídeos e ácido úrico em hamsters chineses alimentados por 45 dias com dieta regular ou dieta enriquecida com colesterol suplementada com extrato líquido obtido de resíduos de frutos de P. cauliflora padronizado em ácido elágico e compostos fenólicos totais. Os resultados mostraram que o extrato concentrado obtido dos resíduos de jabuticaba aumentou a glicemia dos animais alimentados com dieta regular e reduziu os níveis plasmáticos de ácido úrico dos animais alimentados com dieta rica em colesterol. Uma vez que a hiperuricemia é considerada um fator de risco significativo de distúrbios metabólicos e a principal base patológica da gota, o extrato líquido dos resíduos de frutas de P. cauliflora seria um candidato promissor como um novo agente hipouricêmico para investigação posterior.

Effects of liquid extract from Plinia cauliflora fruits residues on Chinese hamsters biochemical parameters / Efeitos do extrato líquido de resíduos de frutos de Plinia cauliflora sobre os parâmetros bioquímicos de hamsters chineses

Plinia cauliflora (Mart.) Kausel, popularly known as jabuticaba, is rich in polyphenols. Phenolic compounds exhibit several biological properties, which reflect on biomarkers such as biochemical parameters. In the present study, we evaluated the plasmatic levels of glucose, total cholesterol, HDL-cholesterol, triglycerides, and uric acid of Chinese hamsters fed for 45 days with a regular diet or cholesterol-enriched diet supplemented with a liquid extract obtained from P. cauliflora fruits residues standardized in ellagic acid and total phenolic compounds. The results showed that the concentrated extract obtained from jabuticaba residues increased the glycemia of animals fed with a regular diet and reduced the plasmatic uric acid levels of animals fed with a cholesterol-enriched diet. Since hyperuricemia is considered to be a significant risk factor of metabolic disorders and the principal pathological basis of gout, the liquid extract from P. cauliflora fruits residues would be a promising candidate as a novel hypouricaemic agent for further investigation.(AU)

Plinia cauliflora (Mart.) Kausel, popularmente conhecida como jabuticaba, é rica em polifenois. Os compostos fenólicos apresentam diversas propriedades biológicas, que refletem em biomarcadores, como os parâmetros bioquímicos. No presente estudo, avaliamos os níveis plasmáticos de glicose, colesterol total, HDL-colesterol, triglicerídeos e ácido úrico em hamsters chineses alimentados por 45 dias com dieta regular ou dieta enriquecida com colesterol suplementada com extrato líquido obtido de resíduos de frutos de P. cauliflora padronizado em ácido elágico e compostos fenólicos totais. Os resultados mostraram que o extrato concentrado obtido dos resíduos de jabuticaba aumentou a glicemia dos animais alimentados com dieta regular e reduziu os níveis plasmáticos de ácido úrico dos animais alimentados com dieta rica em colesterol. Uma vez que a hiperuricemia é considerada um fator de risco significativo de distúrbios metabólicos e a principal base patológica da gota, o extrato líquido dos resíduos de frutas de P. cauliflora seria um candidato promissor como um novo agente hipouricêmico para investigação posterior.(AU)

Enhanced recombinant protein production in CHO cell continuous cultures under growth-inhibiting conditions is associated with an arrested cell cycle in G1/G0 phase.

Low temperature and sodium butyrate (NaBu) are two of the most used productivity-enhancing strategies in CHO cell cultures during biopharmaceutical manufacturing. While these two approaches alter the balance in the reciprocal relationship between cell growth and productivity, we do not fully understand their mechanisms of action beyond a gross cell growth inhibition. Here, we used continuous culture to evaluate the differential effect of low temperature and NaBu supplementation on CHO cell performance and gene expression profile. We found that an increase in cell-productivity under growth-inhibiting conditions was associated with the arrest of cells in the G1/G0 phase. A transcriptome analysis revealed that the molecular mechanisms by which low temperature and NaBu arrested cell cycle in G1/G0 differed from each other through the deregulation of different cell cycle checkpoints and regulators. The individual transcriptome changes in pattern observed in response to low temperature and NaBu were retained when these two strategies were combined, leading to an additive effect in arresting the cell cycle in G1/G0 phase. The findings presented here offer novel molecular insights about the cell cycle regulation during the CHO cell bioprocessing and its implications for increased recombinant protein production. This data provides a background for engineering productivity-enhanced CHO cell lines for continuous manufacturing.

