Challenging the workhorse: Comparative analysis of eukaryotic micro-organisms for expressing monoclonal antibodies.

For commercial protein therapeutics, Chinese hamster ovary (CHO) cells have an established history of safety, proven capability to express a wide range of therapeutic proteins and high volumetric productivities. Expanding global markets for therapeutic proteins and increasing concerns for broadened access of these medicines has catalyzed consideration of alternative approaches to this platform. Reaching these objectives likely will require an order of magnitude increase in volumetric productivity and a corresponding reduction in the costs of manufacture. For CHO-based manufacturing, achieving this combination of targeted improvements presents challenges. Based on a holistic analysis, the choice of host cells was identified as the single most influential factor for both increasing productivity and decreasing costs. Here we evaluated eight wild-type eukaryotic micro-organisms with prior histories of recombinant protein expression. The evaluation focused on assessing the potential of each host, and their corresponding phyla, with respect to key attributes relevant for manufacturing, namely (a) growth rates in industry-relevant media, (b) adaptability to modern techniques for genome editing, and (c) initial characterization of product quality. These characterizations showed that multiple organisms may be suitable for production with appropriate engineering and development and highlighted that yeast in general present advantages for rapid genome engineering and development cycles.

Retinoic acid elicits a coordinated expression of gut homing markers on T lymphocytes of Zambian men receiving oral Vivotif, but not Rotarix, Dukoral or OPVERO vaccines.

All-trans retinoic acid (ATRA) up-regulates, in laboratory animals, the expression of the gut homing markers α4ß7 integrin and CCR9 on lymphocytes, increasing their gut tropism. Here, we show that, in healthy adult volunteers, ATRA induced an increase of these gut homing markers on T cells in vivo in a time dependent manner. The coordinated increase of α4ß7 and CCR9 by ATRA was seen in 57% (12/21) of volunteers and only when given together with an oral Vivotif vaccine. When this coordinated response to ATRA and Vivotif vaccine was present, it was strongly correlated with the gut immunoglobulin A (IgA) specific response to vaccine LPS (ρ = 0.82; P = 0.02). Using RNA-Seq analysis of whole blood transcription, patients receiving ATRA and Vivotif in conjunction showed transcriptomic changes in immune-related pathways, particularly including interferon α/ß signaling pathway, membrane-ECM interactions and immune hubs. These results suggest that exogenous ATRA can be used to manipulate responses to a subclass of oral vaccines, so far limited to a live attenuated Vivotif vaccine.

Different milk diets have substantial effects on the jejunal mucosal immune system of pre-weaning calves, as demonstrated by whole transcriptome sequencing.

There is increasing evidence that nutrition during early mammalian life has a strong influence on health and performance in later life. However, there are conflicting data concerning the appropriate milk diet. This discrepancy particularly applies to ruminants, a group of mammals that switch from monogastric status to rumination during weaning. Little is known regarding how the whole genome expression pattern in the juvenile ruminant gut is affected by alternative milk diets. Thus, we performed a next-generation-sequencing-based holistic whole transcriptome analysis of the jejunum in male pre-weaned German Holstein calves fed diets with restricted or unlimited access to milk during the first 8 weeks of life. Both groups were provided hay and concentrate ad libitum. The analysis of jejunal mucosa samples collected 80 days after birth and four weeks after the end of the feeding regimes revealed 275 differentially expressed loci. While the differentially expressed loci comprised 67 genes encoding proteins relevant to metabolism or metabolic adaptation, the most distinct difference between the two groups was the consistently lower activation of the immune system in calves that experienced restricted milk access compared to calves fed milk ad libitum. In conclusion, different early life milk diets had significant prolonged effects on the intestinal immune system.

Expression and bioactivity of human α-fetoprotein in a Bac-to-Bac system.

α-fetoprotein (AFP) is an early serum growth factor in foetal embryonic development and hepatic oncogenesis. A growing number of investigations of AFP as a tumour-specific biomarker have concluded that AFP is an important target for cancer treatment. AFP also plays an immunomodulatory role in the treatment of several autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, myasthenia gravis and thyroiditis. In an effort to support biochemical screening and drug design and discovery, we attempted to express and purify human AFP in a Bac-to-Bac system. Two key factors affecting the expression of recombinant human AFP (R-AFP), namely the infectious baculovirus inoculum volume and the culturing time post-infection, were optimized to maximize the yield. We achieved a high yield of approximately 1.5 mg/l of harvested medium with a 72-96 h incubation period after infection and an inoculum volume ratio of 1:100. We also assessed the role of R-AFP in the proliferation of the human liver cancer cell line Bel 7402, and the results indicated that R-AFP promoted the growth of hepatoma cells. We concluded that this method can produce high yields of R-AFP, which can be used for studies related to AFP.

