Water Versus Electrolyte Rehydration on Vocal Fold Osmotic and Oxidative Stress Gene Expression.

OBJECTIVES: Systemic dehydration may induce osmotic and oxidative stress in the vocal folds, but our knowledge of the biology and mitigation with rehydration is limited. The purpose of this experiment was to evaluate whether systemic dehydration induces vocal fold oxidative and osmotic stress and to compare the impact of rehydration by water intake versus electrolyte intake on osmotic and oxidative stress-related gene expression. METHODS: Four-month-old male Sprague-Dawley rats (N = 32) underwent water restriction. Rehydration was achieved with ad libitum access to water or electrolytes for 24 hours. Rats were divided into four groups: euhydration control, dehydration-only, dehydration followed by either water or electrolyte rehydration (n = 8/group). Gene expression was assessed via RT2 Gene Expression Profiler arrays. RESULTS: With respect to oxidative stress, 10 genes were upregulated and 2 were downregulated after vocal fold dehydration compared with the euhydrated control. Concerning osmotic stress, six genes were upregulated with dehydration only, six genes were upregulated following rehydration with water, whereas a single gene was upregulated with electrolyte rehydration. All genes with significantly different expression between the rehydration groups showed lower expression with electrolytes compared with water. CONCLUSIONS: The results support a potential role of oxidative and osmotic stresses in vocal folds related to systemic dehydration. The differences in stress-related gene expression in vocal fold tissue between rehydration with electrolytes or water, albeit modest, suggest that both rehydration options offer clinical utility to subjects experiencing vocal fold dehydration with preliminary evidence that electrolytes may be more effective than water in resolving osmotic stress. LEVELS OF EVIDENCE: NA (prospective animal study) Laryngoscope, 2024.

Scaffold-Forming Collagen and Motor-Endplate Expressing Muscle Cells for Porcine Laryngoplasty.

OBJECTIVE: Vocal fold paralysis impairs quality of life, and no curative injectable therapy exists. We evaluated injection of a novel in situ polymerizing (scaffold-forming) collagen in the presence and absence of muscle-derived motor-endplate expressing cells (MEEs) to promote medialization and recurrent laryngeal nerve (RLN) regeneration in a porcine model of unilateral vocal fold paralysis. METHODS: Twelve Yucatan minipigs underwent right RLN transection. Autologous muscle progenitor cells were isolated from muscle biopsies, differentiated, and induced to MEEs. Three weeks after RLN injury, animals received injections of collagen, collagen containing MEEs, or saline into the paralyzed right vocal fold. Stimulated laryngeal electromyography and acoustic vocalization were used for function assessments. Larynges were harvested and underwent histologic, gene expression, and further quantitative analyses. RESULTS: Injections were well-tolerated, with the collagen scaffold showing immunotolerance and collagen-encapsulated MEEs remaining viable. Collagen-treated paralyzed vocal folds showed increased laryngeal adductor muscle volumes relative to that of the uninjured side, with those receiving MEEs and collagen showing the highest volumes. Muscles injected with MEEs and collagen demonstrated increased expression of select neurotrophic (BDNF and NTN1), motor-endplate (DOK7, CHRNA1, and MUSK), and myogenic (MYOG and MYOD) related genes relative to saline controls. CONCLUSION: In a porcine model of unilateral vocal fold paralysis, injection of in situ polymerizing collagen in the absence and presence of MEEs enhanced laryngeal adductor muscle volume, modulated expression of neurotrophic and myogenic factors, and avoided adverse material-mediated immune responses. Further study is needed to determine long-term functional outcomes with this novel therapeutic approach. LEVEL OF EVIDENCE: NA Laryngoscope, 2024.

Design of a Fibroblast Activation Protein-Targeted Radiopharmaceutical Therapy with High Tumor-to-Healthy-Tissue Ratios.