Large Area Microfluidic Bioreactor for Production of Recombinant Protein.

To produce innovative biopharmaceuticals, highly flexible, adaptable, robust, and affordable bioprocess platforms for bioreactors are essential. In this article, we describe the development of a large-area microfluidic bioreactor (LM bioreactor) for mammalian cell culture that works at laminar flow and perfusion conditions. The 184 cm2 32 cisterns LM bioreactor is the largest polydimethylsiloxane (PDMS) microfluidic device fabricated by photopolymer flexographic master mold methodology, reaching a final volume of 2.8 mL. The LM bioreactor was connected to a syringe pump system for culture media perfusion, and the cells' culture was monitored by photomicrograph imaging. CHO-ahIFN-α2b adherent cell line expressing the anti-hIFN-a2b recombinant scFv-Fc monoclonal antibody (mAb) for the treatment of systemic lupus erythematosus were cultured on the LM bioreactor. Cell culture and mAb production in the LM bioreactor could be sustained for 18 days. Moreover, the anti-hIFN-a2b produced in the LM bioreactor showed higher affinity and neutralizing antiproliferative activity compared to those mAbs produced in the control condition. We demonstrate for the first-time, a large area microfluidic bioreactor for mammalian cell culture that enables a controlled microenvironment suitable for the development of high-quality biologics with potential for therapeutic use.

The ß2-Subunit (AMOG) of Human Na+, K+-ATPase Is a Homophilic Adhesion Molecule.

The ß2 subunit of Na+, K+-ATPase was originally identified as the adhesion molecule on glia (AMOG) that mediates the adhesion of astrocytes to neurons in the central nervous system and that is implicated in the regulation of neurite outgrowth and neuronal migration. While ß1 isoform have been shown to trans-interact in a species-specific mode with the ß1 subunit on the epithelial neighboring cell, the ß2 subunit has been shown to act as a recognition molecule on the glia. Nevertheless, none of the works have identified the binding partner of ß2 or described its adhesion mechanism. Until now, the interactions pronounced for ß2/AMOG are heterophilic cis-interactions. In the present report we designed experiments that would clarify whether ß2 is a cell-cell homophilic adhesion molecule. For this purpose, we performed protein docking analysis, cell-cell aggregation, and protein-protein interaction assays. We observed that the glycosylated extracellular domain of ß2/AMOG can make an energetically stable trans-interacting dimer. We show that CHO (Chinese Hamster Ovary) fibroblasts transfected with the human ß2 subunit become more adhesive and make large aggregates. The treatment with Tunicamycin in vivo reduced cell aggregation, suggesting the participation of N-glycans in that process. Protein-protein interaction assay in vivo with MDCK (Madin-Darby canine kidney) or CHO cells expressing a recombinant ß2 subunit show that the ß2 subunits on the cell surface of the transfected cell lines interact with each other. Overall, our results suggest that the human ß2 subunit can form trans-dimers between neighboring cells when expressed in non-astrocytic cells, such as fibroblasts (CHO) and epithelial cells (MDCK).

Cell-surface glycosaminoglycans regulate the cellular uptake of charged polystyrene nanoparticles.

