Collagen-binding integrin alpha11beta1 contributes to joint destruction in arthritic hTNFtg mice

Background: In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) undergo a tumor-like transformation, wherein they develop an aggressive phenotype that is characterized by increased adhesion to components of cartilage extracellular matrix (ECM) and that contributes extensively to joint destruction. The collagen-binding integrin alpha11beta1 was previously shown to be involved in similar processes in cancer-associated fibroblasts mediating tumorigenicity and metastasis in certain tumors. Therefore, this study aimed to study the role of integrin alpha11beta1 in RA and to characterize the effects of alpha11beta1 deficiency on the disease course and severity in arthritic hTNFtg mice. Methods: The expression levels of integrin alpha11beta1 were analyzed by immunohistochemistry, immunofluorescence, and western blot analysis in synovial samples and FLS of patients with RA and osteoarthritis (OA) as well as in samples from wild type (wt) and arthritic hTNFtg mice. Furthermore, the subcellular expression of integrin alpha11beta1 was investigated in co-culture experiments with cartilage explants and analyzed by transmission electron microscopy. To investigate the effects of integrin alpha11beta1 deficiency, itga11-/- mice were interbred with hTNFtg mice and disease severity was assessed by clinical scoring of grip strength and paw swelling over the disease course. Hind paws of 12-weeks-old mice of all genotypes were analyzed by uCT imaging followed by stainings of paraffin-embedded tissue sections with Toluidine-blue and tartrate-resistant acid phosphatase (TRAP) to evaluate established parameters of joint destruction such as inflammation area, cartilage destaining, FLS attachment to the cartilage surface, and bone damage. Results: Expression levels of integrin alpha11beta1 were clearly elevated in synovial tissues and FLS from RA patients and hTNFtg mice, compared to the controls derived from OA patients and wt mice. Interestingly, this expression was shown to be particularly localized in focal adhesions of the FLS. As revealed by transmission electron microscopy, integrin alpha11beta1 expression was particularly evident in areas of direct cellular contact with the ECM of cartilage. Evaluations of clinical scorings and histomorphological analyses demonstrated that itga11-/-hTNFtg displayed alleviated clinical symptoms, higher bone volume, less cartilage destruction, and reduced FLS attachment to the cartilage in comparison to hTNFtg mice. Conclusions: The collagen-binding integrin alpha11beta1 is upregulated in the context of RA and its deficiency in mice with an inflammatory hTNFtg background leads to a significant reduction in the arthritic phenotype which makes integrin alpha11beta1 an interesting target for therapeutical intervention.

Title IgA Nephropathy and Oral Bacterial Species Related to Dental Caries and Periodontitis

A relationship between IgA nephropathy (IgAN) and bacterial infection has been suspected. As IgAN is a chronic disease, bacteria that could cause chronic infection in oral areas might be pathogenetic bacteria candidates. Oral bacterial species related to dental caries and periodontitis should be candidates because these bacteria are well known to be pathogenic in chronic dental disease. Recently, several reports have indicated that collagen-binding protein (cnm)-(+) Streptococcs mutans is relate to the incidence of IgAN and the progression of IgAN. Among periodontal bacteria, Treponema denticola, Porphyromonas gingivalis and Campylobacte rectus were found to be related to the incidence of IgAN. These bacteria can cause IgAN-like histological findings in animal models. While the connection between oral bacterial infection, such as infection with S. mutans and periodontal bacteria, and the incidence of IgAN remains unclear, these bacterial infections might cause aberrantly glycosylated IgA1 in nasopharynx-associated lymphoid tissue, which has been reported to cause IgA deposition in mesangial areas in glomeruli, probably through the alteration of microRNAs related to the expression of glycosylation enzymes. The roles of other factors related to the incidence and progression of IgA, such as genes and cigarette smoking, can also be explained from the perspective of the relationship between these factors and oral bacteria. This review summarizes the relationship between IgAN and oral bacteria, such as cnm-(+) S. mutans and periodontal bacteria.

Cell-derived extracellular matrix enhanced by collagen-binding domain-decorated exosomes to promote neural stem cells neurogenesis

