The Role of Genetic Testing in Avoiding Diagnostic Delays in Inherited Retinal Disease.

PURPOSE: To identify and highlight potential delays in diagnosis and improve the characterization of the providers referring individuals affected with suspected IRDs for specialty care, we performed an analysis of the patients with IRDs seen by an ophthalmic genetics specialty service. In addition, we analyzed the diagnostic yield of genetic testing in patients with IRD in our series and compared this information with other previous studies. METHODS: We analyzed 131 consecutive patients with suspected IRDs referred to an ophthalmic genetics specialty service at a tertiary hospital. Provider referral patterns, delays in diagnosis and the diagnostic yield of genetic testing were evaluated. RESULTS: Mean age in the cohort was 24 years. From the 51 patients that underwent genetic testing, the diagnostic yield was 69%. Of these, genetic testing revealed 51% of patients had an incorrect initial referral clinical diagnosis. The average delay to reach a correct diagnosis was 15 years. Ophthalmologists represented the largest referral base at 80%, followed by neurologists representing 5% of referrals. Pediatric and retinal specialists were the largest referral of ophthalmic subspecialties at 44% and 35%, respectively. CONCLUSION: A significant number of patients experienced a prolonged delay in reaching a correct diagnosis largely due to a delay in initiating the genetic evaluation and testing process. The initial suspected clinical diagnosis was incorrect in a significant number of cases, revealing that affected patients were potentially denied from appropriate recurrence risk counseling, relevant educational resources, specialty referrals in syndromic cases, and clinical trial eligibility in a timely manner.

Canonical Wnt signaling enhances pro-inflammatory response to titanium by macrophages.

Biomaterial characteristics like surface roughness and wettability can determine the phenotype of macrophages following implantation. We have demonstrated that inhibiting Wnt ligand secretion abolishes macrophage polarization in vitro and in vivo; however, the role of canonical Wnt signaling in macrophage activation in response to physical and chemical biomaterial cues is unknown. The aim of this study was to understand whether canonical Wnt signaling affects the response of macrophages to titanium (Ti) surface roughness or wettability in vitro and in vivo. Activating canonical Wnt signaling increased expression of toll-like receptors and interleukin receptors and secreted pro-inflammatory cytokines and reduced anti-inflammatory cytokines on Ti, regardless of surface properties. Inhibiting canonical Wnt signaling reduced pro-inflammatory cytokines on all Ti surfaces and increased anti-inflammatory cytokines on rough or rough-hydrophilic Ti. In vivo, activating canonical Wnt signaling increased total macrophages, pro-inflammatory macrophages, and T cells and decreased anti-inflammatory macrophages on both smooth and rough-hydrophilic implants. Functionally, canonical Wnt activation increases pro-inflammatory macrophage response to cell and cell-extracellular matrix lysates. These results demonstrate that activating canonical Wnt signaling primes macrophages to a pro-inflammatory phenotype that affects their response to Ti implants in vitro and in vivo.

E-cigarette Aerosol Mixtures Inhibit Biomaterial-Induced Osseointegrative Cell Phenotypes.

OBJECTIVES: Smoking is a known contributor to the failure of dental implants. Despite a decline in cigarette use, the popularity of e-cigarettes has exploded. However, little is known about how e-cigarettes affect the biologic response to implants. This study examines the effect of e-cigarette aerosol mixtures (ecig-AM) on macrophage activation and osteoblastogenesis of mesenchymal stem cells (MSCs) in response to titanium (Ti) implant surfaces. METHODS: Ecig-AMs were prepared by bubbling aerosol through PBS. Human-derived MSCs or murine-derived macrophages were plated on smooth, rough-hydrophobic, or rough-hydrophilic Ti surfaces in media supplemented with ecig-AM. In macrophages, expression of inflammatory markers was measured by qPCR and macrophage immunophenotype characterized by flow cytometry after 24 hours of exposure. In MSCs, expression of osteogenic markers and inflammatory cytokines was measured by qPCR and ELISA, while alkaline phosphatase activity (ALP) was determined by colorimetric assay. RESULTS: Ecig-AM polarized primary macrophages into a pro-inflammatory state with higher effect on ecig-AM with flavorants and nicotine. Metabolic activity of MSCs decreased in a concentration dependent fashion and was stronger in ecig-AM containing nicotine. MSCs reduced expression of osteogenic markers in response to ecig-AM, but increased RANKL secretion, particularly at the highest ecig-AM concentrations. The effect of ecig-AM exposure was lessened when macrophages or MSCs were cultured on rough-hydrophilic substrates. SIGNIFICANCE: Ecig-AM activated macrophages into a pro-inflammatory phenotype and impaired MSC-to-osteoblast differentiation in response to Ti implant surfaces. These effects were potentiated by flavorants and nicotine, suggesting that e-cigarette use may compromise the osseointegration of dental implants.

Substrate stiffness induces neutrophil extracellular trap (NET) formation through focal adhesion kinase activation.

Neutrophils predominate the early inflammatory response to tissue injury and implantation of biomaterials. Recent studies have shown that neutrophil activation can be regulated by mechanical cues such as stiffness or surface wettability; however, it is not known how neutrophils sense and respond to physical cues, particularly how they form neutrophil extracellular traps (NET formation). To examine this, we used polydimethylsiloxane (PDMS) substrates of varying physiologically relevant stiffness (0.2-32 kPa) and examined the response of murine neutrophils to untreated surfaces or to surfaces coated with various extracellular matrix proteins recognized by integrin heterodimers (collagen, fibronectin, laminin, vitronectin, synthetic RGD). Neutrophils on higher stiffness PDMS substrates had increased NET formation and higher secretion of pro-inflammatory cytokines and chemokines. Extracellular matrix protein coatings showed that fibronectin induced the most NET formation and this effect was stiffness dependent. Synthetic RGD peptides induced similar levels of NET formation and pro-inflammatory cytokine release than the full-length fibronectin protein. To determine if the observed NET formation in response to substrate stiffness required focal adhesion kinase (FAK) activity, which is down stream of integrin activation, FAK inhibitor PF-573228 was used. Inhibition of FAK using PF-573228 ablated the stiffness-dependent increase in NET formation and pro-inflammatory molecule secretion. These findings demonstrate that neutrophils regulate NET formation in response to physical and mechanical biomaterial cues and this process is regulated through integrin/FAK signaling.

