Dynamic Regulation of the Nexus Between Stress Granules, Roquin, and Regnase-1 Underlies the Molecular Pathogenesis of Warfare Vesicants

The use of chemical warfare agents is prohibited but they have been used in recent Middle Eastern conflicts. Their accidental exposure (e.g. arsenical lewisite) is also known and causes extensive painful cutaneous injury. However, their molecular pathogenesis is not understood. Here, we demonstrate that a nexus of stress granules (SGs), integrated stress, and RNA binding proteins (RBPs) Roquin and Reganse-1 play a key role. Lewisite and its prototype phenylarsine oxide (PAO) induce SG assembly in skin keratinocytes soon after exposure, which associate with various RBPs and translation-related proteins. SG disassembly was detected several hours after exposure. The dynamics of SG assembly-disassembly associates with the chemical insult and cell damage. Enhanced Roquin and Regnase-1 expression occurs when Roquin was recruited to SGs and Regnase-1 to the ribosome while in the disassembling SGs their expression is decreased with consequent induction of inflammatory mediators. SG-targeted protein translational control is regulated by the phosphorylation-dependent activation of eukaryotic initiation factors 2α (eIF2α). Treatment with integrated stress response inhibitor (ISRIB), which blocks eIF2α phosphorylation, impacted SG assembly dynamics. Topical application of ISRIB attenuated the inflammation and tissue disruption in PAO-challenged mice. Thus, the dynamic regulation of these pathways provides underpinning to cutaneous injury and identify translational therapeutic approach for these and similar debilitating chemicals.

Single-Center Experience with Mass Paraquat Exposure in Nine Patients

Paraquat dichloride is a widely used, highly toxic chemical herbicide and a significant cause of fatal poisonings. Toxicity is thought to be secondary to generation of reactive oxygen species. Hours after exposure, patients may experience signs and symptoms ranging from nausea to multi-system organ failure. To mitigate complications and death, immunosuppression with cyclophosphamide and corticosteroid-based therapies have shown to be an effective option in limited studies. Our objective is to report our center’s experience treating patients that had been exposed to paraquat over a two-day period. Patients were identified using our Institutional Burn Center registry, and linked to the clinical and administrative data. Demographics, length of stay, costs and mortality were evaluated. There were nine patients admitted from the exposure. All were male. All survived. Eight were undocumented migrant farmers. The average age was 36 years (25-59 years). The average length of stay was 3.3 days (2-5 days). Seventy-eight percent had cutaneous injury, but only one required debridement and placement of a skin substitute. Thirty-three percent complained of continued shortness of breath after discharge. Average total hospital cost was $28,131 ($9,500-$51,000). Paraquat is a highly toxic herbicide and exposure can be fatal if not treated promptly. Immediate decontamination and repeated pulse therapy with cyclophosphamide and methylprednisolone may be life-saving.

Wound healing properties of a fibrin-based dermal replacement scaffold

When serious cutaneous injury occurs, the innate wound healing process attempts to restore the skin’s appearance and function. Wound healing outcome is affected by factors such as contraction, revascularisation, regeneration versus fibrosis and re-epithelialisation and is also strongly influenced by the pattern and extent of damage to the dermal layer. Dermal replacement scaffolds have been designed to substitute for lost tissue, provide a structure to promote dermal regeneration, and aid skin grafting, resulting in a superior healing outcome. In this study the wound healing properties of a novel fibrin-alginate dermal scaffold were assessed in the porcine wound healing model and also compared to two widely used dermal scaffolds and grafting alone. The fibrin-alginate scaffold, unlike the other scaffolds tested, is not used in combination with an overlying skin graft. Fibrin scaffold treated wounds showed increased, sustained superficial blood flow and reduced contraction during early healing while showing comparable wound closure, re-epithelialisation and final wound outcome to other treatments. The increase in early wound vascularisation coupled with a decrease in contraction and no requirement for a skin graft suggest that the fibrin-based scaffold could provide an effective, distinctive treatment option to improve healing outcomes in human patients.