Enhancing neurogenesis of neural stem cells (NSCs) is crucial in stem cell therapy for neurodegenerative diseases. Within the extracellular microenvironment, extracellular matrix (ECM) plays a pivotal role in modulating cell behaviors. However, a single ECM biomaterial is not sufficient to establish an ideal microenvironment. As multifunctional nanocarriers, exosomes display tremendous advantages for the treatments of various diseases. Herein, collagen binding domain peptide-modified exosomes (CBD-Exo) were obtained from the SH-SY5Y cell line infected with lentivirus particles encoding CBD-lysosome associated membrane glycoprotein 2b (CBD-Lamp2b) to improve the binding efficiency of exosomes and ECM. An exosomes-functionalized ECM (CBD-Exo/ECM) was then constructed via the interaction between CBD and collagen in ECM. Then, CBD-Exo/ECM was employed as a carrier for NSCs culture. The results showed that CBD-Exo/ECM can support the neurogenesis of NSCs with the percentage of proliferation marker EdU-positive (35.8% ± 0.47% vs. 21.9% ± 2.32%) and neuron maker Tuj-1-positive (55.8% ± 0.47% vs. 30.6% ± 2.62%) were both significantly increased in the exosomes-functionalized ECM system. This exosomes-functionalized ECM was capable to promote the cell proliferation and accelerate neuronal differentiation of NSCs, providing a potential biomedical material for stem cell application in tissue engineering and regenerative medicine.

The Extracellular Matrix Enriched With Exosomes for the Treatment on Pulmonary Fibrosis in Mice

Pulmonary fibrosis (PF) is a severe respiratory disease caused by lung microenvironment changes. TGF-β/Smad3 signaling pathway plays a critical role in the fibrotic process. MicroRNA-29 (miR-29) has proved to alleviate the occurrence of PF by downregulating TGF-β/Smad3 signaling pathway. The miRNA application encounters obstacles due to its low stability in body and no targeting to lesions. Exosomes can be used for therapeutic delivery of miRNA due to their favorable delivery properties. However, low efficiency of separation and production impedes the therapeutic application of exosomes. In this study, we developed a liquid natural extracellular matrix (ECM) enriched with miR-29-loaded exosomes for PF treatment. The collagen-binding domain (CBD)-fused Lamp2b (CBD-Lamp2b) and miR-29 were overexpressed in human foreskin fibroblast (HFF) host cells for the entrapment of miR-29-loaded exosomes in ECM of the cells. The repeated freeze-thaw method was performed to prepare the liquid ECM enriched with exosomes without destroying the exosomal membrane. In summary, this study developed a novel functional ECM biomaterial for therapy of PF, and also provided a promising gene therapy platform for different diseases by treatment with liquid ECM that is, enriched with exosomes loaded with different functional miRNAs.

Analyses of the Effects of Arginine, Nicotine, Serotype and Collagen-Binding Proteins on Biofilm Development by 33 Strains of Streptococcus mutans

Streptococcus mutans serotype k strains comprise <3% of oral isolates of S. mutans but are prominent in diseased cardiovascular (CV) tissue. Collagen binding protein (CBP) genes, cbm and cnm, are prevalent in serotype k strains and are associated with endothelial cell invasion. Nicotine increases biofilm formation by serotype c strains of S. mutans, but its effects on serotype k strains and strains with CBP are unknown. Saliva contains arginine which alters certain properties of the extracellular polysaccharides (EPS) in S. mutans biofilm. We examined whether nicotine and arginine affect sucrose-induced biofilm of S. mutans serotypes k (n = 23) and c (n = 10) strains with and without CBP genes. Biofilm mass, metabolism, bacterial proliferation, and EPS production were assessed. Nicotine increased biomass and metabolic activity (p < 0.0001); arginine alone had no effect. The presence of a CBP gene (either cbm or cnm) had a significant effect on biofilm production, but serotype did not. Nicotine increased bacterial proliferation and the effect was greater in CBP + strains compared to strains lacking CBP genes. Addition of arginine with nicotine decreased both bacterial mass and EPS compared to biofilm grown in nicotine alone. EPS production was greater in cnm + than cbm + strains (p < 0.0001). Given the findings of S. mutans in diseased CV tissue, a nicotine induced increase in biofilm production by CBP + strains may be a key link between tobacco use and CV diseases.

Collagen-Binding Nanoparticles: A Scoping Review of Methods and Outcomes

(1) Background: Collagen is the main component of the connective tissue, playing an important role in the histological architecture and function of living organisms. Targeted therapy and improved imaging diagnosis can be obtained through collagen-binding nanoparticles that concentrate in the extracellular matrix. (2) Methods: We performed a scoping review of studies that analyzed the binding capacity of collagen-targeting nanoparticles. The search algorithm and inclusion criteria were based on PRISMA and ARRIVE guidelines. (3) Results: Fourteen studies matched all the inclusion criteria. All studies analyzed the distribution of nanoparticles in the collagen matrix, either by using collagen-targeting nanoparticles or by using unmodified ones. Most studies used collagen-binding nanoparticles for vascular research to target sites of endothelial injury, atherosclerotic plaques, or myocardial infarction. Two studies targeted the exposed collagen in models of liver fibrosis. (4) Conclusions: Our review summarizes the current literature on the methods and outcomes of using nanoparticles to target collagen. The studies reveal that there is high applicability for collagen-binding nanoparticles in cardiac or hepatic pathology and they could prove useful for targeted therapy of neoplastic lesions, which show an abundance of stromal collagen.

