Endothelial cell Piezo1 mediates pressure-induced lung vascular hyperpermeability via disruption of adherens junctions.

Increased pulmonary microvessel pressure experienced in left heart failure, head trauma, or high altitude can lead to endothelial barrier disruption referred to as capillary "stress failure" that causes leakage of protein-rich plasma and pulmonary edema. However, little is known about vascular endothelial sensing and transduction of mechanical stimuli inducing endothelial barrier disruption. Piezo1, a mechanosensing ion channel expressed in endothelial cells (ECs), is activated by elevated pressure and other mechanical stimuli. Here, we demonstrate the involvement of Piezo1 in sensing increased lung microvessel pressure and mediating endothelial barrier disruption. Studies were made in mice in which Piezo1 was deleted conditionally in ECs (Piezo1iΔEC ), and lung microvessel pressure was increased either by raising left atrial pressure or by aortic constriction. We observed that lung endothelial barrier leakiness and edema induced by raising pulmonary microvessel pressure were abrogated in Piezo1iΔEC mice. Piezo1 signaled lung vascular hyperpermeability by promoting the internalization and degradation of the endothelial adherens junction (AJ) protein VE-cadherin. Breakdown of AJs was the result of activation of the calcium-dependent protease calpain and degradation of the AJ proteins VE-cadherin, ß-catenin, and p120-catenin. Deletion of Piezo1 in ECs or inhibition of calpain similarly prevented reduction in the AJ proteins. Thus, Piezo1 activation in ECs induced by elevated lung microvessel pressure mediates capillary stress failure and edema formation secondary to calpain-induced disruption of VE-cadherin adhesion. Inhibiting Piezo1 signaling may be a useful strategy to limit lung capillary stress failure injury in response to elevated vascular pressures.

Thermal injury model in the rabbit ear with quantifiable burn progression and hypertrophic scar.

Hypertrophic scar is a major clinical outcome of deep-partial thickness to full thickness thermal burn injury. Appropriate animal models are a limitation to burn research due to the lack of, or access to, animal models which address the endpoint of hypertrophic scar. Lower species, such as rodents, heal mainly by contracture, which limits the duration of study. Higher species, such as pigs, heal more similarly to humans, but are associated with high cost, long duration for scar development, challenges in quantifying scar hypertrophy, and poor manageability. Here, we present a quantifiable deep-partial thickness burn model in the rabbit ear. Burns were created using a dry-heated brass rod for 10 and 20 seconds at 90 °C. At the time of eschar excision on day 3, excisional wounds were made on the contralateral ear for comparison. Burn wound progression, in which the wound size expands over time is a major distinction between excisional and thermal injuries, was quantified at 1 hour and 3 days after the injuries using calibrated photographs and histology and the size of the wounds was found to be unchanged from the initial wound size at 1 hour, but 10% in the 20 seconds burn wounds at 3 days. A quantifiable hypertrophic scar, measured by histology as the scar elevation index, was present in both 20 seconds burn wounds and excisional wounds at day 35. ImageJ measurements revealed that the 20 seconds burn wound scars were 22% larger than the excisional wound scars and the 20 seconds burn scar area measurements from histology were 26% greater than in the excisional wound scar. The ability to measure both burn progression and scar hypertrophy over a 35-day time frame suits this model to screening early intervention burn wound therapeutics or scar treatments in a burn-specific scar model.

Ultrasound Localization of Resuscitative Endovascular Balloon Occlusion of the Aorta in a Human Cadaver Model.

OBJECTIVE: Resuscitative endovascular balloon occlusion of the aorta (REBOA) is a method of gaining proximal control of noncompressible torso hemorrhage (NCTH). Catheter placement is traditionally confirmed with fluoroscopy, but few studies have evaluated whether ultrasound (US) can be used. METHODS: Using a pressurized human cadaver model, a certified REBOA placer was shown one of four randomized cards that instructed them to place the REBOA either correctly or incorrectly in Zone 1 (the distal thoracic aorta extending from the celiac artery to the left subclavian artery) or Zone 3 (in the distal abdominal aorta, from the aortic bifurcation to the lowest renal artery). Once the REBOA was placed, 10 US-trained locators were asked to confirm balloon placement via US. The participants were given 3 minutes to determine whether the catheter had been correctly placed, repeating this 20 times on two cadavers. RESULTS: Overall, US exhibited an average sensitivity of 83%, specificity of 76%, and accuracy of 80%. For Zone 1, US showed a sensitivity of 78% and specificity of 83%, and for Zone 3, a sensitivity of 88% and specificity of 76%. In addition, US exhibited a likelihood positive ratio (LR+) of 3.73 and a likelihood negative ratio (LR-) of 0.22 for either position, with similar numbers for Zone 1 (+4.57, -0.26) and Zone 3 (+3.16, -0.16). CONCLUSION: Ultrasound could prove to be a useful tool for confirming placement of a REBOA catheter, especially in austere environments.