Naturally occurring homoisoflavonoids and their pharmacological activities.

Homoisoflavonoids, a special subclass of flavonoids, are rarely found in nature, mainly existing in Fabaceae and Asparagaceae families and being less common in Polygonaceae, Portulacaceae, Orchidaceae, and Gentianaceae families. Until now, approximately 240 natural occurring homoisoflavonoids have been identified from roots, barks, heartwood, bulbs, leaves, and seeds of the plants from the above mentioned families, which have often been used in traditional medicine. Homoisoflavonoids have been reported with a broad range of bioactivities, including anti-microbial, anti-mutagenic, anti-oxidant, immunomodulatory, anti-diabetic, cytotoxic, anti-angiogenic, vasorelaxant, and anti-inflammatory effects. To organize this review, the homoisoflavonoids were classified into five groups based on their structures: sappanin-type (I), scillascillin-type (II), brazilin-type (III), caesalpin-type (IV), and protosappanin-type (V). The structures of natural occurring homoisoflavonoids are described, and their proposed biosynthetic pathway and recent pharmacological studies are discussed. The main purpose of this review is to provide a comprehensive and up-to-date state of knowledge from phytochemical and pharmacological studies performed on homoisoflavonoids during the past decades. Homoisoflavonoids might have a large potential for further investigations of their bioactivities in order to identify important leads.

Effects of natural eggshell membrane (NEM) on cytokine production in cultures of peripheral blood mononuclear cells: increased suppression of tumor necrosis factor-α levels after in vitro digestion.

Tumor necrosis factor-α (TNF-α) plays an important role in inflammatory processes. This study examined the effects of natural eggshell membrane (NEM(®)) (ESM Technologies, LLC, Carthage, MO, USA) on interleukin (IL)-2, IL-4, IL-6, IL-10, interferon-γ (IFN-γ), and TNF-α cytokine production by 4-day peripheral blood mononuclear cell (PBMC) cultures exposed to serial dilutions of either an aqueous extract of natural eggshell membrane (NEM-AQ) or NEM subjected to in vitro digestion (NEM-IVD). The effects on cytokine production were also assessed in the presence of phytohemagglutinin (PHA) and pokeweed mitogen (PWM) where exposure to NEM-AQ resulted in reduced levels of proliferation and statistically significant effects on IL-6, IL-10, IFN-γ, and TNF-α cytokine production. NEM-AQ reduced levels of IL-6, IL-10, IFN-γ, and TNF-α in cultures exposed to PHA. In cultures containing PWM, NEM-AQ reduced production of IL-10 and at the highest dose tested increased IL-6 and decreased TNF-α cytokine levels. NEM-IVD, at the two lowest concentrations of product, significantly reduced TNF-α production by PBMC cultures exposed to PWM compared with the in vitro digest control or native NEM. Taken together, these results suggest that NEM-AQ can influence signaling events in response to the T cell-specific mitogen PHA as well as to the mitogen PWM that require cellular cross-talk and that these effects may be partially mediated through a reduction in level of the pro-inflammatory cytokine TNF-α. The suppression of TNF-α production in the presence of NEM-IVD is promising for the use of NEM as a consumable anti-inflammatory product.

Large-scale production of the immunomodulator c-di-GMP from GMP and ATP by an enzymatic cascade.

(3'-5')-Cyclic diguanylate (c-di-GMP) is a bacterial second messenger with immunomodulatory activities in mice suggesting potential applications as a vaccine adjuvant and as a therapeutic agent. Clinical studies in larger animals or humans will require larger doses that are difficult and expensive to generate by currently available chemical or enzymatic synthesis and purification methods. Here we report the production of c-di-GMP at the multi-gram scale from the economical precursors guanosine monophosphate (GMP) and adenosine triphosphate by a "one-pot" three enzyme cascade consisting of GMP kinase, nucleoside diphosphate kinase, and a mutated form of diguanylate cyclase engineered to lack product inhibition. The c-di-GMP was purified to apparent homogeneity by a combination of anion exchange chromatography and solvent precipitation and was characterized by reversed phase high performance liquid chormatography and mass spectrometry, nuclear magnetic resonance spectroscopy, and further compositional analyses. The immunomodulatory activity of the c-di-GMP preparation was confirmed by its potentiating effect on the lipopolysaccharide-induced interleukin 1ß, tumor necrosis factor α, and interleukin 6 messenger RNA expression in J774A.1 mouse macrophages.

