Assessment of Different Expression Strategies for the Production of a Recombinant Lipoprotein Vaccine in Plants

The ability of plants to serve as a production system for bacterial lipoprotein vaccines has been investigated. First, the effect of high-level expression of the Borrelia burgdorferi outer membrane protein A (OspA), a prototype vaccine against Lyme disease, has been examined by a proteomics approach. Analysis by 2D-PAGE of wild type tobacco plants and transplastomic plants accumulating recombinant OspA showed no apparent differences in protein pattern except for OspA. However, presence of the bacterial signal sequence limits transgene accumulation. As an alternative approach OspA was produced in Nicotiana benthamiana plants by transient expression via a deconstructed tobacco mosaic virus-based system. While rapid expression of OspA could be achieved, no palmitoylation occurred with the genuine bacterial sequence. In contrast, modification of the N-terminus with an eukaryotic sequence motif resulted in palmitoylation of OspA. This study shows that plants provide multiple expression strategies and could serve as a versatile production platform for recombinant lipidated subunit vaccines

Effects of Extracellular Vesicles from Osteogenic Differentiated Human BMSCs on Osteogenic and Adipogenic Differentiation Capacity of Naïve Human BMSCs

Osteoporosis, or steroid-induced osteonecrosis of the hip, is accompanied by increased bone marrow adipogenesis. Such a disorder of adipogenic/osteogenic differentiation, affecting bone-marrow-derived mesenchymal stem cells (BMSCs), contributes to bone loss during aging. Here, we investigated the effects of extracellular vesicles (EVs) isolated from human (h)BMSCs during different stages of osteogenic differentiation on the osteogenic and adipogenic differentiation capacity of naïve (undifferentiated) hBMSCs. We observed that all EV groups increased viability and proliferation capacity and suppressed the apoptosis of naïve hBMSCs. In particular, EVs derived from hBMSCs at late-stage osteogenic differentiation promoted the osteogenic potential of naïve hBMSCs more effectively than EVs derived from naïve hBMSCs (naïve EVs), as indicated by the increased gene expression of COL1A1 and OPN. In contrast, the adipogenic differentiation capacity of naïve hBMSCs was inhibited by treatment with EVs from osteogenic differentiated hBMSCs. Proteomic analysis revealed that osteogenic EVs and naïve EVs contained distinct protein profiles, with pro-osteogenic and anti-adipogenic proteins encapsulated in osteogenic EVs. We speculate that osteogenic EVs could serve as an intercellular communication system between bone- and bone-marrow adipose tissue, for transporting osteogenic factors and thus favoring pro-osteogenic processes. Our data may support the theory of an endocrine circuit with the skeleton functioning as a ductless gland

Extracellular Vesicles Derived from Osteogenic-Differentiated Human Bone Marrow-Derived Mesenchymal Cells Rescue Osteogenic Ability of Bone Marrow-Derived Mesenchymal Cells Impaired by Hypoxia

In orthopedics, musculoskeletal disorders, i.e., non-union of bone fractures or osteoporosis, can have common histories and symptoms related to pathological hypoxic conditions induced by aging, trauma or metabolic disorders. Here, we observed that hypoxic conditions (2% O2) suppressed the osteogenic differentiation of human bone marrow-derived mesenchymal cells (hBMSC) in vitro and simultaneously increased reactive oxygen species (ROS) production. We assumed that cellular origin and cargo of extracellular vesicles (EVs) affect the osteogenic differentiation capacity of hBMSCs cultured under different oxygen pressures. Proteomic analysis revealed that EVs isolated from osteogenic differentiated hBMSC cultured under hypoxia (hypo-osteo EVs) or under normoxia (norm-osteo EVs) contained distinct protein profiles. Extracellular matrix (ECM) components, antioxidants and pro-osteogenic proteins were decreased in hypo-osteo EVs. The proteomic analysis in our previous study revealed that under normoxic culture conditions, pro-osteogenic proteins and ECM components have higher concentrations in norm-osteo EVs than in EVs derived from naïve hBMSCs (norm-naïve EVs). When selected for further analysis, five anti-hypoxic proteins were significantly upregulated (response to hypoxia) in norm-osteo EVs. Three of them are characterized as antioxidant proteins. We performed qRT-PCR to verify the corresponding gene expression levels in the norm-osteo EVs’ and norm-naïve EVs’ parent cells cultured under normoxia. Moreover, we observed that norm-osteo EVs rescued the osteogenic ability of naïve hBMSCs cultured under hypoxia and reduced hypoxia-induced elevation of ROS production in osteogenic differentiated hBMSCs, presumably by inducing expression of anti-hypoxic/ antioxidant and pro-osteogenic genes