Speed, Skill Retention, and End User Perceptions of iTClamp Application by Navy Corpsmen on a Manikin Model of Femoral Hemorrhage.

INTRODUCTION: Tactical Combat Casualty Care guidelines recommend packing junctional wounds with gauze, applying direct pressure for 3 minutes, and then securing with an external pressure dressing. This method is time-consuming, which can be problematic in a combat environment. Alternatively, the iTClamp has documented efficacy and rapid application. However, no studies have evaluated device application by military prehospital medical providers, such as Navy corpsmen, or their user experience with the device. MATERIALS AND METHODS: Research data derived from a protocol were approved by the Naval Medical Center Portsmouth's Institutional Review Board in compliance with all applicable federal regulations governing the protection of human subjects. Navy corpsmen with the current Tactical Combat Casualty Care certification applied the iTClamp or standard pressure dressing on a manikin model of femoral hemorrhage in a crossover study design. Each participant used both devices in a randomized fashion. Time to application was recorded, and participants completed Likert scale surveys to evaluate both devices for preference, ease of use, and physical assessment. A repeat assessment was performed 1 month later to assess skill atrophy. Repeated-measures ANOVA was used to compare application time. Likert scale survey data were analyzed using Mann-Whitney and Wilcoxon tests to compare survey data within and between time points, respectively. RESULTS: The application of the iTClamp was more than twice as fast as the application of pressure dressings at both the initial and follow-up evaluations. There was no statistically significant difference in application times between the first evaluation and the 30-day assessment of either device, indicating no atrophy in skill. While 65% and 52% of the participants expressed preference in for the iTClamp in their surveys during the initial and follow-up respective visits, the difference in preference was not statistically significant for either the initial or the follow-up survey. Open-ended survey responses yielded both perceived advantages and disadvantages for each treatment option. CONCLUSIONS: In austere or hostile environments, speed of treatment and extrication can have significant implications for the safety of both the patient and the medical providers. Hemorrhage control interventions must be both effective and easy to use for a prehospital provider to ensure its efficacy in a live battlefield situation. The iTClamp is small, simple, and fast to use, but its wide adoption in the field may be based on limitations perceived by participants, including narrow indications for use. However, based on our findings, it is reasonable to field the iTClamp depending on provider preference.

iTClamp-Mediated Wound Closure Speeds Control of Arterial Hemorrhage With or Without Additional Hemostatic Agents.

BACKGROUND: Exsanguination is the leading cause of preventable posttraumatic death, especially in the prehospital arena. Traditional hemorrhage control methods involve packing the wound with hemostatic agents, providing manual pressure, and then applying a pressure dressing to stabilize the treatment. This is a lengthy process that frequently destabilizes upon patient transport. Conversely, the iTClamp, a compact wound closure device, is designed to rapidly seal wound edges mechanically, expediting clot formation at the site of injury. OBJECTIVES: To determine the efficacy of the iTClamp with and without wound packing in the control of a lethal junction hemorrhage. METHODS: Given the limited available information regarding the efficacy of the iTClamp in conjunction with traditional hemostatic agents, this study used a swine model of severe junctional hemorrhage. The goal was to compare a multiagent strategy using the iTClamp in conjunction with XSTAT to the traditional method of Combat Gauze packing with pressure dressing application. Readouts include application time, blood loss, and rebleed occurrence. RESULTS: Mean application times of the iTClamp treatment alone or in conjunction with other hemostatic agents were at least 75% faster than the application time of Combat Gauze with pressure dressing. Percent blood loss was not significantly different between groups but trended the highest for Combat Gauze treated swine, followed by iTClamp plus XSTAT, iTClamp alone and finally iTClamp plus Combat Gauze. CONCLUSION: The results from this study demonstrate that the iTClamp can be effectively utilized in conjunction with hemostatic packing to control junctional hemorrhages.