Because of upregulated expression on cancer-associated fibroblasts, fibroblast activation protein (FAP) has emerged as an attractive biomarker for the imaging and therapy of solid tumors. Although many FAP ligands have already been developed for radiopharmaceutical therapies (RPTs), most suffer from inadequate tumor uptake, insufficient tumor residence times, or off-target accumulation in healthy tissues, suggesting a need for further improvements. Methods: A new FAP-targeted RPT with a novel ligand (FAP8-PEG3-IP-DOTA) was designed by combining the desirable features of several previous ligand-targeted RPTs. Uptake and retention of [111In]In or [177Lu]Lu-FAP8-PEG3-IP-DOTA were assessed in KB, HT29, MDA-MB-231, and 4T1 murine tumor models by radioimaging or ex vivo biodistribution analyses. Radiotherapeutic potencies and gross toxicities were also investigated by monitoring tumor growth, body weight, and tissue damage in tumor-bearing mice. Results: FAP8-PEG3-IP-DOTA exhibited high affinity (half-maximal inhibitory concentration, 1.6 nM) and good selectivity for FAP relative to its closest homologs, prolyl oligopeptidase (half-maximal inhibitory concentration, ∼14.0 nM) and dipeptidyl peptidase-IV (half-maximal inhibitory concentration, ∼860 nM). SPECT/CT scans exhibited high retention in 2 different solid tumor models and minimal uptake in healthy tissues. Quantitative biodistribution analyses revealed tumor-to-healthy-tissue ratios of more than 5 times for all major organs, and live animal studies demonstrated 65%-93% suppression of tumor growth in all 4 models tested, with minimal or no evidence of systemic toxicity. Conclusion: We conclude that [177Lu]Lu-FAP8-PEG3-IP-DOTA constitutes a promising and safe RPT candidate for FAPα-targeted radionuclide therapy of solid tumors.

Yeast and filamentous Candida auris stimulate distinct immune responses in the skin.

Candida auris, an emerging multidrug-resistant fungal pathogen, predominately colonizes the human skin long term leading to subsequent life-threatening invasive infections. Fungal morphology is believed to play a critical role in modulating mucocutaneous antifungal immunity. In this study, we used an intradermal mouse model of C. auris infection to examine fungal colonization and the associated innate and adaptive immune response to yeast and filamentous C. auris strains. Our results indicate that mice infected with a filamentous C. auris had significantly decreased fungal load compared to mice infected with the yeast form. Mice infected with yeast and filamentous forms of C. auris stimulated distinct innate immune responses. Phagocytic cells (CD11b+Ly6G+ neutrophils, CD11b+Ly6Chi inflammatory monocytes, and CD11b+MHCII+CD64+ macrophages) were differentially recruited to mouse skin tissue infected with yeast and filamentous C. auris. The percentage and absolute number of interleukin 17 (IL-17) producing innate lymphoid cells, TCRγδ+, and CD4+ T cells in the skin tissue of mice infected with filamentous C. auris were significantly increased compared to the wild-type of yeast strain. Furthermore, complementation of filamentous mutant strain of C. auris (Δelm1 + ELM1) strain exhibited wild-type yeast morphology in vivo and induced comparable level of skin immune responses similar to mice infected with yeast strain. Collectively, our findings indicate that yeast and filamentous C. auris induce distinct local immune responses in the skin. The decreased fungal load observed in mouse skin infected with filamentous C. auris is associated with a potent IL-17 immune response induced by this morphotype.IMPORTANCECandida auris is a globally emerging fungal pathogen that transmits among individuals in hospitals and nursing home residents. Unlike other Candida species, C. auris predominantly colonizes and persists in skin tissue resulting in outbreaks of systemic infections. Understanding the factors that regulate C. auris skin colonization and host immune response is critical to develop novel preventive and therapeutic approaches against this emerging pathogen. We identified that yeast and filamentous forms of C. auris induce distinct skin immune responses in the skin. These findings may help explain the differential colonization and persistence of C. auris morphotypes in skin tissue. Understanding the skin immune responses induced by yeast and filamentous C. auris is important to develop novel vaccine strategies to combat this emerging fungal pathogen.

Generating prophylactic immunity against arboviruses in vertebrates and invertebrates.