Farnesol-Loaded Liposomes Protect the Epidermis and Dermis from PM2.5-Induced Cutaneous Injury

Particulate matter with aerodynamic diameter ≤2.5 μm (PM2.5) increases oxidative stress through free radical generation and incomplete volatilization. In addition to affecting the respiratory system, PM2.5 causes aging- and inflammation-related damage to skin. Farnesol (Farn), a natural benzyl semiterpene, possesses anti-inflammatory, antioxidative, and antibacterial properties. However, because of its poor water solubility and cytotoxicity at high concentrations, the biomedical applications of Farn have been limited. This study examined the deleterious effects of PM2.5 on the epidermis and dermis. In addition, Farn-encapsulated liposomes (Lipo-Farn) and gelatin/HA/xanthan gel containing Lipo-Farn were prepared and applied in vivo to repair and alleviate PM2.5-induced damage and inflammation in skin. The prepared Lipo-Farn was 342 ± 90 nm in diameter with an encapsulation rate of 69%; the encapsulation significantly reduced the cytotoxicity of Farn. Lipo-Farn exhibited a slow-release rate of 35% after 192 h of incubation. The half-maximal inhibitory concentration of PM2.5 was approximately 850 μg/mL, and ≥400 μg/mL PM2.5 significantly increased IL-6 production in skin fibroblasts. Severe impairment in the epidermis and hair follicles and moderate impairment in the dermis were found in the groups treated with post-PM2.5 and continuous subcutaneous injection of PM2.5. Acute and chronic inflammation was observed in the skin in both experimental categories in vivo. Treatment with 4 mM Lipo-Farn largely repaired PM2.5-induced injury in the epidermis and dermis, restored injured hair follicles, and alleviated acute and chronic inflammation induced by PM2.5 in rat skin. In addition, treatment with 4 mM pure Farn and 2 mM Lipo-Farn exerted moderate reparative and anti-inflammatory effects on impaired skin. The findings of the current study indicate the therapeutic and protective effects of Lipo-Farn against various injuries caused by PM2.5 in the pilosebaceous units, epidermis, and dermis of skin.

P313 Long-term durability and safety of anti-TNF agents compared with change to a different mechanism of action as a second-line biological treatment after first anti-TNF failure in Crohn′s disease: results from the Andalusian CambiaCrohn study

Background After failure to a first aTNF agent in Crohn’s disease (CD), a second aTNF shows higher rates of failure and discontinuation. Initiating a therapy with a different mechanism of action (MoA) such as ustekinumab (UST) or vedolizumab (VDZ) could lead to a greater durability of second-line biological treatment with a higher safety profile. Methods A retrospective and multicenter study (10 hospitals in Andalusia). We included patients with active CD (Harvey-Bradsaw index >4) who had failed a first aTNF agent and started a second-line biological with other aTNF or other MoA (UST or VDZ) between July 2017 and February 2020. The aim was to evaluate the long-term durability and safety of aTNF agents compared with other MoA as a second-line biological treatment. Results 249 CD patients were included; There were no significant differences between both groups in age, sex, disease duration, location, CD behavior, perianal disease, smoking habit or concomitant corticosteroid use. Whereas there were significant differences in the proportion of patients with abdominal surgery (29.5% aTNF group vs 42.5% othermMoA, p=0.032), and concomitant immunomodulators (41.9% aTNF vs 25.8% other MoA, p=0.008). Second-line biological treatment was aTNF in 129 patients (57 IFX and 72 ADA) and other MoA in 120 (97 UST and 23 VDZ). Second aTNF was discontinued in 81/129 patients (62.8%) after a median follow-up of 21 months (mo). Whereas 24/120 patients (20%) discontinued other MoA after a median follow-up of 41mo (p< 0.001). The rate of discontinuation per patient-year of follow-up was 20.9% for aTNF and 6.7% for other MoA. The probability of maintaining aTNF or other MoA was 64.4% vs 88.3% at 12mo, 46.5% vs 81.7% at 24mo and 31% vs 80% at 36mo (p<0.001). Discontinuation rates during follow-up were 68.4% for IFX, 58.3% for ADA, 39.1% for VDZ and 15.5% for UST (p<0.001). Reasons for discontinuation were 32.4% primary non-response (63.6% aTNF and 36.4% other MoA), 51% loss of response (82.7% aTNF and 17.3% other MoA) and 16.7% intolerance or adverse events (82.3% aTNF and 17.7% other MoA). Adverse events were reported in 31/249 patients (12%), 25/31 with aTNF and 6/31 with other MoA (5 vs 0 infusion reactions, 4 vs 1 mild infections, 1 vs 0 severe infections, 10 vs 2 cutaneous injury, 2 vs 1 arthralgias and 3 vs 2 other event). Conclusion In our clinical practice, a second-line aTNF associated with significantly lower long-term drug survival compared to changing to a different MoA. Lower rates of discontinuation were observed with change to a different MoA, especially to ustekinumab. Ustekinumab and vedolizumab showed a better safety profile than infliximab or adalimumab as second-line biologic in CD.