Subcellular localization of PD‐L1 and cell‐cycle‐dependent expression of nuclear PD‐L1 variants: implications for head and neck cancer cell functions and therapeutic efficacy

The programmed cell death 1 ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) axis is primarily associated with immunosuppression in cytotoxic T lymphocytes (CTLs). However, mounting evidence is supporting the thesis that PD-L1 not only functions as a ligand but mediates additional cellular functions in tumor cells. Moreover, it has been demonstrated that PD-L1 is not exclusively localized at the cellular membrane. Subcellular fractionation revealed the presence of PD-L1 in various cellular compartments of six well-characterized head and neck cancer (HNC) cell lines, including the nucleus. Via Western blotting, we detected PD-L1 in its well-known glycosylated/deglycosylated state at 40–55 kDa. In addition, we detected previously unknown PD-L1 variants with a molecular weight at approximately 70 and > 150 kDa exclusively in nuclear protein fractions. These in vitro findings were confirmed with primary tumor samples from head and neck squamous cell carcinoma (HNSCC) patients. Furthermore, we demonstrated that nuclear PD-L1 variant expression is cell-cycle-dependent. Immunofluorescence staining of PD-L1 in different cell cycle phases of synchronized HNC cells supported these observations. Mechanisms of nuclear PD-L1 trafficking remain less understood; however, proximity ligation assays showed a cell-cycle-dependent interaction of the cytoskeletal protein vimentin with PD-L1, whereas vimentin could serve as a potential shuttle for nuclear PD-L1 transportation. Mass spectrometry after PD-L1 co-immunoprecipitation, followed by gene ontology analysis, indicated interaction of nuclear PD-L1 with proteins involved in DNA remodeling and messenger RNA (mRNA) splicing. Our results in HNC cells suggest a highly complex regulation of PD-L1 and multiple tumor cell-intrinsic functions, independent of immune regulation. These observations bear significant implications for the therapeutic efficacy of immune checkpoint inhibition

Impact of platelet-rich plasma on cell migration processes after external radiation

BACKGROUND: To overcome the compromised wound healing in radiation induced chronic wounds platelet-rich plasma (PRP), as therapeutic agent, is current subject of studies. PRP is associated with pro-angiogenic effects. Nevertheless, effects of platelet-rich plasma in cutaneous wound healing processes are poorly understood so far. Methods: In this study, the migration of endothelial cells, fibroblasts and keratinocytes in conjunction with platelet-rich plasma treatment is investigated in the context of radiation effects. Additionally, cell proliferation and viability after external radiation was analyzed regarding treatment by platelet-rich plasma. RESULTS: All cell cultures showed a trend towards decreasing proliferation and viability after irradiation irrespective of PRP. Upon PRP treatment, irradiated fibroblasts as well as endothelial cells showed an enhanced proliferation whereas proliferation and viability of keratinocytes was reduced after PRP treatment. Scratch assays support the positive effect of PRP on fibroblast and endothelial cell migration, whereas a negative effect on keratinocytes was observed after PRP treatment. CONCLUSIONS: The present study documents both deleterious effects of external radiation as well as the protective effect of PRP. In summary, increased viability, proliferation and migration are indeed a consequence of the pro-proliferative effect exerted by PRP. Therefore, treatment with PRP products might be useful in the management of chronic radiogenic wounds