Cric in the Dark: Surgical Cricothyrotomy in Low Light Tactical Environments.

BACKGROUND: Surgical cricothyrotomy (SC) is a difficult procedure with high failure rates in the battlefield environment. The difficulty of this procedure is compounded in a low-light tactical environment in which white light cannot be used. This study compared the use of red-green (RG) light and red (R) light in the performance of SC in a low-light environment. MATERIALS AND METHODS: Tactical Combat Casualty Care-certified navy corpsmen (n = 33) were provided 15 minutes of standardized instruction followed by hands-on practice with the Tactical CricKit and the H&H bougie-assisted Emergency Cricothyrotomy Kit. Participants acclimated to a dark environment for 30 minutes before performing SC on a mannequin with both devices using both R and RG light in a randomized order. Application time, success, participant preference, and participant confidence were analyzed. RESULTS: There were similarly high levels of successful placement (>87.5%) in all four cohorts. Light choice did not appear to affect placement time with either of the two kits. On Likert-scale surveys, participants reported that RG decreased difficulty (p < .0001) and increased confidence (p < .0001) in performing the procedure. CONCLUSION: RG light increased confidence and decreased perceived difficulty when performing SC, though no differences in placement time or success were observed.

Serine 129 phosphorylation reduces the ability of alpha-synuclein to regulate tyrosine hydroxylase and protein phosphatase 2A in vitro and in vivo.

Alpha-synuclein (a-Syn), a protein implicated in Parkinson disease, contributes significantly to dopamine metabolism. a-Syn binding inhibits the activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. Phosphorylation of TH stimulates its activity, an effect that is reversed by protein phosphatase 2A (PP2A). In cells, a-Syn overexpression activates PP2A. Here we demonstrate that a-Syn significantly inhibited TH activity in vitro and in vivo and that phosphorylation of a-Syn serine 129 (Ser-129) modulated this effect. In MN9D cells, a-Syn overexpression reduced TH serine 19 phosphorylation (Ser(P)-19). In dopaminergic tissues from mice overexpressing human a-Syn in catecholamine neurons only, TH-Ser-19 and TH-Ser-40 phosphorylation and activity were also reduced, whereas PP2A was more active. Cerebellum, which lacks excess a-Syn, had PP2A activity identical to controls. Conversely, a-Syn knock-out mice had elevated TH-Ser-19 phosphorylation and activity and less active PP2A in dopaminergic tissues. Using an a-Syn Ser-129 dephosphorylation mimic, with serine mutated to alanine, TH was more inhibited, whereas PP2A was more active in vitro and in vivo. Phosphorylation of a-Syn Ser-129 by Polo-like-kinase 2 in vitro reduced the ability of a-Syn to inhibit TH or activate PP2A, identifying a novel regulatory role for Ser-129 on a-Syn. These findings extend our understanding of normal a-Syn biology and have implications for the dopamine dysfunction of Parkinson disease.

Alpha-synuclein aggregation alters tyrosine hydroxylase phosphorylation and immunoreactivity: lessons from viral transduction of knockout mice.

Tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine synthesis, is frequently used as a marker of dopaminergic neuronal loss in animal models of Parkinson's disease (PD). We have been exploring the normal function of the PD-related protein alpha-synuclein (alpha-Syn) with regard to dopamine synthesis. TH is activated by the phosphorylation of key seryl residues in the TH regulatory domain. Using in vitro models, our laboratory discovered that alpha-Syn inhibits TH by acting to reduce TH phosphorylation, which then reduces dopamine synthesis [X.-M. Peng, R. Tehranian, P. Dietrich, L. Stefanis, R.G. Perez, Alpha-synuclein activation of protein phosphatase 2A reduces tyrosine hydroxylase phosphorylation in dopaminergic cells, J. Cell. Sci. 118 (2005) 3523-3530; R.G. Perez, J.C. Waymire, E. Lin, J.J. Liu, F. Guo, M.J. Zigmond, A role for alpha-synuclein in the regulation of dopamine biosynthesis, J. Neurosci. 22 (2002) 3090-3099]. We recently began exploring the impact of alpha-Syn on TH in vivo, by transducing dopaminergic neurons in alpha-Syn knockout mouse (ASKO) olfactory bulb using wild type human alpha-Syn lentivirus. At 3.5-21 days after viral delivery, alpha-Syn expression was transduced primarily in periglomerular dopaminergic neurons. Cells with modest levels of alpha-Syn consistently co-labeled for Total-TH. However, cells bearing aggregated alpha-Syn, as revealed by proteinase K or Thioflavin-S treatment had significantly reduced Total-TH immunoreactivity, but high phosphoserine-TH labeling. On immunoblots, we noted that Total-TH immunoreactivity was equivalent in all conditions, although tissues with alpha-Syn aggregates again had higher phosphoserine-TH levels. This suggests that aggregated alpha-Syn is no longer able to inhibit TH. Although the reason(s) underlying reduced Total-TH immunoreactivity on tissue sections await(s) confirmation, the dopaminergic phenotype was easily verified using phosphorylation-state-specific TH antibodies. These findings have implications not only for normal alpha-Syn function in TH regulation, but also for measuring cell loss that is associated with synucleinopathy.

