Xylose is a general component of O-glycans in mammals. Core-xylosylation of N-glycans is only found in plants and helminth. Consequently, xylosylated N-glycans cause immunological response in humans. We have used the F-protein of the human respiratory syncytial virus (RSV), one of the main causes of respiratory tract infection in infants and elderly, as a model protein for vaccination. The RSV-F protein was expressed in CHO-DG44 cells, which were further modified by co-expression of β1,2-xylosyltransferase from Nicotiana tabacum. Xylosylation of RSV-F N-glycans was shown by monosaccharide analysis and MALDI-TOF mass spectrometry. In immunogenic studies with a human artificial lymph node model, the engineered RSV-F protein revealed improved vaccination efficacy.
Subunit vaccines often require adjuvants to elicit sustained immune activity. Here, a method is described to evaluate the efficacy of single vaccine candidates in the preclinical stage based on cytokine and gene expression analysis. As a model, the recombinant human respiratory syncytial virus (RSV) fusion protein (RSV-F) was produced in CHO cells. For comparison, wild-type and glycoengineered, afucosylated RSV-F were established. Both glycoprotein vaccines were tested in a commercial Human Artificial Lymph Node in vitro model (HuALN®). The analysis of six key cytokines in cell culture supernatants showed well-balanced immune responses for the afucosylated RSV-F, while immune response of wild-type RSV-F was more Th1 accentuated. In particular, stronger and specific secretion of interleukin-4 after each round of re-stimulation underlined higher potency and efficacy of the afucosylated vaccine candidate. Comprehensive gene expression analysis by nCounter gene expression assay confirmed the stronger onset of the immunologic reaction in stimulation experiments with the afucosylated vaccine in comparison to wild-type RSV-F and particularly revealed prominent activation of Th17 related genes, innate immunity, and comprehensive activation of humoral immunity. We, therefore, show that our method is suited to distinguish the potency of two vaccine candidates with minor structural differences.
Pituitary neuroendocrine tumors (PitNET) do not only belong to the most common intracranial neoplasms but seem to be generally more common than has been thought. Minimally invasive liquid biopsies have the potential to improve their early screening efficiency as well as monitor prognosis by facilitating the diagnostic procedures. This review aims to assess the potential of using liquid biopsies of different kinds of biomarker species that have only been obtained from solid pituitary tissues so far. Numerous molecules have been associated with the development of a PitNET, suggesting that it often develops from the cumulative effects of many smaller genetic or epigenetic changes. These minor changes eventually pile up to switch critical molecules into tumor-promoting states, which may be the key regulatory nodes representing the most potent marker substances for a diagnostic test. Drugs targeting these nodes may be superior for the therapeutic outcome and therefore the identification of such pituitary-specific cellular key nodes will help to accelerate their application in medicine. The ongoing genetic degeneration in pituitary adenomas suggests that repeated tumor profiling via liquid biopsies will be necessary for personalized and effective treatment solutions.
Insulin-like growth factor 1 (IGF-1) is the standard biochemical marker for the diagnosis and treatment control of acromegaly and growth hormone deficiency (GHD). However, its limitations necessitate the screening for new specific and sensitive biomarkers. The elonginB/C-cullin5-SOCS-box-complex (ECS-complex) (an intracellular five-protein complex) is stimulated by circulating growth hormone (GH) and regulates GH receptor levels through a negative feedback loop. It mediates the cells’ sensitivity for GH and therefore, represents a potent new biomarker for those diseases. In this study, individual ECS-complex proteins were measured in whole blood samples of patients with acromegaly (n = 32) or GHD (n = 12) via ELISA and compared to controls. Hierarchical clustering of the results revealed that by combining the three ECS-complex proteins suppressor of cytokine signaling 2 (SOCS2), cullin-5 and ring-box protein 2 (Rbx-2), 93% of patient samples could be separated from controls, despite many patients having a normal IGF-1 or not receiving medical treatment. SOCS2 showed the best individual diagnostic performance with an overall accuracy of 0.93, while the combination of the three proteins correctly identified all patients and controls. This resulted in perfect sensitivity and specificity for all patient groups, which demonstrates potential benefits of the ECS-complex proteins as clinical biomarkers for the diagnostics of GH-related diseases and substantiates their important role in GH metabolism.
Conventional Bioreactor systems for cultivating cells in Life Science have been widely used for decades. An in vitro cell cultivation bioreactor should reliably and reproducibly mimic the in vivo microenvironment of the cultured cells. Normally, mammalian cell cultures are performed in conventional bioreactor devices such as culture flasks and culture-dishes. However, these tools have fundamental limitations due to being inappropriate for high throughput screening and consume a considerable amount of resources and time [1]. Therefore, there is a trend towards miniaturization, disposables and even micro platforms that fulfill increasing demands strongly aiming for production and testing of novel pharmaceutical products. Here we present the development and manufacture of a disposable miniaturized flow-through bioreactor system that can be produced in large numbers at low costs.
nanoporous hollow fibers are located at the fluidic sources and drains of the miniaturized bioreactors and retain cells. The necessary mixture of oxygen and carbon dioxide is provided via diffusion through a semi-permeable membrane. Fluidic connections allow the continuous feeding of the cells adding nutrient solution at constant rates at the inlet of the micro bioreactor and removing the solution at the same rate at the outlet. This medium can be collected and used for subsequent analysis. Different designs and concepts for such bioreactors were carried out with varying numbers of plates, and integrated or joined miniaturized reactor chambers. First tests show full technical and biological functionality, cells could successfully be cultivated at high viability rates for some days.
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