876 Intratumoral administration and local retention of IL-2/IL-12 fusion proteins drive a potent systemic anti-tumor immune response

BackgroundIL-2 and IL-12 synergistically trigger the stimulation and proliferation of T and NK cells to mediate anti-tumor immunity. Although aldesleukin, a high-dose IL-2 intravenous (IV) infusion regimen, has been approved for the treatment of melanoma and renal cell carcinoma, adoption has been hindered by frequent grade 3 and 4 severe adverse events. No IL-12 therapy has been approved yet due to toxicity. Cullinan Amber is developing a fusion protein that uniquely combines in one polypeptide both IL-2 and IL-12 with a collagen-binding domain to reduce toxicity and increase efficacy following intra-tumoral (IT) administration via retention in the tumor microenvironment.MethodsProteins were expressed in HEK293 cells. Collagen binding was measured by ELISA. IL-2 and IL-12 bioactivity was evaluated by CTLL-2 proliferation and HEK-Blue IL-12 reporter cells. In vivo studies were conducted in B16F10, MC38, and CT26 syngeneic tumor models. Systemic Amber construct concentrations were determined by ELISA.Results”Amber” constructs, comprised of IL-2, IL-12, and a collagen-binding domain, were produced and confirmed to retain bioactivity. B16F10 tumor-bearing mice injected with Amber IT had systemic Amber levels <5% as compared to mice administered the same dose IV. When IL-2/IL-12 fusion proteins lacking a collagen-binding domain were injected IT in B16F10-bearing mice, 60% of mice needed to be euthanized due to severe body weight loss, while Amber-treated mice did not lose body weight. In the checkpoint-refractory B16F10 and MC38 models, Amber demonstrated 95% tumor growth inhibition (figure 1a) and 100% CRs (figure 1b), respectively. 90% of the mice cured of their primary MC38 tumors were protected from re-challenge (figure 1b). Notably, 70% CRs were observed in the MC38 model even after a single-dose treatment of Amber. Similar data was obtained in the CT26 model. Amber treatment of mice bearing large 500 mm3 MC38 tumors resulted in dramatic tumor shrinkage (figure 1c). In mice bearing two MC38 tumors, only one of which was treated IT, 100% of treated tumors and 90% of distal untreated tumors were eliminated when Amber was combined with an anti-PD1 antibody (figure 1d), demonstrating a robust abscopal response.Abstract 876 Figure 1Efficacy of amber constructs in syngeneic tumor modelsConclusionsThe use of collagen-binding domains for tumor retention enables the safe and effective delivery of IL-2 and IL-12 in a single multifunctional molecule. Taken together, the preclinical data suggests that Amber constructs may show robust single-agent activity in clinical trials against checkpoint-refractory tumors with minimal toxicity, as well as the potential to significantly deepen anti-tumor responses in combination with checkpoint inhibitor therapy.

Urethral Tissue Reconstruction Using the Acellular Dermal Matrix Patch Modified with Collagen-Binding VEGF in Beagle Urethral Injury Models

Objectives. Urethral tissue reconstruction for hypospadias is challenging for urologists. In this study, bovine acellular dermal matrix (ADM) patch loading with collagen-binding vascular endothelial growth factor (CBD-VEGF) was used to repair the urethral injury in beagles. Methods. The safety and effectiveness of the scaffold implantation were carefully evaluated by comparing among the urethral injury control group, ADM implantation group, and ADM modified with CBD-VEGF implantation group during 6 months. Urodynamic examination, urethral angiography, and pathological examination were performed to evaluate the recovery of urethral tissue. Results. Stricture, urethral diverticulum, and increased urethral closure pressure were observed in the control group. Fistula was observed in one animal in the ADM group. By contrast, no related complications or other adverse situations were observed in animals treated with ADM patch modified with CBD-VEGF. The average urethra diameter was significantly smaller in the control animals than in scaffold implantation groups. Pathological examination revealed more distribution of proliferative blood vessels in the animals treated with ADM modified with CBD-VEGF. Conclusions. Overall, ADM patches modified with CBD-VEGF demonstrated an optimized tissue repair performance in a way to increase tissue angiogenesis and maintain urethral function without inducing severe inflammation and scar formation.

Amyloid Aggregation of Streptococcus mutans Cnm Influences Its Collagen-Binding Activity

Streptococcus mutans is a keystone pathogen that promotes caries by acidifying the dental biofilm milieu. The collagen- and laminin-binding glycoprotein Cnm is a virulence factor of S. mutans . Expression of Cnm by S. mutans is hypothesized to contribute to niche expansion, allowing colonization of multiple sites in the body, including collagen-rich surfaces such as dentin and heart valves.