Engineered nanoparticles approaching the cell body will first encounter and interact with cell-surface glycosaminoglycans (GAGs) before reaching the plasma membrane and becoming internalized. However, how surface GAGs may regulate the cellular entry of nanoparticles remains poorly understood. Herein, it is shown that the surface GAGs of Chinese hamster ovary cells perform as a charge-based barrier against the cellular internalization of anionic polystyrene nanoparticles (PS NPs). In contrast, cationic PS NPs interact favorably with the surface GAGs and thereby are efficiently internalized. Anionic PS NPs eventually reaching the plasma membrane bind to scavenger receptors and are endocytosed by clathrin-mediated and lipid raft/cholesterol-dependent mechanisms, whereas cationic PS NPs are primarily internalized via clathrin-mediated endocytosis and macropinocytosis. Upon the enzymatic shedding of surface GAGs, the uptake of anionic PS NPs increases while that of cationic PS NPs is dramatically reduced. Interestingly, the diminished uptake of cationic PS NPs is observed only when heparan sulfate, but not chondroitin sulfate, is cleaved from the cell surface. Heparan sulfate therefore serves as anchors/first receptors to facilitate the cellular entry of cationic PS NPs. These findings contribute to advance the basic science of nanoparticle endocytosis while also having important implications for the use of engineered nanocarriers as intracellular drug-delivery systems.

A comprehensive comparison of mixing and mass transfer in shake flasks and their relationship with MAb productivity of CHO cells.

The selection of highly recombinant protein (RP)-productive Chinese hamster ovary (CHO) cell lines is widely carried out in shake flasks. It is assumed that increases in the operating parameters in shake flasks lead to impairments in cell growth and RP production. These effects in cells metabolism are widely associated with high mass transfers and hydrodynamic stress. This study examined the impact of commonly used operational parameters on growth and specific productivity (qP) of two CHO cell lines differentially secreting a humanized anti-hIL8 monoclonal antibody (mAb) and cultured in 250 ml flasks. The evaluated parameters are filling volume (10, 15, and 20%), shaking frequency (60 and 120 revolutions per minute -rpm-), and orbital diameter (25.4 and 19 mm). The analysis of the oxygen transfer was done in terms of the measured volumetric mass transfer coefficient (kLa) and of the hydrodynamics in terms of power input per unit volume of liquid (P/V), the turbulent eddy length scale measured by the Kolmogorov's microscale of turbulence, the energy dissipation rate, the average shear stress, and the shear rate. Though almost all measured kinetic and stoichiometric parameters remained unchanged, mAb titer included, significant differences were found in maximum cell concentration, 10-45% higher in conditions with lower values of kLa and P/V. Changes in glucose metabolism contributing to qP were only shown in the higher producer cell line. Non-lethal responses to elevated oxygen transfer and shear stress might be present and must be considered when evaluating CHO cell cultures in shake flasks.

Nanoencapsulation of buriti oil (Mauritia flexuosa L.f.) in porcine gelatin enhances the antioxidant potential and improves the effect on the antibiotic activity modulation.

The present study evaluated the cytotoxicity, antioxidant potential, and antimicrobial effect on the antibiotic activity modulation of gelatin nanoparticles containing buriti oil (OPG). The cytotoxicity analysis was performed on Chinese Hamster Ovary Cells (CHO) using a MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] test. The antioxidant potential of buriti oil and OPG was determined by total antioxidant capacity, reducing power, and the ABTS (2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid) test. The modulating antimicrobial activity was evaluated by determining the minimum inhibitory concentration (MIC) concentration against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, gentamicin and norflaxacillin. The nanoformulation of OPG did not show a cytotoxic effect on CHO cells and had a higher antioxidant potential than free buriti oil (p<0.05). The combination of antibiotics with free buriti oil and OPG was more efficient in inhibiting E. coli and P. aeruginosa than isolated norfloxacillin and gentamicin (p<0.05). Regarding the inhibition of S. aureus, OPG in combination with norfloxacillin reduced MIC by 50%. Nanoencapsulation was a viable alternative to enhance functionality and adding commercial value to buriti oil.