A Trifecta of New Insights into Ovine Footrot for Infection Drivers, Immune Response, and Host-Pathogen Interactions.

Footrot is a polymicrobial infectious disease in sheep causing severe lameness, leading to one of the industry's largest welfare problems. The complex etiology of footrot makes in situ or in vitro investigations difficult. Computational methods offer a solution to understanding the bacteria involved and how they may interact with the host, ultimately providing a way to identify targets for future hypothesis-driven investigative work. Here, we present the first combined global analysis of bacterial community transcripts together with the host immune response in healthy and diseased ovine feet during a natural polymicrobial infection state using metatranscriptomics. The intratissue and surface bacterial populations and the most abundant bacterial transcriptomes were analyzed, demonstrating that footrot-affected skin has reduced diversity and increased abundances of not only the causative bacterium Dichelobacter nodosus but also other species such as Mycoplasma fermentans and Porphyromonas asaccharolytica. Host transcriptomics reveals the suppression of biological processes related to skin barrier function, vascular functions, and immunosurveillance in unhealthy interdigital skin, supported by histological findings that type I collagen (associated with scar tissue formation) is significantly increased in footrot-affected interdigital skin compared to outwardly healthy skin. Finally, we provide some interesting indications of host and pathogen interactions associated with virulence genes and the host spliceosome, which could lead to the identification of future therapeutic targets.

Recapitulation of physiological spatiotemporal signals promotes in vitro formation of phenotypically stable human articular cartilage.

Standard isotropic culture fails to recapitulate the spatiotemporal gradients present during native development. Cartilage grown from human mesenchymal stem cells (hMSCs) is poorly organized and unstable in vivo. We report that human cartilage with physiologic organization and in vivo stability can be grown in vitro from self-assembling hMSCs by implementing spatiotemporal regulation during induction. Self-assembling hMSCs formed cartilage discs in Transwell inserts following isotropic chondrogenic induction with transforming growth factor ß to set up a dual-compartment culture. Following a switch in the basal compartment to a hypertrophic regimen with thyroxine, the cartilage discs underwent progressive deep-zone hypertrophy and mineralization. Concurrent chondrogenic induction in the apical compartment enabled the maintenance of functional and hyaline cartilage. Cartilage homeostasis, chondrocyte maturation, and terminal differentiation markers were all up-regulated versus isotropic control groups. We assessed the in vivo stability of the cartilage formed under different induction regimens. Cartilage formed under spatiotemporal regulation in vitro resisted endochondral ossification, retained the expression of cartilage markers, and remained organized following s.c. implantation in immunocompromised mice. In contrast, the isotropic control groups underwent endochondral ossification. Cartilage formed from hMSCs remained stable and organized in vivo. Spatiotemporal regulation during induction in vitro recapitulated some aspects of native cartilage development, and potentiated the maturation of self-assembling hMSCs into stable and organized cartilage resembling the native articular cartilage.

Antibodies Isolated from Rheumatoid Arthritis Patients against Lysine-Containing Proteus mirabilis O3 (S1959) Lipopolysaccharide May React with Collagen Type I.

Most rheumatic diseases, including rheumatoid arthritis (RA), are characterized by immune disorders that affect antibody activity. In the present study, using Dot blot and ELISA assay, we showed that patients with rheumatic disease produced significantly more antibodies against lipopolysaccharide (LPS) P. mirabilis O3 compared to healthy donors (p < 0.05), and affinity purified antibodies against LPS O3 may cross-react with collagen type I. It was demonstrated that purified of antibodies isolated from RA patients sera, reacted stronger with the collagen than healthy donors (p = 0.015), and cross-reaction was correlated with level of anti-citrullinated peptide antibodies (r = 0.7, p = 0.003). Moreover, using six different lipopolysaccharides were demonstrated the significant correlations in sera reactivity among lysine-containing lipopolysaccharides observed in patients' sera (p < 0.05). Using Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) it was shown that unique wavenumbers of sera spectra correlate with reactivity with lipopolysaccharides allowing distinguish patients from healthy blood donors. Antibodies adsorption by synthetic antigens shows that in patients' group anti-LPS O3 antibodies can be adsorbed by both amides of galacturonic acid and lysine or threonine, which suggests less specificity of antibodies binding with non-carbohydrate LPS component. The observed correlations suggest that non-carbohydrate components of LPS may be an important epitope for less specific anti-LPS antibodies, which might lead to cross-reactions and affect disease development.