The World Health Organization recently declared a global initiative to control arboviral diseases. These are mainly caused by pathogenic flaviviruses (such as dengue, yellow fever and Zika viruses) and alphaviruses (such as chikungunya and Venezuelan equine encephalitis viruses). Vaccines represent key interventions for these viruses, with licensed human and/or veterinary vaccines being available for several members of both genera. However, a hurdle for the licensing of new vaccines is the epidemic nature of many arboviruses, which presents logistical challenges for phase III efficacy trials. Furthermore, our ability to predict or measure the post-vaccination immune responses that are sufficient for subclinical outcomes post-infection is limited. Given that arboviruses are also subject to control by the immune system of their insect vectors, several approaches are now emerging that aim to augment antiviral immunity in mosquitoes, including Wolbachia infection, transgenic mosquitoes, insect-specific viruses and paratransgenesis. In this Review, we discuss recent advances, current challenges and future prospects in exploiting both vertebrate and invertebrate immune systems for the control of flaviviral and alphaviral diseases.

Listeria adhesion protein orchestrates caveolae-mediated apical junctional remodeling of epithelial barrier for Listeria monocytogenes translocation.

The cellular junctional architecture remodeling by Listeria adhesion protein-heat shock protein 60 (LAP-Hsp60) interaction for Listeria monocytogenes (Lm) passage through the epithelial barrier is incompletely understood. Here, using the gerbil model, permissive to internalin (Inl) A/B-mediated pathways like in humans, we demonstrate that Lm crosses the intestinal villi at 48 h post-infection. In contrast, the single isogenic (lap- or ΔinlA) or double (lap-ΔinlA) mutant strains show significant defects. LAP promotes Lm translocation via endocytosis of cell-cell junctional complex in enterocytes that do not display luminal E-cadherin. In comparison, InlA facilitates Lm translocation at cells displaying apical E-cadherin during cell extrusion and mucus expulsion from goblet cells. LAP hijacks caveolar endocytosis to traffic integral junctional proteins to the early and recycling endosomes. Pharmacological inhibition in a cell line and genetic knockout of caveolin-1 in mice prevents LAP-induced intestinal permeability, junctional endocytosis, and Lm translocation. Furthermore, LAP-Hsp60-dependent tight junction remodeling is also necessary for InlA access to E-cadherin for Lm intestinal barrier crossing in InlA-permissive hosts. IMPORTANCE: Listeria monocytogenes (Lm) is a foodborne pathogen with high mortality (20%-30%) and hospitalization rates (94%), particularly affecting vulnerable groups such as pregnant women, fetuses, newborns, seniors, and immunocompromised individuals. Invasive listeriosis involves Lm's internalin (InlA) protein binding to E-cadherin to breach the intestinal barrier. However, non-functional InlA variants have been identified in Lm isolates, suggesting InlA-independent pathways for translocation. Our study reveals that Listeria adhesion protein (LAP) and InlA cooperatively assist Lm entry into the gut lamina propria in a gerbil model, mimicking human listeriosis in early infection stages. LAP triggers caveolin-1-mediated endocytosis of critical junctional proteins, transporting them to early and recycling endosomes, facilitating Lm passage through enterocytes. Furthermore, LAP-Hsp60-mediated junctional protein endocytosis precedes InlA's interaction with basolateral E-cadherin, emphasizing LAP and InlA's cooperation in enhancing Lm intestinal translocation. This understanding is vital in combating the severe consequences of Lm infection, including sepsis, meningitis, encephalitis, and brain abscess.

α-Gal Nanoparticles in CNS Trauma: II. Immunomodulation Following Spinal Cord Injury (SCI) Improves Functional Outcomes.

BACKGROUND: Previous investigations have shown that local application of nanoparticles presenting the carbohydrate moiety galactose-α-1,3-galactose (α-gal epitopes) enhance wound healing by activating the complement system and recruiting pro-healing macrophages to the injury site. Our companion in vitro paper suggest α-gal epitopes can similarly recruit and polarize human microglia toward a pro-healing phenotype. In this continuation study, we investigate the in vivo implications of α-gal nanoparticle administration directly to the injured spinal cord. METHODS: α-Gal knock-out (KO) mice subjected to spinal cord crush were injected either with saline (control) or with α-gal nanoparticles immediately following injury. Animals were assessed longitudinally with neurobehavioral and histological endpoints. RESULTS: Mice injected with α-gal nanoparticles showed increased recruitment of anti-inflammatory macrophages to the injection site in conjunction with increased production of anti-inflammatory markers and a reduction in apoptosis. Further, the treated group showed increased axonal infiltration into the lesion, a reduction in reactive astrocyte populations and increased angiogenesis. These results translated into improved sensorimotor metrics versus the control group. CONCLUSIONS: Application of α-gal nanoparticles after spinal cord injury (SCI) induces a pro-healing inflammatory response resulting in neuroprotection, improved axonal ingrowth into the lesion and enhanced sensorimotor recovery. The data shows α-gal nanoparticles may be a promising avenue for further study in CNS trauma.