Inhibition of H1N1 influenza A virus growth and induction of inflammatory mediators by the isoquinoline alkaloid berberine and extracts of goldenseal (Hydrastis canadensis).

In this study we tested whether the isoquinoline alkaloid berberine can inhibit the growth of influenza A. Our experiments showed strong inhibition of the growth of H1N1 influenza A strains PR/8/34 or WS/33 in RAW 264.7 macrophage-like cells, A549 human lung epithelial-derived cells and murine bone marrow derived macrophages, but not MDCK canine kidney cells. Studies of the mechanism underlying this effect suggest that berberine acts post-translationally to inhibit virus protein trafficking/maturation which in turn inhibits virus growth. Berberine was also evaluated for its ability to inhibit production of TNF-α and PGE(2) from A/PR/8/34 infected-RAW 264.7 cells. Our studies revealed strong inhibition of production of both mediators and suggest that this effect is distinct from the anti-viral effect. Finally, we asked whether berberine-containing ethanol extracts of goldenseal also inhibit the growth of influenza A and production of inflammatory mediators. We found strong effectiveness at high concentrations, although upon dilution extracts were somewhat less effective than purified berberine. Taken together, our results suggest that berberine may indeed be useful for the treatment of infections with influenza A.

Immunomodulatory activity of an extract of the novel fungal endophyte Entrophospora infrequens isolated from Nothapodytes foetida (Wight) Sleumer.

A novel camptothecin-producing endophytic fungus viz., Entrophospora infrequens was isolated from an important Indian medicinal plant Nothapodytes foetida. The present study reports evaluation ofbioactivities of two novel extracts viz., chloroform (CEEI) and methanolic (MEEI) extracts of Entrophospora infrequens with respect to their immunomodulatory potential in vitro and in vivo (in Balb/c mice). The endophyte E. infrequens was found to synthesize camptothecin, which tested positive in CEEI. The immunomodulatory potential of CEEI and MEEI was compared with standard camptothecin (CPT). Doses of the chloroform extract (CEEI) ranging from 12.5-100 mg/kg body weight, significantly (p < 0.05) stimulated the humoral and cell-mediated immune responses in a dose-dependent manner. MEEI on the other hand significantly (p < 0.05) stimulated the delayed type hypersensitivity (DTH) reaction (by nearly 80%), plaque forming cell (PFC) assay (33%), phagocytic response (38%) and haemagglutination antibody (HA) titre [IgM by 79.07% and IgG by 62.05%] at a dose of 12.5 mg/kg body weight. The present study is the first report of the immunomodulatory potential of this neoteric camptothecin-producing endophyte from Nothapodytes foetida.

Biosynthesis of 15-deoxy-delta12,14-PGJ2 and the ligation of PPARgamma.

15-deoxy-Delta12,14-PGJ2 (15d-PGJ2) has been identified as an endogenous ligand for PPARgamma, inducing adipogenesis in vitro. Additional roles for this molecule in the propagation and resolution of inflammation, ligation of NF-kappaB, and mediation of apoptosis have been proposed. However, quantitative, physiochemical evidence for the formation of 15d-PGJ2 in vivo is lacking. We report that 15d-PGJ2 is detectable using liquid chromatography-mass spectrometry-mass spectrometry at low picomolar concentrations in the medium of 3T3-L1 preadipocytes. However, despite induction of COX-2, production of PGs, including 15d-PGJ2, does not increase during adipocyte differentiation, a process unaltered by COX inhibition. 15d-PGJ2 is detectable as a minor product of COX-2 in human urine. However, its biosynthesis is unaltered during or after COX activation in vivo by LPS. Furthermore, the biosynthesis of 15d-PGJ2 is not augmented in the joint fluid of patients with arthritis, nor is its urinary excretion increased in patients with diabetes or obesity. 15d-PGJ2 is not the endogenous mediator of PPARgamma-dependent adipocyte activation and is unaltered in clinical settings in which PPARgamma activation has been implicated.