Ultrastructural Location and Interactions of the Immunoglobulin Receptor Binding Sequence within Fibrillar Type I Collagen.

Collagen type I is a major constituent of animal bodies. It is found in large quantities in tendon, bone, skin, cartilage, blood vessels, bronchi, and the lung interstitium. It is also produced and accumulates in large amounts in response to certain inflammations such as lung fibrosis. Our understanding of the molecular organization of fibrillar collagen and cellular interaction motifs, such as those involved with immune-associated molecules, continues to be refined. In this study, antibodies raised against type I collagen were used to label intact D-periodic type I collagen fibrils and observed with atomic force microscopy (AFM), and X-ray diffraction (XRD) and immunolabeling positions were observed with both methods. The antibodies bind close to the C-terminal telopeptide which verifies the location and accessibility of both the major histocompatibility complex (MHC) class I (MHCI) binding domain and C-terminal telopeptide on the outside of the collagen fibril. The close proximity of the C-telopeptide and the MHC1 domain of type I collagen to fibronectin, discoidin domain receptor (DDR), and collagenase cleavage domains likely facilitate the interaction of ligands and receptors related to cellular immunity and the collagen-based Extracellular Matrix.

The Commensal Microbiota Enhances ADP-Triggered Integrin αIIbß3 Activation and von Willebrand Factor-Mediated Platelet Deposition to Type I Collagen.

The commensal microbiota is a recognized enhancer of arterial thrombus growth. While several studies have demonstrated the prothrombotic role of the gut microbiota, the molecular mechanisms promoting arterial thrombus growth are still under debate. Here, we demonstrate that germ-free (GF) mice, which from birth lack colonization with a gut microbiota, show diminished static deposition of washed platelets to type I collagen compared with their conventionally raised (CONV-R) counterparts. Flow cytometry experiments revealed that platelets from GF mice show diminished activation of the integrin αIIbß3 (glycoprotein IIbIIIa) when activated by the platelet agonist adenosine diphosphate (ADP). Furthermore, washed platelets from Toll-like receptor-2 (Tlr2)-deficient mice likewise showed impaired static deposition to the subendothelial matrix component type I collagen compared with wild-type (WT) controls, a process that was unaffected by GPIbα-blockade but influenced by von Willebrand factor (VWF) plasma levels. Collectively, our results indicate that microbiota-triggered steady-state activation of innate immune pathways via TLR2 enhances platelet deposition to subendothelial matrix molecules. Our results link host colonization status with the ADP-triggered activation of integrin αIIbß3, a pathway promoting platelet deposition to the growing thrombus.

Comparison of the Effects of KE and AED Peptides on Functional Activity of Human Skin Fibroblasts during Their Replicative Aging.

We studied the effect of KE and AED peptides on the expression of sirtuin-1, sirtuin-6, collagen I, cytokines (IL-1, TGF-ß), and transcription factor NF-κB in human skin fibroblasts during their replicative aging. Immunocytochemical analysis and confocal microscopy showed that KE peptide reduces the synthesis of factors of the inflammatory response IL-1, NF-κB, and TGF-ß and stimulates the synthesis of sirtuin-6. KE peptide normalizes the immunological function of human skin fibroblasts during their aging. AED peptide activates the synthesis of sirtuin-1, sirtuin-6, and collagen I in human skin fibroblasts during their replicative aging, which attests to its geroprotective effect.

Interleukin-17 and -22 synergy linking inflammation and EMT-dependent fibrosis in Sjögren's syndrome.

