Identification and Monitoring of Host Cell Proteins by Mass Spectrometry Combined with High Performance Immunochemistry Testing

Biotherapeutics are often produced in non-human host cells like Escherichia coli, yeast, and various mammalian cell lines. A major focus of any therapeutic protein purification process is to reduce host cell proteins to an acceptable low level. In this study, various E. coli host cell proteins were identified at different purifications steps by HPLC fractionation, SDS-PAGE analysis, and tryptic peptide mapping combined with online liquid chromatography mass spectrometry (LC-MS). However, no host cell proteins could be verified by direct LC-MS analysis of final drug substance material. In contrast, the application of affinity enrichment chromatography prior to comprehensive LC-MS was adequate to identify several low abundant host cell proteins at the final drug substance level. Bacterial alkaline phosphatase (BAP) was identified as being the most abundant host cell protein at several purification steps. Thus, we firstly established two different assays for enzymatic and immunological BAP monitoring using the cobas® technology. By using this strategy we were able to demonstrate an almost complete removal of BAP enzymatic activity by the established therapeutic protein purification process. In summary, the impact of fermentation, purification, and formulation conditions on host cell protein removal and biological activity can be conducted by monitoring process-specific host cell proteins in a GMP-compatible and high-throughput (> 1000 samples/day) manner.     

Dental follicle progenitor cells responses to Porphyromonas gingivalis LPS

Periodontitis is a bacterially induced chronic inflammatory disease. Dental follicle progenitor cells (DFPCs) have been proposed as biological graft for periodontal regenerative therapies. The potential impact of bacterial toxins on DFPCs properties is still poorly understood. The aim of this study was to investigate whether DFPCs are able to sense and respond to lipopolysaccharide (LPS) from Porphyromonas gingivalis, a major periopathogenic bacterium. Specifically, we hypothesized that LPS could influence the migratory capacity and IL‐6 secretion of DFPCs. DFPCs properties were compared to bone marrow mesenchymal stem cells (BMSCs), a well‐studied class of adult stem cells. The analysis by flow cytometry indicated that DFPCs, similar to BMSCs, expressed low levels of both toll‐like receptor (TLR) 2 and 4. The TLR4 mRNA expression was down‐regulated in response to LPS in both cell populations, while on protein level TLR4 was significantly up‐regulated on BMSCs. The TLR2 expression was not influenced by the LPS treatment in both DFPCs and BMSCs. The migratory efficacy of LPS‐treated DFPCs was evaluated by in vitro scratch wound assays and found to be significantly increased. Furthermore, we assayed the secretion of interleukin‐6 (IL‐6), a potent stimulator of cell migration. Interestingly, the levels of IL‐6 secretion of DFPCs and BMSCs remained unchanged after the LPS treatment. Taken together, these results suggest that DFPCs are able to sense and respond to P. gingivalis LPS. Our study provides new insights into understanding the physiological role of dental‐derived progenitor cells in sites of periodontal infection.     

MMP-9 and CXCL8/IL-8 Are Potential Therapeutic Targets in Epidermolysis Bullosa Simplex

Epidermolysis bullosa refers to a group of genodermatoses that affects the integrity of epithelial layers, phenotypically resulting in severe skin blistering. Dowling-Meara, the major subtype of epidermolysis bullosa simplex, is inherited in an autosomal dominant manner and can be caused by mutations in either the keratin-5 (K5) or the keratin-14 (K14) gene. Currently, no therapeutic approach is known, and the main objective of this study was to identify novel therapeutic targets. We used microarray analysis, semi-quantitative real-time PCR, western blot and ELISA to identify differentially regulated genes in two K14 mutant cell lines carrying the mutations K14 R125P and K14 R125H, respectively. We found kallikrein-related peptidases and matrix metalloproteinases to be upregulated. We also found elevated expression of chemokines, and we observed deregulation of the Cdc42 pathway as well as aberrant expression of cytokeratins and junction proteins. We further demonstrated, that expression of these genes is dependent on interleukin-1 β signaling. To evaluate these data in vivo we analysed the blister fluids of epidermolysis bullosa simplex patients vs. healthy controls and identified matrix metalloproteinase-9 and the chemokine CXCL8/IL-8 as potential therapeutic targets.     

Cooperation in Health: Mapping Collaborative Networks on the Web

Objective

To map and investigate the relationships established on the web between leading health-research institutions around the world.

Methods

Sample selection was based on the World Health Organization (WHO) Collaborating Centres (CCs). Data on the 768 active CCs in 89 countries were retrieved from the WHO''s database. The final sample consisted of 190 institutions devoted to health sciences in 42 countries. Data on each institution''s website were retrieved using webometric techniques (interlinking), and an asymmetric matrix was generated for social network analysis.

Findings

The results showed that American and European institutions, such as the Centers for Disease Control and Prevention (CDC), the National Institutes of Health (NIH) and the National Institute of Health and Medical Research (INSERM), are the most highly connected on the web and have a higher capacity to attract hyperlinks. The Karolinska Institute (KI-SE) in Sweden is well placed as an articulation point between several integrants of the network and the component''s core but lacks general recognition on the web by hyperlinks. Regarding the north-south divide, Mexico and Brazil appear to be key southern players on the web. The results showed that the hyperlinks exchanged between northern and southern countries present an abysmal gap: 99.49% of the hyperlinks provided by the North are directed toward the North itself, in contrast to 0.51% that are directed toward the South. Regarding the South, its institutions are more connected to its northern partners, with 98.46% of its hyperlinks directed toward the North, and mainly toward the United States, compared with 1.54% toward southern neighbors.

