EPCR deficiency ameliorates inflammatory arthritis in mice by suppressing the activation and migration of T cells and dendritic cells.

OBJECTIVES: Endothelial protein C receptor (EPCR) is highly expressed in synovial tissues of patients with RA, but the function of this receptor remains unknown in RA. This study investigated the effect of EPCR on the onset and development of inflammatory arthritis and its underlying mechanisms. METHODS: CIA was induced in EPCR gene knockout (KO) and matched wild-type (WT) mice. The onset and development of arthritis was monitored clinically and histologically. T cells, dendritic cells (DCs), EPCR and cytokines from EPCR KO and WT mice, RA patients and healthy controls (HCs) were detected by flow cytometry and ELISA. RESULTS: EPCR KO mice displayed >40% lower arthritis incidence and 50% less disease severity than WT mice. EPCR KO mice also had significantly fewer Th1/Th17 cells in synovial tissues with more DCs in circulation. Lymph nodes and synovial CD4 T cells from EPCR KO mice expressed fewer chemokine receptors CXCR3, CXCR5 and CCR6 than WT mice. In vitro, EPCR KO spleen cells contained fewer Th1 and more Th2 and Th17 cells than WT and, in concordance, blocking EPCR in WT cells stimulated Th2 and Th17 cells. DCs generated from EPCR KO bone marrow were less mature and produced less MMP-9. Circulating T cells from RA patients expressed higher levels of EPCR than HC cells; blocking EPCR stimulated Th2 and Treg cells in vitro. CONCLUSION: Deficiency of EPCR ameliorates arthritis in CIA via inhibition of the activation and migration of pathogenic Th cells and DCs. Targeting EPCR may constitute a novel strategy for future RA treatment.

Skin Barrier Dysregulation in Psoriasis.

The skin barrier is broadly composed of two elements-a physical barrier mostly localised in the epidermis, and an immune barrier localised in both the dermis and epidermis. These two systems interact cooperatively to maintain skin homeostasis and overall human health. However, if dysregulated, several skin diseases may arise. Psoriasis is one of the most prevalent skin diseases associated with disrupted barrier function. It is characterised by the formation of psoriatic lesions, the aberrant differentiation and proliferation of keratinocytes, and excessive inflammation. In this review, we summarize recent discoveries in disease pathogenesis, including the contribution of keratinocytes, immune cells, genetic and environmental factors, and how they advance current and future treatments.

Activated Protein C in Cutaneous Wound Healing: From Bench to Bedside.

Independent of its well-known anticoagulation effects, activated protein C (APC) exhibits pleiotropic cytoprotective properties. These include anti-inflammatory actions, anti-apoptosis, and endothelial and epithelial barrier stabilisation. Such beneficial effects have made APC an attractive target of research in a plethora of physiological and pathophysiological processes. Of note, the past decade or so has seen the emergence of its roles in cutaneous wound healing-a complex process involving inflammation, proliferation and remodelling. This review will highlight APC's functions and mechanisms, and detail its pre-clinical and clinical studies on cutaneous wound healing.

Functional characterization of Gram-negative bacteria from different genera as multiplex cadmium biosensors.

Widespread presence of cadmium in soil and water systems is a consequence of industrial and agricultural processes. Subsequent accumulation of cadmium in food and drinking water can result in accidental consumption of dangerous concentrations. As such, cadmium environmental contamination poses a significant threat to human health. Development of microbial biosensors, as a novel alternative method for in situ cadmium detection, may reduce human exposure by complementing traditional analytical methods. In this study, a multiplex cadmium biosensing construct was assembled by cloning a single-output cadmium biosensor element, cadRgfp, and a constitutively expressed mrfp1 onto a broad-host range vector. Incorporation of the duplex fluorescent output [green and red fluorescence proteins] allowed measurement of biosensor functionality and viability. The biosensor construct was tested in several Gram-negative bacteria including Pseudomonas, Shewanella and Enterobacter. The multiplex cadmium biosensors were responsive to cadmium concentrations ranging from 0.01 to 10µgml-1, as well as several other heavy metals, including arsenic, mercury and lead at similar concentrations. The biosensors were also responsive within 20-40min following exposure to 3µgml-1 cadmium. This study highlights the importance of testing biosensor constructs, developed using synthetic biology principles, in different bacterial genera.

Development and Application of a Synthetically-Derived Lead Biosensor Construct for Use in Gram-Negative Bacteria.