Primary Sjögren's syndrome (pSS) is a chronic inflammatory, autoimmune and systemic disorder commonly associated with dry eyes and a dry mouth. Recently, the hypothetical link between epithelial-mesenchymal transition (EMT)-dependent salivary gland (SG) fibrosis and chronic inflammatory conditions has been suggested. In this study, we present data demonstrating a negative correlation of the epithelial marker E-cadherin expression and a positive correlation of mesenchymal vimentin and collagen type I expression with increasing degrees of tissue inflammation in pSS SG specimens. In addition, as it is not clear whether dysregulated cytokines in pSS, interleukin (IL)-17 and IL-22 may also contribute to the EMT-dependent fibrosis process, the effect of IL-17 and IL-22 treatment on EMT-dependent SG fibrosis was evaluated in primary human salivary gland epithelial cells (SGEC) isolated from healthy subjects. Here we present data demonstrating that IL-17 and IL-22 can induce SGEC to undergo a morphological and phenotypical transition to a mesenchymal phenotype. In support of this, vimentin and collagen type I were up-regulated while a decreased expression of E-cadherin occurs after interleukin treatment, and co-operation between IL-17 and Il-22 was required to induce the EMT.

Defect of Interferon γ Leads to Impaired Wound Healing through Prolonged Neutrophilic Inflammatory Response and Enhanced MMP-2 Activation.

Interferon (IFN)-γ is mainly secreted by CD4+ T helper 1 (Th1), natural killer (NK) and NKT cells after skin injury. Although IFN-γ is well known regarding its inhibitory effects on collagen synthesis by fibroblasts in vitro, information is limited regarding its role in wound healing in vivo. In the present study, we analyzed how the defect of IFN-γ affects wound healing. Full-thickness wounds were created on the backs of wild type (WT) C57BL/6 and IFN-γ-deficient (KO) mice. We analyzed the percent wound closure, wound breaking strength, accumulation of leukocytes, and expression levels of COL1A1, COL3A1, and matrix metalloproteinases (MMPs). IFN-γKO mice exhibited significant attenuation in wound closure on Day 10 and wound breaking strength on Day 14 after wound creation, characteristics that are associated with prolonged neutrophil accumulation. Expression levels of COL1A1 and COL3A1 mRNA were lower in IFN-γKO than in WT mice, whereas expression levels of MMP-2 (gelatinase) mRNA were significantly greater in IFN-γKO than in WT mice. Moreover, under neutropenic conditions created with anti-Gr-1 monoclonal antibodies, wound closure in IFN-γKO mice was recovered through low MMP-2 expression levels. These results suggest that IFN-γ may be involved in the proliferation and maturation stages of wound healing through the regulation of neutrophilic inflammatory responses.

Endogenous IL-10 maintains immune tolerance but IL-10 gene transfer exacerbates autoimmune cholangitis.

The immunomodulatory effect of IL-10 as an immunosuppressive and anti-inflammatory cytokine is well known. Taking advantage of our established mouse model of autoimmune cholangitis using 2-octynoic acid conjugated ovalbumin (2-OA-OVA) induction, we compared liver pathology, immune cell populations and antimitochondrial antibodies between IL-10 knockout and wild type mice immunized with 2-OA-OVA. At 10 weeks post immunization, portal inflammation and fibrosis were more severe in 2-OA-OVA immunized IL-10 knockout mice than in wild type mice. This was accompanied by significant higher levels of collagen I and III expression, T, NK and NKT subsets in liver and IgG anti-mitochondrial autoantibodies (AMAs) compared to 2-OA-OVA immunized wild type mice, suggesting that endogenous IL-10 is necessary for the maintenance of immune tolerance in primary biliary cholangitis (PBC). Further, we investigated whether administration of exogenous IL-10 could prevent PBC by administration of IL-10 expressing recombinant adeno-associated virus (AAV-IL-10) either 3 days before or 3 weeks after the establishment of liver pathology. Interestingly, administration of AAV-IL-10 resulted in increased liver inflammation and fibrosis, accompanied by increases in IFN-γ in liver CD4+ T cell, granzyme B, FasL, and CD107a in liver CD8+ T and NKT cells, and granzyme B and FasL in liver NK cells of AAV-IL-10 administered mice compared with control mice. Furthermore, administration of AAV-IL-10 significantly increased levels of proinflammatory cytokines and chemokines (IFN-γ, TNF-α, CXCL9 and CXCL10) and collagen I and III production in naïve mice, together with increase in immune cell infiltration and collagen deposition in the liver, suggesting a role of IL-10 in fibrosis. In conclusion, our data demonstrate that endogenous IL-10 is critical in the maintenance of immune tolerance but exogenous administration of IL-10 exacerbates liver inflammation and fibrosis. Furthermore, the distinctive presence of inflammatory immune cell populations and collagen expression in AAV-IL-10 treated naïve mice cautions against the clinical use of exogenous IL-10 in patients with autoimmune cholangitis.