Surface characteristics on commercial dental implants differentially activate macrophages in vitro and in vivo.

OBJECTIVES: Biomaterial implantation provokes an inflammatory response that controls integrative fate. M2 macrophages regulate the response to implants by resolving the inflammatory phase and recruiting progenitor cells to aid healing. We have previously shown that modified titanium (Ti) disks directly induce M2 macrophage polarization. The aim of this study was to examine macrophage response to commercially available Ti or Ti alloy implants with comparable roughness and varying hydrophilicity. MATERIAL AND METHODS: Eleven commercially available Ti (A-F) or Ti alloy (G-K) dental implants were examined in this study. Surface topography, chemistry, and hydrophilicity were characterized for each implant. To compare the immune response in vitro, human monocyte-derived macrophages were seeded on implants and secreted pro- and anti-inflammatory proteins measured. To evaluate the inflammatory response in vivo, mice were subcutaneously instrumented with clinical implants, and implant adherent macrophage populations were characterized by flow cytometry. RESULTS: Macrophages on hydrophobic Implant C produced the highest level of pro-inflammatory proteins in vitro. In contrast, hydrophilic Implant E produced the second-highest pro-inflammatory response. Implants F and K, both hydrophilics, produced the highest anti-inflammatory protein secretions. Likewise, pro-inflammatory CD80hi macrophages predominated in vivo on implants C and E, and M2 CD206 + macrophages predominated on implants F and K. CONCLUSIONS: These findings show that hydrophilicity alone is insufficient to predict the anti-inflammatory effect on macrophage polarization and that other properties-surface composition or topography-determine immune modulation. This in vivo model may be a useful screening method to compare the immunomodulatory response to clinical implants of disparate geometry or size.

Wnt signaling modulates macrophage polarization and is regulated by biomaterial surface properties.

Macrophages are among the first cells to interact with biomaterials and ultimately determine their integrative fate. Biomaterial surface characteristics like roughness and hydrophilicity can activate macrophages to an anti-inflammatory phenotype. Wnt signaling, a key cell proliferation and differentiation pathway, has been associated with dysregulated macrophage activity in disease. However, the role Wnt signaling plays in macrophage activation and response to biomaterials is unknown. The aim of this study was to characterize the regulation of Wnt signaling in macrophages during classical pro- and anti-inflammatory polarization and in their response to smooth, rough, and rough-hydrophilic titanium (Ti) surfaces. Peri-implant Wnt signaling in macrophage-ablated (MaFIA) mice instrumented with intramedullary Ti rods was significantly attenuated compared to untreated controls. Wnt ligand mRNA were upregulated in a surface modification-dependent manner in macrophages isolated from the surface of Ti implanted in C57Bl/6 mice. In vitro, Wnt mRNAs were regulated in primary murine bone-marrow-derived macrophages cultured on Ti in a surface modification-dependent manner. When macrophageal Wnt secretion was inhibited, macrophage sensitivity to both physical and biological stimuli was abrogated. Loss of macrophage-derived Wnts also impaired recruitment of mesenchymal stem cells and T-cells to Ti implants in vivo. Finally, inhibition of integrin signaling decreased surface-dependent upregulation of Wnt genes. These results suggest that Wnt signaling regulates macrophage response to biomaterials and that macrophages are an important source of Wnt ligands during inflammation and healing.

Hydrophilic titanium surfaces reduce neutrophil inflammatory response and NETosis.

Biomaterial implantation triggers an immune response initially predominated by neutrophils, which activate an inflammatory cascade by producing cytokines, enzymes, immune cell recruitment chemokines, and DNA fiber networks called neutrophil extracellular traps (NETs). While the role of neutrophils has been studied extensively in infection, little is known of their role in the response to biomaterials, in this case titanium (Ti) implants. Furthermore, while implant surface modifications have been shown to attenuate pro-inflammatory polarization in other immune cells, their effects on neutrophil behavior is unknown. The aim of this study was to characterize the neutrophil response to Ti surface topography and hydrophilicity and understand how the products of biomaterial-induced neutrophil activation alters macrophage polarization. Murine neutrophils were isolated by density gradient centrifugation and plated on smooth, rough, and rough hydrophilic (rough-hydro) Ti surfaces. Neutrophils on rough-hydro Ti decreased pro-inflammatory cytokine and enzyme production as well as decreased NET formation compared to neutrophils on smooth and rough Ti. Conditioned media (CM) from neutrophils on smooth Ti enhanced pro-inflammatory macrophage polarization compared to CM from neutrophils on rough or rough-hydro Ti; pretreatment of neutrophils with a pharmacological NETosis inhibitor impaired this macrophage stimulation. Finally, co-culture of neutrophils and macrophages on Ti surfaces induced pro-inflammatory macrophage polarization compared to macrophages alone on surfaces, but this effect was ablated when neutrophils were pretreated with the NETosis inhibitor. These findings demonstrate that neutrophils are sensitive to changes in biomaterial surface properties and exhibit differential activation in response to Ti surface cues. Additionally, inhibition of NETosis enhanced anti-inflammatory macrophage polarization, suggesting NETosis as a possible therapeutic target for enhancing implant integration.