Residual sodium dodecyl sulfate in decellularized muscle matrices leads to fibroblast activation in vitro and foreign body response in vivo.

Detergents such as sodium dodecyl sulfate (SDS) are commonly used to extract cells from tissues in a process called "decellularization". Residual SDS is difficult to completely remove and may lead to an undesirable host response towards an implanted biomaterial. In this study, we developed a modification for SDS cell extraction from muscle equally efficient to previous methods but leading to significantly less residual SDS remnants in the matrices. Muscle-derived matrices were prepared via 2 SDS-based decellularization methods, which led to removal of either 81.4% or 98.4% of the SDS. In vitro, matrices were seeded with thp1 macrophages and primary human foreskin fibroblasts. By Day 2, both matrices demonstrated similar macrophage polarization; however, fibroblasts cultured on matrices with greater residual SDS expressed higher levels of mRNA associated with fibroblast activation: α-smooth muscle actin and connective tissue growth factor. In vivo, Collagen I gels spiked with increasing concentrations of SDS displayed a corresponding decrease in cell infiltration when implanted subcutaneously in rats after 4 days. Finally, as a model for muscle regeneration, matrices produced by each method were implanted in rat latissimus dorsi defects. At POD 30 greater levels of IL-1ß mRNA were present in defects treated with matrices containing higher levels of SDS, indicating a more severe inflammatory response. Although matrices containing higher levels of residual SDS became encapsulated by POD 30 and showed evidence of a foreign body response, matrices with the lower levels of SDS integrated into the defect area with lower levels of inflammatory and fibrosis-related gene expression.

Transport patterns of anti-TNF-α in burn wounds: Therapeutic implications of hyaluronic acid conjugation.

A central complication in burn injuries is progression of the zone of necrosis, which is associated with intense inflammatory responses. Conjugation of monoclonal antibodies against tumor necrosis factor-α (TNF-α), a central mediator of inflammation, to high molecular weight hyaluronic acid (HA) has been shown to be an effective treatment in reducing secondary necrosis in rodent models of deep partial-thickness burns. Here the transport of conjugated and non-conjugated antibodies in burn injuries was investigated to explore the effects of antibody tethering on the spatiotemporal distribution of anti-TNF-α. Diffusion constants were measured in solution and in type I collagen gels in vitro using fluorescence correlation spectroscopy to provide quantitative comparisons of the effects of conjugation. It is shown that the HA significantly increased the antibody residence time in the superficial region at 24 h in burn injuries, which strongly correlated with the pattern of inflammatory cell infiltrate in the tissue. A transport model was used to fit the results of antibody distribution in the tissue based on fluorescence correlation spectroscopy measurements, resulting in estimates for effective diffusion constants that demonstrate the effects of HA conjugation on the biodistribution of therapeutic proteins. These results demonstrate that tuning residence time of therapeutic proteins can be an effective strategy in regulating the inflammatory response associated with acute injuries.

S100A12 Induced in the Epidermis by Reduced Hydration Activates Dermal Fibroblasts and Causes Dermal Fibrosis.