Periostin in kidney diseases.

Chronic kidney disease is an incurable to date pathology, with renal replacement therapy through dialysis or transplantation being the only available option for end-stage patients. A deeper understanding of the molecular mechanisms governing the progression of kidney diseases will permit the identification of unknown mediators and potential novel markers or targets of therapy which promise more efficient diagnostic and therapeutic applications. Over the last years, periostin was established by several studies as a novel key player in the progression of renal disease. Periostin is de novo expressed focally by the injured kidney cells during the development of renal disease. In diverse cohorts of renal disease patients, the expression levels of periostin in the kidney and urine were highly correlated with the stage of the pathology and the decline of renal function. Subsequent studies in animal models demonstrated that periostin is centrally involved in mediating renal inflammation and fibrosis, contributing to the deterioration of renal structure and function. Genetic or pharmaco-genetic inhibition of periostin in animal models of renal disease was efficient in arresting the progression of the pathology. This review will summarize the recent advances on periostin in the field of kidney diseases and will discuss its utility of as a novel target of therapy for chronic kidney disease.

The role of periostin in lung fibrosis and airway remodeling.

Periostin is a protein that plays a key role in development and repair within the biological matrix of the lung. As a matricellular protein that does not contribute to extracellular matrix structure, periostin interacts with other extracellular matrix proteins to regulate the composition of the matrix in the lung and other organs. In this review, we discuss the studies exploring the role of periostin to date in chronic respiratory diseases, namely asthma and idiopathic pulmonary fibrosis. Asthma is a major health problem globally affecting millions of people worldwide with significant associated morbidity and mortality. Periostin is highly expressed in the lungs of asthmatic patients, contributes to mucus secretion, airway fibrosis and remodeling and is recognized as a biomarker of Th2 high inflammation. Idiopathic pulmonary fibrosis is a fatal interstitial lung disease characterized by progressive aberrant fibrosis of the lung matrix and respiratory failure. It predominantly affects adults over 50 years of age and its incidence is increasing worldwide. Periostin is also highly expressed in the lungs of idiopathic pulmonary fibrosis patients. Serum levels of periostin may predict clinical progression in this disease and periostin promotes myofibroblast differentiation and type 1 collagen production to contribute to aberrant lung fibrosis. Studies to date suggest that periostin is a key player in several pathogenic mechanisms within the lung and may provide us with a useful biomarker of clinical progression in both asthma and idiopathic pulmonary fibrosis.

Periostin in the pathogenesis of skin diseases.

Skin is an organ that is susceptible to damage by external injury, chronic inflammation, and autoimmunity. Tissue damage causes alterations in both the configuration and type of cells in lesional skin. This phenomenon, called tissue remodeling, is a universal biological response elicited by programmed cell death, inflammation, immune disorders, and tumorigenic, tumor proliferative, and cytoreductive activity. In this process, changes in the components of the extracellular matrix are required to provide an environment that facilitates tissue remodeling. Among these extracellular matrix components, periostin, a glycoprotein that is predominantly secreted from dermal fibroblasts, has attracted attention. Periostin localizes in the papillary dermis of normal skin, and is aberrantly expressed in the dermis of lesional skin in atopic dermatitis, scar, systemic/limited scleroderma, melanoma, cutaneous T cell lymphoma, and skin damage caused by allergic/autoimmune responses. Periostin induces processes that result in the development of dermal fibrosis, and activate or protract the immune response. The aim of this review was to summarize recent knowledge of the role of periostin in the pathogenesis of dermatoses, and to explore whether periostin is a potential therapeutic target for skin diseases.