Proteomic analysis of cathepsin B- and L-deficient mouse brain lysosomes

Cathepsins B and L are lysosomal cysteine proteases which have been implicated in a variety of pathological processes such as cancer, tumor angiogenesis, and neurodegeneration. However, only a few protein substrates have thus far been described and the mechanisms by which cathepsins B and L regulate cell proliferation, invasion, and apoptosis are poorly understood. Combined deficiency of both cathepsins results in early-onset neurodegeneration in mice reminiscent of neuronal ceroid lipofuscinoses in humans. Therefore, we intended to quantify accumulated proteins in brain lysosomes of double deficient mice. A combination of subcellular fractionation and LC-MS/MS using isobaric tagging for relative and absolute quantitation (iTRAQ™) allowed us to simultaneously assess wildtype and cathepsin B−/−L−/− cerebral lysosomes. Altogether, 19 different proteins were significantly increased in cathepsin B−/−L−/− lysosomes. Most elevated proteins had previously been localized to neuronal biosynthetic, recycling/endocytic or lysosomal compartments. A more than 10-fold increase was observed for Rab14, the Delta/Notch-like epidermal growth factor-related receptor (DNER), calcyon, and carboxypeptidase E. Intriguingly, immunohistochemistry demonstrated that Rab14 and DNER specifically stain swollen axons in double deficient brains. Since dense accumulations of expanded axons are the earliest phenotypic and pathognomonic feature of cathepsin B−/−L−/− brains, our data suggest a role for cathepsins B and L in recycling processes during axon outgrowth and synapse formation in the developing postnatal central nervous system

Impact of Platelet-Rich Plasma on Viability and Proliferation in Wound Healing Processes after External Radiation

Platelet-rich plasma is a current subject of studies on chronic wound healing therapy due to possible pro-angiogenic effects. Microvascular compromise represents the major component in radiogenic wound healing complications. The effects of platelet-rich plasma on irradiated cells of the cutaneous wound healing process are poorly understood so far. In this study, the interaction of endothelial cells and adipose-derived stem cells in conjunction with treatment with platelet-rich plasma is investigated in the context of radiation effects. Therefore, the expression of surface-marker CD90 and CD31 was determined. Moreover, cell proliferation and viability after external radiation was analyzed with and without treatment by platelet-rich plasma

Causal Modeling of Cancer-Stromal Communication Identifies PAPPA as a Novel Stroma-Secreted Factor Activating NFκB Signaling in Hepatocellular Carcinoma

Inter-cellular communication with stromal cells is vital for cancer cells. Molecules involved in the communication are potential drug targets. To identify them systematically, we applied a systems level analysis that combined reverse network engineering with causal effect estimation. Using only observational transcriptome profiles we searched for paracrine factors sending messages from activated hepatic stellate cells (HSC) to hepatocellular carcinoma (HCC) cells. We condensed these messages to predict ten proteins that, acting in concert, cause the majority of the gene expression changes observed in HCC cells. Among the 10 paracrine factors were both known and unknown cancer promoting stromal factors, the former including Placental Growth Factor (PGF) and Periostin (POSTN), while Pregnancy-Associated Plasma Protein A (PAPPA) was among the latter. Further support for the predicted effect of PAPPA on HCC cells came from both in vitro studies that showed PAPPA to contribute to the activation of NFκB signaling, and clinical data, which linked higher expression levels of PAPPA to advanced stage HCC. In summary, this study demonstrates the potential of causal modeling in combination with a condensation step borrowed from gene set analysis [Model-based Gene Set Analysis (MGSA)] in the identification of stromal signaling molecules influencing the cancer phenotype

Mono-ADP-ribosylation sites of human CD73 inhibit its adenosine-generating enzymatic activity

CD73-derived adenosine plays a major role in damage-induced tissue responses by inhibiting inflammation. Damage-associated stimuli, such as hypoxia and mechanical stress, induce the cellular release of ATP and NA