Clinical Trial on Solid Second-Generation Platelet-Concentrates in the Management of the Chronic Osteomyelitis: Advanced Bioregenerative Surgeries

The supposition is that the usage of fibrin rich in leukocytes and platelets advanced (A-PRF) in ulcerative osteomyelitis of the diabetic foot allows rehabilitation from this critical illness. In this investigation, the focus was to normalize the use of platelet-rich fibrin (PRF) in patients with osteomyelitis not amputated, to use this second-generation platelet concentrate as a regeneration enabler. The researchers submitted and utilized A-PRF membranes (1300 g × 8 min) in 7 patients (all diabetics) with osteomyelitis and cutaneous injury for 6 months. The membranes, in combination with the supernatant fluid produced by stress, have been integrated into the skin lesion down to the bone after surgical debridement. The advancement of the lesions after some period of time has been analyzed. All 7 subjects had a probe-to-bone positive assay; magnetic resonance imaging indicated cortico-periosteal coagulation and/or foci of cortico-spongeous osteolysis contiguous to the lesion. Gram-positive bacteria were identified in our procedures in 52% of cases. Gram + Cocci, for example, Staphylococcus aureus (15.6%), β-hemolytic Streptococci (12.1%), Streptococcus viridans (7.1%), and Gram-negative bacteria, for example, Pseudomonas (10.6%), Proteus (7.8%), Enterobacter (5.7%) were present. Candida albicans was active in 2.8% of cases. The blood count showed no relevant differences. To date, cutaneous lesions have been cured in 6 of the 7 subjects treated (1 patient for more than 6 years) without any evidence of infection or recurrence. The results obtained on our subjects indicate that PRF membranes may be a therapeutic option in this problematic disease. In fact, this clinical approach may have the potential to promote the healing of diabetic skin lesions with osteomyelitis.

566 Single-Center Experience with Mass Paraquat Exposure

Introduction Paraquat dichloride is a widely used, highly toxic chemical herbicide and a leading cause of fatal poisonings. Toxicity is thought to be related to lipid peroxidation. Hours after exposure, patients may experience signs and symptoms ranging from nausea to multi-system organ failure. To prevent pulmonary complications and death, it is recommended to give patients repeated pulse therapy with cyclophosphamide and methylprednisolone administered over a 72-hour period. Our objective is to report our center’s experience treating patients who had been exposed to paraquat over a two-day period. Methods Patients were identified using Institutional Burn Center registry, and linked to the clinical and administrative data. Demographics, length of stay, costs and mortality were evaluated. Results There were nine patients admitted from the exposure. All were male. All survived. Eight were undocumented migrant farmer workers. The average age was 36 years (range 25–59 years). The average length of stay was 3.3 days (range 2–5 days). Fifty-six percent had cutaneous injury, but only one required debridement and placement of a skin substitute. Thirty-three percent complained of continued shortness of breath after discharge. Average total hospital cost was $28,131 ($9,500–51,000). Conclusions Paraquat is a highly toxic herbicide and exposure can be fatal if not treated promptly. Immediate decontamination and repeated pulse therapy with cyclophosphamide and methylprednisolone may be lifesaving.

Genetic Background and Kinetics Define Wound Bed Extracellular Vesicles in a Mouse Model of Cutaneous Injury

Extracellular vesicles (EVs) have an important role in mediating intercellular signaling in inflammation and affect the kinetics of wound healing, however, an understanding of the mechanisms regulating these responses remains limited. Therefore, we have focused on the use of cutaneous injury models in which to study the biology of EVs on the inflammatory phase of wound healing. For this, the foreign body response using sterile subcutaneous polyvinylalcohol (PVA) sponges is ideally suited for the parallel analysis of immune cells and EVs without the need for tissue dissociation, which would introduce additional variables. We have previously used this model to identify mediators of EV biogenesis, establishing that control of how EVs are made affects their payload and biological activity. These studies in normal mice led us to consider how conditions such as immunodeficiency and obsesity affect the profile of immune cells and EVs in this model using genetically defined mutant mice. Since EVs are intrinsically heterogenous in biological fluids, we have focused our studies on a novel technology, vesicle flow cytometry (vFC) to quantify changes in EVs in mouse models. Here, we show that myeloid-derived immune cells and EVs express proteins relevant in antigen presentation in PVA sponge implants that have distinct profiles in wildtype, immune-deficient (NOD scid) vs. diabetic (Leprdb) mice. Together, these results establish a foundation for the parallel analysis of both immune cells and EVs with technologies that begin to address the heterogeneity of intercellular communication in the wound bed.