Autoantibodies to post-translationally modified type I and II collagen in Charcot neuroarthropathy in subjects with type 2 diabetes mellitus.

AIMS: Charcot neuroarthropathy (CN) is a disabling complication, culminating in bone destruction and involving joints and articular cartilage with high inflammatory environment. Its real pathogenesis is as yet unknown. In autoinflammatory diseases, such as rheumatoid arthritis, characterized by inflammation and joint involvement, autoantibodies against oxidative post-translationally modified (oxPTM) collagen type I (CI) and type II (CII) were detected. Therefore, the aim of our study was to assess the potential involvement of autoimmunity in charcot neuroarthropathy, investigating the presence of autoantibodies oxPTM-CI and oxPTM-CII, in participants with charcot neuroarthropathy. METHODS: In this case-control study, we enrolled 124 participants with type 2 diabetes mellitus (47 with charcot neuroarthropathy, 37 with diabetic peripheral neuropathy without charcot neuroarthropathy, and 40 with uncomplicated diabetes), and 32 healthy controls. The CI and CII were modified with ribose and other oxidant species, and the modifications were evaluated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Binding of sera from the participants was analyzed with enzyme-linked immunosorbent assay. RESULTS: Age, body mass index, waist and hip circumferences, and lipid profile were similar across the 4 groups, as well as glycated hemoglobin and duration of diabetes among people with diabetes. An increased binding to both native and all oxidation-modified forms of CII was found in participants with CN and diabetic neuropathy. Conversely, for CI, an aspecific increased reactivity was noted. CONCLUSIONS: Our results detected the presence of autoantibodies against oxidative post-translational modified collagen, particularly type 2 collagen, in participants with charcot neuroarthropathy and diabetic neuropathy, suggesting the possible involvement of autoimmunity. Further studies are required to understand the role of autoimmunity in the pathogenesis of charcot neuroarthropathy.

EBI3 Downregulation Contributes to Type I Collagen Overexpression in Scleroderma Skin.

IL-12 family cytokines are implicated in the pathogenesis of various autoimmune diseases, but their role in the regulation of extracellular matrix expression and its contribution to the phenotype of systemic sclerosis (SSc) remain to be elucidated. Among the IL-12 family members, IL-35 decreases type I collagen expression in cultured dermal fibroblasts. IL-35 consists of p35 and EBI3 subunits, and EBI3 alone could downregulate the protein and mRNA expression of type I or type III collagen in the presence or absence of TGF-ß costimulation. We found that collagen mRNA stability was reduced by EBI3 via the induction of miR-4500. The IL-35 levels in the sera or on the surface of T cells were not altered in SSc patients, while EBI3 expression was decreased in the keratinocytes of the epidermis and regulatory T cells of the dermis in SSc skin compared with normal skin, which may induce collagen synthesis in SSc dermal fibroblasts. We also found that gp130, the EBI3 receptor, was expressed in both normal and SSc fibroblasts. Moreover, we revealed that EBI3 supplementation by injection into the skin improves mice skin fibrosis. Decreased EBI3 in SSc skin may contribute to an increase in collagen accumulation and skin fibrosis. Clarifying the mechanism regulating the extracellular matrix expression by EBI3 in SSc skin may lead to better understanding of this disease and new therapeutic strategies using ointment or microinjection of the subunit.

Silencing of Long Non-Coding RNA NONHSAT009968 Ameliorates the Staphylococcal Protein A-Inhibited Osteogenic Differentiation in Human Bone Mesenchymal Stem Cells.

