Hyaluronic Acid-Binding, Anionic, Nanoparticles Inhibit ECM Degradation and Restore Compressive Stiffness in Aggrecan-Depleted Articular Cartilage Explants

Joint trauma results in the production of inflammatory cytokines that stimulate the secretion of catabolic enzymes, which degrade articular cartilage. Molecular fragments of the degraded articular cartilage further stimulate inflammatory cytokine production, with this process eventually resulting in post-traumatic osteoarthritis (PTOA). The loss of matrix component aggrecan occurs early in the progression of PTOA and results in the loss of compressive stiffness in articular cartilage. Aggrecan is highly sulfated, associates with hyaluronic acid (HA), and supports the compressive stiffness in cartilage. Presented here, we conjugated the HA-binding peptide GAHWQFNALTVRGSG (GAH) to anionic nanoparticles (hNPs). Nanoparticles conjugated with roughly 19 GAH peptides, termed 19 GAH-hNP, bound to HA in solution and increased the dynamic viscosity by 94.1% compared to an HA solution treated with unconjugated hNPs. Moreover, treating aggrecan-depleted (AD) cartilage explants with 0.10 mg of 19 GAH-hNP restored the cartilage compressive stiffness to healthy levels six days after a single nanoparticle treatment. Treatment of AD cartilage with 0.10 mg of 19 GAH-hNP inhibited the degradation of articular cartilage. Treated AD cartilage had 409% more collagen type II and 598% more GAG content than untreated-AD explants. The 19 GAH-hNP therapeutic slowed ECM degradation in AD cartilage explants, restored the compressive stiffness of damaged cartilage, and showed promise as a localized treatment for PTOA.

Nonmicrobial Activation of TLRs Controls Intestinal Growth, Wound Repair, and Radioprotection

TLRs, key components of the innate immune system, recognize microbial molecules. However, TLRs also recognize some nonmicrobial molecules. In particular, TLR2 and TLR4 recognize hyaluronic acid, a glycosaminoglycan in the extracellular matrix. In neonatal mice endogenous hyaluronic acid binding to TLR4 drives normal intestinal growth. Hyaluronic acid binding to TLR4 in pericryptal macrophages results in cyclooxygenase2- dependent PGE2 production, which transactivates EGFR in LGR5+ crypt epithelial stem cells leading to increased proliferation. The expanded population of LGR5+ stem cells leads to crypt fission and lengthening of the intestine and colon. Blocking this pathway at any point (TLR4 activation, PGE2 production, EGFR transactivation) results in diminished intestinal and colonic growth. A similar pathway leads to epithelial proliferation in wound repair. The repair phase of dextran sodium sulfate colitis is marked by increased epithelial proliferation. In this model, TLR2 and TLR4 in pericryptal macrophages are activated by microbial products or by host hyaluronic acid, resulting in production of CXCL12, a chemokine. CXCL12 induces the migration of cyclooxygenase2-expressing mesenchymal stem cells from the lamina propria of the upper colonic crypts to a site adjacent to LGR5+ epithelial stem cells. PGE2 released by these mesenchymal stem cells transactivates EGFR in LGR5+ epithelial stem cells leading to increased proliferation. Several TLR2 and TLR4 agonists, including hyaluronic acid, are radioprotective in the intestine through the inhibition of radiation-induced apoptosis in LGR5+ epithelial stem cells. Administration of exogenous TLR2 or TLR4 agonists activates TLR2/TLR4 on pericryptal macrophages inducing CXCL12 production with migration of cyclooxygenase2-expressing mesenchymal stem cells from the lamina propria of the villi to a site adjacent to LGR5+ epithelial stem cells. PGE2 produced by these mesenchymal stem cells, blocks radiation-induced apoptosis in LGR5+ epithelial stem cells by an EGFR mediated pathway.

Retinal and Lens Degeneration in New Zealand White Rabbits Administered Intravitreal TSG-6 Link Domain-Rabbit FAb Fusion Proteins

Fusion of biologic therapeutics to hyaluronic acid binding proteins, such as the link domain (LD) of Tumor necrosis factor (TNF)-Stimulated Gene-6 (TSG-6), is expected to increase vitreous residence time following intravitreal injection and provide for long-acting delivery. The toxicity of a single intravitreal dose of free TSG-6-LD and fusion proteins of TSG-6-LD and a nonbinding rabbit antibody fragment (RabFab) were assessed in New Zealand White rabbits. Animals administered free TSG-6-LD exhibited extensive lens opacities and variable retinal vascular attenuation, correlated with microscopic findings of lens and retinal degeneration. Similar but less severe findings were present in animals dosed with the RabFab-TSG-6-LD fusion proteins. In-life ocular inflammation was noted in all animals from 7-days postdose and was associated with high anti-RabFab antibody titers in animals administered fusion proteins. Inflammation and retinal degeneration were multifocally associated with evidence of retinal detachment, and hypertrophy and migration of vimentin, glial fibrillary acidic protein, and glutamine synthetase positive Müller cells to the outer nuclear layer. Further assessment of alternative hyaluronic acid binding protein fusions should consider the potential for retinal degeneration and enhanced immune responses early in development.

Validation and evaluation of a common biomarker in human cancers sera protein detected by a monoclonal antibody UNIVMab

Abstract Objective: Management and diagnosis of multiple human cancers remain a challenge and search for a common biomarker are still debatable. We described a method and evaluated the use of monoclonal antibody UNIVMab, to detect the protein (H11) as a common biomarker for all cancer irrespective of grade and origin . H11 protein identified as a unique Hyaluronan binding protein not detected earlier. We applied this test both with ELISA, Western blot, fractionated in anion exchange, cibacron gel exclusion, b-Hyaluronan interaction and HA-Oligo competition from various grades of Human cancers sera and processed for the detection of hyaluronan binding protein H11, reacted with Monoclonal antibody UNIVMab and with b-HA. Results: Studies from ELISA, Western blot and b-Hyaluronan interactions showed a definitive positive over-expression of UNIVMab reacted H11 antigen in all fortyfive cancer sera compared to normal sera and can be used as a common biomarker. We believe, UNIVMab detected H11 antigen, a unique hyaluronan binding protein, is a common biomarker for all cancer sera. Keywords: ELISA, Western blot, Hyaluronic acid binding protein, H11 (sera antigen), UNIVMab. Common Biomarker, Cancers Sera