Conclusion

It is advisable to strengthen integration policies on the web and to increase web networking through hyperlink exchange. In this way, the web could actually reflect international cooperation in health and help to legitimize and enhance the visibility of the many existing south-south collaboration networks.     

Anaerobic Co-Culture of Mesenchymal Stem Cells and Anaerobic Pathogens - A New In Vitro Model System

Background

Human mesenchymal stem cells (hMSCs) are multipotent by nature and are originally isolated from bone marrow. In light of a future application of hMSCs in the oral cavity, a body compartment with varying oxygen partial pressures and an omnipresence of different bacterial species i.e. periodontitis pathogens, we performed this study to gain information about the behavior of hMSC in an anaerobic system and the response in interaction with oral bacterial pathogens.

Methodology/Principal Findings

We established a model system with oral pathogenic bacterial species and eukaryotic cells cultured in anaerobic conditions. The facultative anaerobe bacteria Fusobacterium nucleatum, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans were studied. Their effects on hMSCs and primary as well as permanent gingival epithelial cells (Ca9-22, HGPEC) were comparatively analyzed. We show that hMSCs cope with anoxic conditions, since 40% vital cells remain after 72 h of anaerobic culture. The Ca9-22 and HGPEC cells are significantly more sensitive to lack of oxygen. All bacterial species reveal a comparatively low adherence to and internalization into hMSCs (0.2% and 0.01% of the initial inoculum, respectively). In comparison, the Ca9-22 and HGPEC cells present better targets for bacterial adherence and internalization. The production of the pro-inflammatory chemokine IL-8 is higher in both gingival epithelial cell lines compared to hMSCs and Fusobacterium nucleatum induce a time-dependent cytokine secretion in both cell lines. Porphyromonas gingivalis is less effective in stimulating secretion of IL-8 in the co-cultivation experiments.

Conclusions/significance

HMSCs are suitable for use in anoxic regions of the oral cavity. The interaction with local pathogenic bacteria does not result in massive pro-inflammatory cytokine responses. The test system established in this study allowed further investigation of parameters prior to set up of oral hMSC in vivo studies.     

Expression patterns of peroxisome proliferator-activated receptor gamma 1 versus gamma 2, and their association with intramuscular fat in goat tissues

Intramuscular fat (IMF) shortage causes the lack of juiciness and tenderness of goat meat, while peroxisome proliferator-activated receptor gamma 1 (PPARγ1) and gamma 2 (PPARγ2) play key roles in lipid metabolism. Nevertheless, their expression patterns and the relationship with IMF have been poorly exposed. Using quantitative polymerase chain reaction (qPCR), classical Soxhlet extraction, and in situ hybridization, we demonstrated that among 13 goat tissues, expression of PPARγ1 was dramatically higher than that of PPARγ2 except for lung. We further demonstrated the expression patterns of PPARγ1 and PPARγ2 and their negative association with intramuscular fat content in three goat muscles with kids growing. Meanwhile, PPARγ expression was located in the connective tissues. These results suggest that PPARγ1 is rather active for most tissues of goat, and closely related with the muscular fat metabolism during early postnatal life, but a more direct proof remains to be provided.     

Identification of Serpin Determinants of Specificity and Selectivity for Furin Inhibition through Studies of α1PDX (α1-Protease Inhibitor Portland)-Serpin B8 and Furin Active-site Loop Chimeras

α₁-Protease inhibitor Portland (α₁PDX) is an engineered serpin family inhibitor of the proprotein convertase (PC), furin, that exhibits high specificity but limited selectivity for inhibiting furin over other PC family proteases. Here, we characterize serpin B8, a natural inhibitor of furin, together with α₁PDX-serpin B8 and furin-PC chimeras to identify determinants of serpin specificity and selectivity for furin inhibition. Replacing reactive center loop (RCL) sequences of α₁PDX with those of serpin B8 demonstrated that both the P4–P1 RXXR recognition sequence as well as the P1′–P5′ sequence are critical determinants of serpin specificity for furin. Alignments of PC catalytic domains revealed four variable active-site loops whose role in furin reactivity with serpin B8 was tested by engineering furin-PC loop chimeras. The furin(298–300) loop but not the other loops differentially affected furin reactivity with serpin B8 and α₁PDX in a manner that depended on the serpin RCL-primed sequence. Modeling of the serpin B8-furin Michaelis complex identified serpin exosites in strand 3C close to the 298–300 loop whose substitution in α₁PDX differentially affected furin reactivity depending on the furin loop and serpin RCL-primed sequences. These studies demonstrate that RCL-primed residues, strand 3C exosites, and the furin(298–300) loop are critical determinants of serpin reactivity with furin, which may be exploited in the design of specific and selective α₁PDX inhibitors of PCs.     

Effects of actinobacteria on plant disease suppression and growth promotion

Biological control and plant growth promotion by plant beneficial microbes has been viewed as an alternative to the use of chemical pesticides and fertilizers. Bacteria and fungi that are naturally associated with plants and have a beneficial effect on plant growth by the alleviation of biotic and abiotic stresses were isolated and developed into biocontrol (BCA) and plant growth-promoting agents (PGPA). Actinobacteria are a group of important plant-associated spore-forming bacteria, which have been studied for their biocontrol, plant growth promotion, and interaction with plants. This review summarizes the effects of actinobacteria as BCA, PGPA, and its beneficial associations with plants.