In Vivo Visualization and Quantification of Rat Laryngeal Blood Supply After Hydration Challenge.

OBJECTIVES: Systemic dehydration decreases total body blood volume; however, hemodynamic alterations at the level of local organs, such as the larynx, remain unclear. Here we sought to quantify superior thyroid artery (STA) blood flow after dehydration and rehydration using in vivo magnetic resonance angiography (MRA) and ultrasound imaging in a rat model. METHODS: Male Sprague-Dawley rats (N = 17) were included in this prospective, repeated measures design. Rats first underwent MRA to determine baseline STA cross-sectional area, followed by high-frequency in vivo ultrasound imaging to measure STA blood velocity at baseline. Next, rats were systemically dehydrated (water withholding), followed by rehydration (water ad-lib). Ultrasound imaging was repeated immediately after dehydration and following rehydration. The STA blood velocity and STA cross-sectional area were used to compute STA blood flow. Three rats served as temporal controls for ultrasound imaging. To determine if the challenges to hydration status affected the STA cross-sectional area, four rats underwent only MRA at baseline, dehydration, and rehydration. RESULTS: Systemic dehydration resulted in 10.5% average body weight loss. Rehydration resulted in average body weight gain of 10.9%. Statistically significant reductions were observed in STA mean blood flow rate after dehydration. Rehydration reversed these changes to pre-dehydration levels. No significant differences were observed in STA cross-sectional area with dehydration or rehydration. CONCLUSION: Systemic dehydration decreased blood flow in the superior thyroid artery. Rehydration restored blood flow in the STA. Change in hydration status did not alter the STA cross-sectional area. These preliminary findings demonstrate the feasibility of using ultrasound and MRA to quantify hemodynamic changes and visualize laryngeal blood vessels. LEVEL OF EVIDENCE: NA Laryngoscope, 134:779-785, 2024.

Aortic dissection detection and thrombus structure quantification using volumetric ultrasound, histology, and scanning electron microscopy.

Aortic dissection occurs when a weakened portion of the intima tears, and a separation of layers propagates along the aortic wall to form a false lumen filled with active blood flow or intramural thrombus. The unpredictable nature of aortic dissection formation and need for immediate intervention leaves limited serial human image data to study the formation and morphological changes that follow dissection. We used volumetric ultrasound examination, histology, and scanning electron microscopy (SEM) to examine intramural thrombi at well-defined timepoints after dissection occurs in apolipoprotein E-deficient mice infused with angiotensin II (n = 71). Stratification of red blood cell (RBC) morphologies (biconcave, intermediate biconcave, intermediate polyhedrocyte, and polyhedrocyte) in the thrombi with scanning electron microscopy (n = 5) was used to determine degree of thrombus deposition/contraction. Very few biconcave RBCs (1.2 ± 0.6%) were in the thrombi, and greater amounts of intermediate biconcave RBCs (25.8 ± 6.7%) were located in the descending thoracic portion of the dissection while more polyhedrocytes (14.6 ± 5.1%) and fibrin (42.3 ± 4.5%; P < .05) were found in the distal suprarenal aorta. Thrombus deposition likely plays some role in patient outcomes, and this multimodality technique can help investigate thrombus deposition and characteristics in experimental animal models and human tissue samples.

Sea squirt-inspired bio-derived tissue sealants.

Sea squirts' or tunicates' bodies are composed of cellulose nanofibers and gallol- functionalized proteins. These sea creatures are known to heal their injuries under seawater by forming crosslinks between gallols and functional groups from other proteins in their bodies. Inspired by their wound healing mechanism, herein, we have developed a tissue sealant using zein (a plant-based protein) and tannic acid (gallol-containing polyphenol). Except for fibrin- based sealants, most commercial surgical adhesives, and sealants available today are derived from petroleum products that compromise their biodegradability. They often have complicated and multi-step synthesis processes that ultimately affect their affordability. To overcome this challenge, we ensured that these sea squirt-inspired tissue sealants are bio-based, easily synthesized, and low-cost. The sealants were studied on their own and with a food-grade enzyme transglutaminase. The adhesion performances of the sealants were found to be higher than physiological pressures in seven out of nine different tissue substrates studied here. Their performance was also better than or on par with the FDA-approved fibrin sealant Tisseel. Ex vivo models demonstrate instant sealing of leaking wounds in less than a minute. The sealants were not only cytocompatible but also showed complete wound healing on par with sutures and Tisseel when applied in vivo on skin incisions in rats. Overall, these sea squirt-inspired bio-based sealants show great potential to replace currently available wound closure methods.