BACKGROUND/AIMS: Osteomyelitis is defined as an inflammation of the bones and bone marrow. The inflammatory microenvironment attenuates the osteogenic differentiation capacity of stem cells and inhibits osteoblast-mediated bone formation, leading to net bone loss. However, the whole expression profile, function and side effect of long non-coding RNAs (lncRNAs) on osteogenic differentiation of stem cells in an inflammatory microenvironment of osteomyelitis are not known. METHODS: In the present study, human bone mesenchymal stem cells (hBMSCs) were treated with different concentrations of Staphylococcal protein A (SpA) to trigger an inflammatory microenvironment in vitro to partly duplicate the inflammatory microenvironment of osteomyelitis, which was confirmed using ELISA for detecting the inflammatory cytokines. The complete expression profiles of lncRNAs and mRNA during osteogenic differentiation of hBMSCs in an inflammatory microenvironment triggered by SpA were analyzed using a lncRNA microarray. LncRNA expression levels were verified by quantitative reverse transcription PCR analysis (qRT-PCR). The expression of NONHSAT009968 in hB-MSCs was silenced by infection with lentivirus expressing NONHSAT009968-shRNA. The expression of Runx2, OCN, OPN, COL1A1, and alkaline phosphatase (ALP) activity was detected by western blot. Alizarin red staining and ALP activity detection were carried out. RESULTS: The results of ELISA showed that SpA treatment induced secretion of inflammatory cytokines IL-1A, IL-6, and TNFA. The results of alizarin red staining and ALP detection showed that SpA treatment suppressed the osteogenic differentiation of hBMSCs. A total of 2033 lncRNAs were found with aberrant expression in SpA-treated hBMSCs compared to controls. Among these lncRNAs, 641 were down-regulated and 1392 were up-regulated. Based on the results of qRT-PCR, lncRNA NONHSAT009968 was chosen for further investigation. The results of alizarin red staining, ALP activity detection, and western blot detection of Runx2, OCN, OPN, COL1A1, and ALP indicated that NONHSAT009968 silencing ameliorates SpA-inhibited osteogenic differentiation in hBMSCs. CONCLUSION: Our present study provides a basis for future analyses of the role of lncRNAs in osteoblastic differentiation in an inflammatory environment triggered by SpA, and lncRNA NONHSAT009968 might be a new target for promoting osteoblast formation.

Anti-tumor effects of DNA vaccine targeting human fibroblast activation protein α by producing specific immune responses and altering tumor microenvironment in the 4T1 murine breast cancer model.

Fibroblast activation protein α (FAPα) is a tumor stromal antigen overexpressed by cancer-associated fibroblasts (CAFs). CAFs are genetically more stable compared with the tumor cells and immunosuppressive components of the tumor microenvironment, rendering them excellent targets for cancer immunotherapy. DNA vaccines are widely applied due to their safety. To specifically destroy CAFs, we constructed and examined the immunogenicity and anti-tumor immune mechanism of a DNA vaccine expressing human FAPα. This vaccine successfully reduced 4T1 tumor growth through producing FAPα-specific cytotoxic T lymphocyte responses which could kill CAFs, and the decrease in FAPα-expressing CAFs resulted in markedly attenuated expression of collagen I and other stromal factors that benefit the tumor progression. Based on these results, a DNA vaccine targeting human FAPα may be an attractive and effective cancer immunotherapy strategy.

The fibronectin-binding protein EfbA contributes to pathogenesis and protects against infective endocarditis caused by Enterococcus faecalis.

EfbA is a PavA-like fibronectin adhesin of Enterococcus faecalis previously shown to be important in experimental urinary tract infection. Here, we expressed and purified the E. faecalis OG1RF EfbA and confirmed that this protein binds with high affinity to immobilized fibronectin, collagen I, and collagen V. We constructed an efbA deletion mutant and demonstrated that its virulence was significantly attenuated (P < 0.0006) versus the wild type in a mixed inoculum rat endocarditis model. Furthermore, efbA deletion resulted in diminished ability to bind fibronectin (P < 0.0001) and reduced biofilm (P < 0.001). Reintroduction of efbA into the original chromosomal location restored virulence, adherence to fibronectin, and biofilm formation to wild-type levels. Finally, vaccination of rats with purified recombinant EfbA protein protected against OG1RF endocarditis (P = 0.008 versus control). Taken together, our results demonstrate that EfbA is an important factor involved in E. faecalis endocarditis and that rEfbA immunization is effective in preventing such infection, likely by interfering with bacterial adherence.