Disruption of the barrier function of skin increases transepidermal water loss and up-regulates inflammatory pathways in the epidermis. Consequently, sustained expression of proinflammatory cytokines from the epidermis is associated with dermal scarring. We found increased expression of S100A12 in the epidermis of human hypertrophic and keloid scar. Exposing a stratified keratinocyte culture to a reduced-hydration environment increased the expression and secretion of S100A12 by nearly 70%, which in turn activated dermal fibroblasts in vitro. Direct treatment of fibroblasts with conditioned medium collected from stratified keratinocyte culture under reduced-hydration conditions activated fibroblasts, shown by up-regulation of α-smooth muscle actin, pro-collagen 1, and F-actin expression. However, this fibroblast activation was not found when S100A12 was knocked down by RNA interference in keratinocytes. Pharmacological blockade of S100A12 receptors, RAGE, or TLR4 inhibited S100A12-induced fibroblast activation. Local delivery of S100A12 resulted in a marked hypertrophic scar formation in a validated rabbit hypertrophic scar model compared with saline control. Our findings indicate that S100A12 functions as a proinflammatory cytokine and suggest that S100A12 is a potential therapeutic target for dermal scarring.

Tip-Loaded Dissolvable Microneedle Arrays Effectively Deliver Polymer-Conjugated Antibody Inhibitors of Tumor-Necrosis-Factor-Alpha Into Human Skin.

Autoinflammatory skin diseases are characterized by a disequilibrium of cytokines in the local skin microenvironment, suggesting that local delivery of therapeutics, including anticytokine antibodies, may provide benefit without the unwanted off-target effects of systemically delivered therapies. Rapid diffusion of therapeutics away from the target site has been a challenge to the development of local therapies. Conjugation of high molecular weight hydrophilic polymers to cytokine neutralizing mAbs has been shown to be an effective strategy for local control of inflammation in healing burn wounds. However, the burn application is unique because the skin barrier is already breached. For the treatment of autoinflammatory skin diseases, the major challenge for local delivery lies in penetrating the stratum corneum. Here, we investigate a new therapeutic approach combining the use of tip-loaded dissolvable microneedle arrays (TL-dMNAs) for local application of polymer-conjugated antibody inhibitors of tumor-necrosis-factor-alpha (TNF-α). Specifically, intradermal delivery and pharmacokinetics of (anti-TNF-α-Ab)-(high molecular weight hyaluronic acid [HA]) conjugates from tip-loaded, obelisk-shaped dissolvable microneedle arrays were investigated in living human skin. The results indicate (1) TL-dMNAs can be successfully fabricated to integrate (anti-TNF-α-Ab)-HA at the tip portion of the microneedles while preserving the biological activity necessary for antibody ligand binding; (2) (anti-TNF-α-Ab)-HA can be effectively delivered into human skin using obelisk-shaped TL-dMNAs; and (3) polymer conjugation effectively inhibits antibody diffusion from the delivery site. Taken together, these results support the evaluation of microneedle array-based delivery of varying polymer-antibody conjugates for the treatment of inflammatory skin diseases.

Local delivery of antitumor necrosis factor-α through conjugation to hyaluronic acid: dosing strategies and early healing effects in a rat burn model.

The objective of this study was to measure dose-response effects of topical delivery of inhibitors of tumor necrosis factor-α (TNF-α) through conjugation to hyaluronic acid in a rat burn model to determine effects on inflammatory responses, burn progression, and early stages of healing. Monoclonal antibodies against TNF-α were conjugated to hyaluronic acid and applied topically in a rat partial-thickness burn model. Metrics of inflammatory responses and tissue necrosis were measured as well as the quantitative analysis of collagen composition and organization. The minimum effective conjugated antibody dose was found to be 100 µg with three applications 48 hours apart. Nonviable tissue thicknesses decreased with increasing dose and dose frequency. Free antibody retarded macrophage infiltration in the periphery but not at the surface, while the conjugated antibody was able to hinder macrophage infiltration at both the periphery and the surface. Quantification of collagen I and III staining ratios at days 4, 7, and 14 and quantitative image analysis of collagen organization at day 14 demonstrated differences between saline and conjugate treatment. This correlated with increases in re-epithelialization observed in conjugate-treated sites. Reductions in inflammatory markers and secondary tissue necrosis under treatment with the conjugates were understood in terms of differences in antibody transport compared to nonconjugated antibody. Differences in collagen composition and organization at Day 14 suggested that the reductions in inflammatory responses altered early healing responses. These results indicate anti-TNF-α conjugated to hyaluronic acid can be an effective treatment for reducing secondary necrosis and improving healing outcomes in burns.

Therapeutic intradermal delivery of tumor necrosis factor-alpha antibodies using tip-loaded dissolvable microneedle arrays.