Maize near-isogenic lines with enhanced flavonoids alleviated dextran sodium sulfate-induced murine colitis via modulation of the gut microbiota.

The rising incidence of inflammatory bowel disease (IBD) has necessitated the search for safe and effective novel therapeutic strategies. Dietary flavonoids exhibited antioxidant, antiproliferative, and anticarcinogenic activities in several model systems with proven abilities to reduce inflammation and oxidative stress, thus they could be promising therapeutic agents for IBD prevention/treatment. However, understanding the role of a specific class of compounds in foods that promote health is difficult because of the chemically complex food matrices. This study aimed to utilize four maize near-isogenic lines to determine the anti-colitis effects of specific classes of flavonoids, anthocyanins and/or phlobaphenes, in a whole-food matrix. Results showed that the intake of anthocyanin and phlobaphene-enriched maize diets effectively alleviated dextran sodium sulfate (DSS)-induced colitis in mice via reducing the intestinal permeability and restoring the barrier function. Anthocyanin diets were more effective in maintaining the crypt structure and muc2 protein levels and reducing inflammation. Bacterial communities of mice consuming diets enriched with anthocyanins and phlobaphenes were more similar to the healthy control compared to the DSS control group, suggesting the role of flavonoids in modulating the gut microbiota to retrieve intestinal homeostasis. Microbiota depletion rendered these compounds ineffective against colitis. Lower serum concentrations of several phenolic acids were detected in the microbiota-depleted mice, indicating that gut microbiota plays a role in flavonoid metabolism and bioavailability.

Thrombin cleavage of the hepatitis E virus polyprotein at multiple conserved locations is required for genome replication.

The genomes of positive-sense RNA viruses encode polyproteins that are essential for mediating viral replication. These viral polyproteins must undergo proteolysis (also termed polyprotein processing) to generate functional protein units. This proteolysis can be performed by virally-encoded proteases as well as host cellular proteases, and is generally believed to be a key step in regulating viral replication. Hepatitis E virus (HEV) is a leading cause of acute viral hepatitis. The positive-sense RNA genome is translated to generate a polyprotein, termed pORF1, which is necessary and sufficient for viral genome replication. However, the mechanism of polyprotein processing in HEV remains to be determined. In this study, we aimed to understand processing of this polyprotein and its role in viral replication using a combination of in vitro translation experiments and HEV sub-genomic replicons. Our data suggest no evidence for a virally-encoded protease or auto-proteolytic activity, as in vitro translation predominantly generates unprocessed viral polyprotein precursors. However, seven cleavage sites within the polyprotein (suggested by bioinformatic analysis) are susceptible to the host cellular protease, thrombin. Using two sub-genomic replicon systems, we demonstrate that mutagenesis of these sites prevents replication, as does pharmacological inhibition of serine proteases including thrombin. Overall, our data supports a model where HEV uses host proteases to support replication and could have evolved to be independent of a virally-encoded protease for polyprotein processing.

Impact of Rehydration Following Systemic Dehydration on Vocal Fold Gene Expression.