The use of lead in manufacturing has decreased significantly over the last few decades. However, previous widespread use of lead-containing products and their incorrect disposal has resulted in environmental contamination. Accumulation of harmful quantities of lead pose a threat to all living organisms, through inhalation, ingestion, or direct contact, resulting in lead poisoning. This study utilized synthetic biology principles to develop plasmid-based whole-cell bacterial biosensors for detection of lead. The genetic element of the lead biosensor construct consists of pbrR, which encodes the regulatory protein, together with its divergent promoter region and a promoterless gfp. GFP expression is controlled by PbrR in response to the presence of lead. The lead biosensor genetic element was cloned onto a low-copy number broad host range plasmid, which can stably exist in a range of laboratory and environmental isolates, including Pseudomonas, Shewanella, and Enterobacter. The biosensors constructed were found to be sensitive, rapid, and specific and could, as such, serve as monitoring tools for lead-contaminated water.

Uptake of milk with and without solid feed during the monogastric phase: Effect on fibrolytic and methanogenic microorganisms in the gastrointestinal tract of calves.

Microbial communities are affected by diet and play a role in the successful transition from milk to a solid diet. The response of microorganisms in the gastrointestinal tract of Holstein bull calves to the uptake of milk with solid feed (control treatment; CT), or milk without solid feed (milk-only treatment; MT) during the first 3 weeks of life was investigated. Samples were collected from the rumen (fluid and tissue), abomasum (fluid), cecum (fluid and tissue) and feces at 7, 14 and 20 days of age. Calf weight was higher on days 14 and 20 in the MT than the CT. In the rumen at 14 days, the fibrolytic bacteria Fibrobacter succinogenes and Prevotella ruminicola increased in the CT and Ruminococcus flavefaciens increased in the MT. This suggests that R. flavefaciens is not strictly fibrolytic and that it might use milk as a substrate or other microbial species might supply a substrate. Diet affected methanogens, but this may have been due to an indirect effect via an association with Geobacter spp. or other syntrophic partners. The treatments also affected microorganisms in the abomasum, cecum and feces. Our results contribute to an understanding of diet, microbes in the gastrointestinal tract and weaning.

Presence of Selected Methanogens, Fibrolytic Bacteria, and Proteobacteria in the Gastrointestinal Tract of Neonatal Dairy Calves from Birth to 72 Hours.

The microbial communities in the gastrointestinal tract of a young calf are essential for the anatomical and physiological development that permits a transition from milk to solid feed. Selected methanogens, fibrolytic bacteria, and proteobacteria were quantified in the rumen fluid and tissue, abomasum fluid, cecum fluid and tissue, and feces of Holstein bull calves on day 0 (0-20 mins after birth), day 1 (24 ± 1 h after birth), day 2 (48 ± 1 h after birth), and day 3 (72 ± 1 h after birth). Methanogens, fibrolytic bacteria, and Geobacter spp. were found to be already present from birth, indicating that microbial colonization of the gastrointestinal tract occurred before or during delivery. The abundance of methanogens and Geobacter spp. differed between the days tested and between compartments of the digestive tract and feces, but such difference was not observed for fibrolytic bacteria. Our findings suggests that methanogens might have an alternative hydrogen provider such as Geobacter spp. during these early stages of postnatal development. In addition, fibrolytic bacteria were present in the rumen well before the availability of fibrous substrates, suggesting that they might use nutrients other than cellulose and hemicellose.

Environmental sensing of heavy metals through whole cell microbial biosensors: a synthetic biology approach.

Whole cell microbial biosensors are offering an alternative means for rapid, on-site heavy metal detection. Based in microorganisms, biosensing constructs are designed and constructed to produce both qualitative and quantitative outputs in response to heavy metal ions. Previous microbial biosensors designs are focused on single-input constructs; however, development of multiplexed systems is resulting in more flexible designs. The movement of microbial biosensors from laboratory based designs toward on-site, functioning heavy metal detectors has been hindered by the toxic nature of heavy metals, along with the lack of specificity of heavy metals promoter elements. Applying a synthetic biology approach with alternative microbial chassis may increase the robustness of microbial biosensors and mitigate these issues. Before full applications are achieved, further consideration has to be made regarding the risk and regulations of whole cell microbial biosensor use in the environment. To this end, a standard framework for future whole cell microbial biosensor design and use is proposed.