Neurotensin-loaded collagen dressings reduce inflammation and improve wound healing in diabetic mice.

Impaired wound healing is an important clinical problem in diabetes mellitus and results in failure to completely heal diabetic foot ulcers (DFUs), which may lead to lower extremity amputations. In the present study, collagen based dressings were prepared to be applied as support for the delivery of neurotensin (NT), a neuropeptide that acts as an inflammatory modulator in wound healing. The performance of NT alone and NT-loaded collagen matrices to treat wounds in streptozotocin (STZ) diabetic induced mice was evaluated. Results showed that the prepared dressings were not-cytotoxic up to 72h after contact with macrophages (Raw 264.7) and human keratinocyte (HaCaT) cell lines. Moreover, those cells were shown to adhere to the collagen matrices without noticeable change in their morphology. NT-loaded collagen dressings induced faster healing (17% wound area reduction) in the early phases of wound healing in diabetic wounded mice. In addition, they also significantly reduced inflammatory cytokine expression namely, TNF-α (p<0.01) and IL-1ß (p<0.01) and decreased the inflammatory infiltrate at day 3 post-wounding (inflammatory phase). After complete healing, metalloproteinase 9 (MMP-9) is reduced in diabetic skin (p<0.05) which significantly increased fibroblast migration and collagen (collagen type I, alpha 2 (COL1A2) and collagen type III, alpha 1 (COL3A1)) expression and deposition. These results suggest that collagen-based dressings can be an effective support for NT release into diabetic wound enhancing the healing process. Nevertheless, a more prominent scar is observed in diabetic wounds treated with collagen when compared to the treatment with NT alone.

Induction of a type I interferon signature in normal human monocytes by gadolinium-based contrast agents: comparison of linear and macrocyclic agents.

The gadolinium-based contrast agent (GdBCA) Omniscan activates human macrophages through Toll-like receptor (TLR)-4 and TLR-7 signalling. To explore the mechanisms responsible we compared the ability of linear and macrocyclic GdBCA to induce a type I interferon signature and a proinflammatory/profibrotic phenotype in normal human monocytes in vitro. Expression of genes associated with type I interferon signalling and inflammation and production of their corresponding proteins were determined. Both linear and macrocyclic GdBCA stimulated expression of multiple type I interferon-regulated genes and the expression of numerous chemokines, cytokines and growth factors in normal human peripheral blood monocytes. There was no correlation between the magnitude of the measured response and the Gd chelate used. To explore the mechanisms responsible for GdBCA induction of fibrosis in nephrogenic systemic fibrosis (NSF) in vitro, normal human dermal fibroblasts were incubated with GdBCA-treated monocyte culture supernatants and the effects on profibrotic gene expression were examined. Supernatants from monocytes exposed to all GdBCA stimulated types I and III collagen, fibronectin and α-smooth muscle actin (α-SMA) expression in normal dermal fibroblasts. The results indicate that the monocyte activation induced by GdBCA may be the initial step in the development of GdBCA associated fibrosis in NSF.

Testing the anti-fibrotic potential of the single-chain Fv antibody against the α2 C-terminal telopeptide of collagen I.

Abstract This study focuses on the single-chain fragment variable (scFv) variant of the original IgA-type antibody, recognizing the α2 C-terminal telopeptide (α2Ct) of human collagen I, designed to inhibit post-traumatic localized fibrosis via blocking the formation of collagen-rich deposits. We have demonstrated that the scFv construct expressed in yeast cells was able to fold into an immunoglobulin-like conformation, but it was prone to forming soluble aggregates. Functional assays, however, indicate that the scFv construct specifically binds to the α2Ct epitope and inhibits collagen fibril formation both in vitro and in a cell culture model representing tissues that undergo post-traumatic fibrosis. Thus, the presented study demonstrates the potential of the scFv variant to serve as an inhibitor of the excessive formation of collagen-rich fibrotic deposits, and it reveals certain limitations associated with the current stage of development of this antibody construct.