OBJECTIVE: Biological data on the beneficial effects of vocal fold rehydration are lacking. This study aimed to examine the effects of acute systemic dehydration on vocal fold gene expression and determine whether rehydration would reverse these changes. METHODS: Male New Zealand White rabbits (N = 24, n = 8/group) provided the animal model. Systemic dehydration was induced by 5 days of water volume restriction. Rehydration was provided by ad-lib water for 3 days following dehydration. Euhydrated rabbits were used as the control group. Vocal fold tissue was dissected. Seventeen genes were selected based on physiological function and role in supporting vocal fold structure, oxidative stress, hemodynamics, and extracellular matrix turnover. Relative gene expression was assessed by RT-qPCR. RESULTS: Rehydration following systemic dehydration can modulate gene expression, with expression patterns suggesting that rehydration reverses dehydration-induced changes in over half of the tested genes. CLIC5 (chloride intracellular channel 5) and EFEMP1 (EGF containing fibulin extracellular matrix protein 1) genes were significantly upregulated in the dehydration group compared with the euhydrated control. A1BG (alpha-1B-glycoprotein) and IL1RAP (interleukin 1 receptor accessory protein) were downregulated by rehydration compared with the dehydration group. CONCLUSION: This study provides molecular evidence for a transcriptional response to rehydration following acute systemic dehydration in the vocal folds. These data are the first to study gene expression following realistic dehydration and rehydration paradigms and provide biological data to support clinical recommendations to increase water intake after acute dehydration. LEVEL OF EVIDENCE: NA Laryngoscope, 133:3499-3505, 2023.

Cell-surface anchoring of Listeria adhesion protein on L. monocytogenes is fastened by internalin B for pathogenesis.

Listeria adhesion protein (LAP) is a secreted acetaldehyde alcohol dehydrogenase (AdhE) that anchors to an unknown molecule on the Listeria monocytogenes (Lm) surface, which is critical for its intestinal epithelium crossing. In the present work, immunoprecipitation and mass spectrometry identify internalin B (InlB) as the primary ligand of LAP (KD ∼ 42 nM). InlB-deleted and naturally InlB-deficient Lm strains show reduced LAP-InlB interaction and LAP-mediated pathology in the murine intestine and brain invasion. InlB-overexpressing non-pathogenic Listeria innocua also displays LAP-InlB interplay. In silico predictions reveal that a pocket region in the C-terminal domain of tetrameric LAP is the binding site for InlB. LAP variants containing mutations in negatively charged (E523S, E621S) amino acids in the C terminus confirm altered binding conformations and weaker affinity for InlB. InlB transforms the housekeeping enzyme, AdhE (LAP), into a moonlighting pathogenic factor by fastening on the cell surface.

Biocompatibility and Safety Assessment of Combined Topical Ozone and Antibiotics for Treatment of Infected Wounds.

Wound infections with antibiotic-resistant bacteria, particularly the Gram-negative strains, pose a substantial health risk for patients with limited treatment options. Recently topical administration of gaseous ozone and its combination with antibiotics through portable systems has been demonstrated to be a promising approach to eradicate commonly found Gram-negative strains of bacteria in wound infections. However, despite the significant impact of ozone in treating the growing number of antibiotic-resistant infections, uncontrolled and high concentrations of ozone can cause damage to the surrounding tissue. Hence, before such treatments could advance into clinical usage, it is paramount to identify appropriate levels of topical ozone that are effective in treating bacterial infections and safe for use in topical administration. To address this concern, we have conducted a series of in vivo studies to evaluate the efficacy and safety of a portable and wearable adjunct ozone and antibiotic wound therapy system. The concurrent ozone and antibiotics are applied through a wound interfaced gas permeable dressing coated with water-soluble nanofibers containing vancomycin and linezolid (traditionally used to treat Gram-positive infections) and connected to a portable ozone delivery system. The bactericidal properties of the combination therapy were evaluated on an ex vivo wound model infected with Pseudomonas aeruginosa, a common Gram-negative strain of bacteria found in many skin infections with high resistance to a wide range of currently available antibiotics. The results indicated that the optimized combination delivery of ozone (4 mg h-1) and topical antibiotic (200 µg cm-2) provided complete bacteria eradication after 6 h of treatment while having minimum cytotoxicity to human fibroblast cells. Furthermore, in vivo local and systemic toxicity studies (e.g., skin monitoring, skin histopathology, and blood analysis) on pig models showed no signs of adverse effects of ozone and antibiotic combination therapy even after 5 days of continuous administration. The confirmed efficacy and biosafety profile of the adjunct ozone and antibiotic therapy places it as a strong candidate for treating wound infection with antimicrobial-resistant bacteria and further pursuing human clinical trials.

Fibroblast Activation Protein-Targeted Radioligand Therapy for Treatment of Solid Tumors.