Tumor necrosis factor-alpha (TNF-α) specific antibodies (anti-TNF-α Ab) have been shown to be potent TNF inhibitors and effective therapeutics for a range of inflammatory diseases. Typically, these drugs are administered systemically, but systemic dosing sufficient to achieve locally effective concentrations in peripheral tissues has been associated with systemic immunosuppression and related adverse events. Here, we evaluated the use of tip-loaded dissolvable microneedle arrays (MNAs) for localized intradermal delivery of anti-TNF-α Ab. MNAs with obelisk shape microneedles that incorporate the antibody cargo in the needle tips were created from carboxymethylcellulose (CMC) using a micromilling/spin-casting fabrication method. We found that anti-TNF-α Ab integrated into MNAs using this room temperature fabrication process maintained conformationally dependent TNF-α binding activity. Further, these MNAs efficiently delivered anti-TNF-α antibodies to the dermis of human skin with clinically applicable release profiles. To evaluate MNA delivered anti-TNF-α Ab function, we applied anti-TNF-α Ab containing MNAs to established psoriasiform lesions on the skin of mice. MNA anti-TNF-α Ab treatment reduced key biomarkers of psoriasiform inflammation including epidermal thickness and IL-1ß expression. Taken together, these results demonstrate efficient and biologically effective MNA delivery of anti-TNF-α Ab to the intradermal microenvironment of the skin in mice and humans, and support the development of MNA mediated antibody delivery for clinical applications. STATEMENT OF SIGNIFICANCE: Tumor necrosis factor-alpha (TNF-α) specific antibodies (anti-TNF-α Ab) have been shown to be potent TNF inhibitors and effective therapeutics for a range of inflammatory diseases. Typically, these drugs are administered systemically, but systemic dosing sufficient to achieve locally effective concentrations in peripheral tissues has been associated with systemic immunosuppression and related adverse events. Here we demonstrate efficient and biologically effective MNA delivery of anti-TNF-α Ab to the intradermal microenvironment of the skin in mice and humans. These results support the development of MNA mediated antibody delivery of therapeutic antibodies for clinical applications.

Effects of hyaluronic acid conjugation on anti-TNF-α inhibition of inflammation in burns.

Biomaterials capable of neutralizing specific cytokines could form the basis for treating a broad range of conditions characterized by intense, local inflammation. Severe burns, spanning partial- to full-thickness of the dermis, can result in complications due to acute inflammation that contributes to burn progression, and early mediation may be a key factor in rescuing thermally injured tissue from secondary necrosis to improve healing outcomes. In this work, we examined the effects on burn progression and influence on the inflammatory microenvironment of topical application of anti-tumor necrosis factor-α (anti-TNF-α) alone, mixed with hyaluronic acid (HA) or conjugated to HA. We found that non-conjugated anti-TNF-α decreased macrophage infiltration to a greater extent than that conjugated to HA; however, there was little effect on the degree of progression or IL-1ß levels. A simple transport model is proposed to analyze the results, which predicts qualitative and quantitative differences between untreated burn sites and those treated with the conjugates. Our results indicate that conjugation of anti-TNF-α to high molecular weight HA provides sustained, local modulation of the post-injury inflammatory responses compared to direct administration of non-conjugated antibodies.

Polymer-conjugated inhibitors of tumor necrosis factor-α for local control of inflammation.

Burns, chronic wounds, osteoarthritis, and uveitis are examples of conditions characterized by local, intense inflammatory responses that can impede healing or even further tissue degradation. The most powerful anti-inflammatory drugs available are often administered systemically, but these carry significant side effects and are not compatible for patients that have underlying complications associated with their condition. Conjugation of monoclonal antibodies that neutralize pro-inflammatory cytokines to high molecular weight hydrophilic polymers has been shown to be an effective strategy for local control of inflammation. Lead formulations are based on antibody inhibitors of tumor necrosis factor-α conjugated to hyaluronic acid having molecular weight greater than 1 MDa. This review will discuss fundamental aspects of medical conditions that could be treated with these conjugates and design principles for preparing these cytokine-neutralizing polymer conjugates. Results demonstrating that infliximab, an approved inhibitor of tumor necrosis factor-α, can be incorporated into the conjugates using a broad range of water-soluble polymers are also presented, along with a prospectus for clinical translation.