Fibroblast activation protein (FAP) has received increasing attention as an oncologic target because of its prominent expression in solid tumors but virtual absence from healthy tissues. Most radioligand therapies (RLTs) targeting FAP, however, suffer from inadequate tumor retention or clearance from healthy tissues. Herein we report a FAP-targeted RLT comprising an FAP6 ligand conjugated to DOTA and an albumin binder (4-p-iodophenylbutyric acid, or IP) for enhanced pharmacokinetics. We evaluated the performance of the resulting FAP6-IP-DOTA conjugate in 4 tumor models, 3 of which express FAP only on cancer-associated fibroblasts, that is, analogously to human tumors. Methods: Single-cell RNA-sequencing data were analyzed from 34 human breast, ovarian, colorectal, and lung cancers to quantify FAP-overexpressing cells. FAP6-DOTA conjugates were synthesized with or without an albumin binder (IP) and investigated for binding to human FAP-expressing cells. Accumulation of 111In- or 177Lu-labeled conjugates in KB, HT29, U87MG, and 4T1 murine tumors was also assessed by radioimaging or biodistribution analyses. Radiotherapeutic potency was quantitated by measuring tumor volumes versus time. Results: Approximately 5% of all cells in human tumors overexpressed FAP (cancer-associated fibroblasts comprised ∼77% of this FAP-positive subpopulation, whereas ∼2% were cancer cells). FAP6 conjugates bound to FAP-expressing cells with high affinity (dissociation constant, ∼1 nM). 177Lu-FAP6-IP-DOTA achieved an 88-fold higher tumor dose than 177Lu-FAP6-DOTA and improved all tumor-to-healthy-organ ratios. Single doses of 177Lu-FAP6-IP-DOTA suppressed tumor growth by about 45% in all tested tumor models without causing reproducible toxicities. Conclusion: We conclude that 177Lu-FAP6-IP-DOTA constitutes a promising candidate for FAP-targeted RLT of solid tumors.

Engineered collagen polymeric materials create noninflammatory regenerative microenvironments that avoid classical foreign body responses.

The efficacy and longevity of medical implants and devices is largely determined by the host immune response, which extends along a continuum from pro-inflammatory/pro-fibrotic to anti-inflammatory/pro-regenerative. Using a rat subcutaneous implantation model, along with histological and transcriptomics analyses, we characterized the tissue response to a collagen polymeric scaffold fabricated from polymerizable type I oligomeric collagen (Oligomer) in comparison to commercial synthetic and collagen-based products. In contrast to commercial biomaterials, no evidence of an immune-mediated foreign body reaction, fibrosis, or bioresorption was observed with Oligomer scaffolds for beyond 60 days. Oligomer scaffolds were noninflammatory, eliciting minimal innate inflammation and immune cell accumulation similar to sham surgical controls. Genes associated with Th2 and regulatory T cells were instead upregulated, implying a novel pathway to immune tolerance and regenerative remodeling for biomaterials.

Hydration State and Hyaluronidase Treatment Significantly Affect Porcine Vocal Fold Biomechanics.

OBJECTIVES: The understanding of vocal fold hydration state, including dehydrated, euhydrated, rehydrated tissue, and how hydration affects vocal fold biomechanical properties is still evolving. Although clinical observations support the benefits of increasing vocal fold hydration after dehydrating events, more mechanistic information on the effects of vocal fold dehydration and the beneficial effects of rehydration are needed. Alterations to hyaluronic acid (HA), an important component of the vocal fold extracellular matrix, are likely to influence the biomechanical properties of vocal folds. In this study, we investigated the influence of hydration state and HA on vocal fold tissue stiffness via biomechanical testing. STUDY DESIGN: Prospective, ex vivo study design. METHODS: Fresh porcine vocal folds (N = 18) were examined following sequential immersion in hypertonic (dehydration) and isotonic solutions (rehydration). In a separate experiment, vocal folds were incubated in hyaluronidase (Hyal) to remove HA. Control tissues were not exposed to any challenges. A custom micromechanical system with a microforce sensing probe was used to measure the force-displacement response. Optical strain was calculated, and ultrasound imaging was used to measure tissue cross-sectional area to obtain stress-strain curves. RESULTS: Significant increases (P ≤ 0.05) were found in tangent moduli between dehydrated and rehydrated vocal folds at strains of ε = 0.15. The tangent moduli of Hyal-digested tissues significantly increased at both ε = 0.15 and 0.3 (P ≤ 0.05). CONCLUSION: Vocal fold dehydration increased tissue stiffness and rehydration reduced the stiffness. Loss of HA increased vocal fold stiffness, suggesting a potential mechanical role for HA in euhydrated vocal folds.

Comparative proteomic changes in rabbit vocal folds undergoing systemic dehydration and systemic rehydration.

BACKGROUND: A considerable body of clinical evidence suggests that systemic dehydration can negatively affect voice production, leading to the common recommendation to rehydrate. Evidence for the corrective benefits of rehydration, however, is limited with mixed conclusions, and biological data on the underlying tissue changes with rehydration is lacking. In this study, we used a rabbit model (n = 24) of acute (5 days) water restriction-induced systemic dehydration with subsequent rehydration (3 days) to explore the protein-level changes underlying the molecular transition from euhydration to dehydration and following rehydration using LC-MS/MS protein quantification in the vocal folds. We show that 5-day water restriction led to an average 4.3% decrease in body weight with relative increases in anion gap, Cl-, creatinine, Na+, and relative decreases in BUN, iCa2+, K+, and tCO2 compared to control (euhydrated) animals. A total of 309 differentially regulated (p < 0.05) proteins were identified between the Control and Dehydration groups. We observed a noteworthy similarity between the Dehydration and Rehydration groups, both well differentiated from the Control group, highlighting the distinct timelines of resolution of the clinical symptoms of systemic dehydration and the underlying molecular changes. SIGNIFICANCE: Voice disorders are a ubiquitous problem with considerable economic and psychological impact. Maintenance of proper hydration is commonly prescribed as a general vocal hygiene practice. There is evidence that dehydration negatively impacts phonation, but our understanding of the state of vocal folds in the context of systemic dehydration are limited, particular from a molecular perspective. Further, ours is a novel molecular study of the short-term impact of rehydration on the tissue. Given the relatively minimal difference in vocal fold proteomic profiles between the Dehydration and Rehydration groups, our data demonstrate a complex physiological response to acute systemic dehydration, and highlight the importance of considering persistent underlying molecular pathology despite the rapid resolution of clinical measures. This study sets a foundation for future research to confirm the nature of potential beneficial outcomes of clinical recommendations related to hydration.

Canary Seed (Phalaris canariensis L.) Peptides Prevent Obesity and Glucose Intolerance in Mice Fed a Western Diet.

Previous research showed that canary seed (Phalaris canariensis L.) peptides (CSP) possess robust in vitro antiobesity properties via inhibition of pancreatic lipase (PL). Nevertheless, no studies have yet explored their antiobesity properties in vivo. Consequently, we investigated the effects of CSP in C57BL/6J mice under a Western diet (WD). Mice were assigned into groups and fed a normal diet (ND) or a WD accompanied by an oral dose of CSP (250 or 500 mg/kg/day), orlistat (40 mg/kg/day), or distilled water. The results showed that consuming CSP can provide metabolic benefits, including preventing weight gain by up to 20%, increasing glucose tolerance, and reducing insulin, leptin, and LDL/VLDL levels in plasma. Conversely, total ghrelin was unaffected by CSP-500, but decreased by CSP-250, and amplified by orlistat. Surprisingly, CSP-250 was more effective in preventing weight gain and promoting satiety than CSP-500. Parallel to this, protein absorption in CSP-500 was decreased, supported by a rise in fecal crude protein (+3.5%). Similarly, fecal fat was increased by orlistat (38%) and was unaffected by CSP-250 (3.0%) and CSP (3.0%), comparatively to WD (2.5%). Despite this, both CSP treatments were equally effective in decreasing hepatic steatosis and avoiding hyperlipidemia. Furthermore, the enzymatic analysis showed that CSP-PL complexes dissociated faster (15 min) than orlistat-PL complexes (41 min). Lastly, CSP did not affect expression of hepatic lipid oxidation genes ACO and PPAR-α, but reduced the expression of the hydrolase gene LPL, and lipogenesis related genes FAS and ACC. Taken together, these results suggest that CSP antiobesity mechanism relies on lipid metabolism retardation to increase fat transit time and subsequently suppress hunger.