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The corneal epithelium is an avascular structure that has a unique wound healing mechanism, which allows for rapid wound closure without compromising vision. This wound healing mechanism is attenuated in diabetic patients, resulting in poor clinical outcomes and recurrent non-healing erosion. We investigated changes in cellular calcium signaling activity during the wound response in murine diabetic tissue using live cell imaging from both ex vivo and in vitro models. The calcium signaling propagation in diabetic cells was significantly decreased and displayed altered patterns compared to non-diabetic controls. Diabetic cells and tissue display distinct expression of the purinergic receptor, P2X7, which mediates the wound healing response. We speculate that alterations in P2X7 expression, interactions with other proteins, and calcium signaling activity significantly impact the wound healing response. This may explain aberrations in the diabetic wound response.

The cornea is exposed daily to a number of mechanical stresses including shear stress from tear film and blinking. Over time, these stressors can lead to changes in the extracellular matrix that alter corneal stiffness, cell-substrate structures, and the integrity of cell-cell junctions. We hypothesized that changes in tissue stiffness of the cornea with age may alter calcium signaling between cells after injury, and the downstream effects of this signaling on cellular motility and wound healing. Nanoindentation studies revealed that there were significant differences in the stiffness of the corneal epithelium and stroma between corneas of 9- and 27-week mice. These changes corresponded to differences in the timeline of wound healing and in cell signaling. Corneas from 9-week mice were fully healed within 24 h. However, the wounds on corneas from 27-week mice remained incompletely healed. Furthermore, in the 27-week cohort there was no detectable calcium signaling at the wound in either apical or basal corneal epithelial cells. This is in contrast to the young cohort, where there was elevated basal cell activity relative to background levels. Cell culture experiments were performed to assess the roles of P2Y2, P2X7, and pannexin-1 in cellular motility during wound healing. Inhibition of P2Y2, P2X7, or pannexin-1 all significantly reduce wound closure. However, the inhibitors all have different effects on the trajectories of individual migrating cells. Together, these findings suggest that there are several significant differences in the stiffness and signaling that underlie the decreased wound healing efficacy of the cornea in older mice.

Epithelial wound healing is essential to repair the corneal barrier function after injury and requires coordinated epithelial sheet movement over the wounded region. The presence and role of pannexin1 on multilayered epithelial sheet migration was examined in unwounded and wounded corneal epithelium from C57BL/6J (B6) control and diet-induced obese (DiO) mice, a pretype 2 diabetic model. We hypothesize that pannexin1 is dysregulated, and the interaction of two ion-channel proteins (P2X7 and pannexin1) is altered in pretype 2 diabetic tissue. Pannexin1 was found to be present along cell borders in unwounded tissue, and no significant difference was observed between DiO and B6 control. However, an epithelial debridement induced a striking difference in pannexin1 localization. The B6 control epithelium displayed intense staining near the leading edge, which is the region where calcium mobilization was detected, whereas the staining in the DiO corneal epithelium was diffuse and lacked distinct gradation in intensity back from the leading edge. Cells distal to the wound in the DiO tissue were irregular in shape, and the morphology was similar to that of epithelium inhibited with 10Panx, a pannexin1 inhibitor. Pannexin1 inhibition reduced mobilization of calcium between cells near the leading edge, and MATLAB scripts revealed a reduction in cell-cell communication that was also detected in cultured cells. Proximity ligation was performed to determine if P2X7 and pannexin1 interaction was a necessary component of motility and communication. While there was no significant difference in the interaction in unwounded DiO and B6 control corneal epithelium, there was significantly less interaction in the wounded DiO corneas both near the wound and back from the edge. The results demonstrate that pannexin1 contributes to the healing response, and P2X7 and pannexin1 coordination may be a required component of cell-cell communication and an underlying reason for the lack of pathologic tissue migration.

Epithelial wound healing requires the coordination of cells to migrate as a unit over the basement membrane after injury. To understand the process of this coordinated movement, it is critical to study the dynamics of cell-cell communication. We developed a method to characterize the injury-induced sustained Ca2+ mobilizations that travel between cells for periods of time up to several hours. These events of communication are concentrated along the wound edge and are reduced in cells further away from the wound. Our goal was to delineate the role and contribution of these sustained mobilizations and using MATLAB analyses, we determined the probability of cell-cell communication events in both in vitro models and ex vivo organ culture models. We demonstrated that the injury response was complex and represented the activation of a number of receptors. In addition, we found that pannexin channels mediated the cell-cell communication and motility. Furthermore, the sustained Ca2+ mobilizations are associated with changes in cell morphology and motility during wound healing. The results demonstrate that both purinoreceptors and pannexins regulate the sustained Ca2+ mobilization necessary for cell-cell communication in wound healing.

The cornea is avascular, which makes it an excellent model to study matrix protein expression and tissue stiffness. The corneal epithelium adheres to the basement zone and the underlying stroma is composed of keratocytes and an extensive matrix of collagen and proteoglycans. Our goal was to examine changes in corneas of 8- and 15-week mice and compare them to 15-week pre-Type 2 diabetic obese mouse. Nanoindentation was performed on corneal epithelium in situ and then the epithelium was abraded, and the procedure repeated on the basement membrane and stroma. Confocal imaging was performed to examine the localization of proteins. Stiffness was found to be age and obesity dependent. Young’s modulus was greater in the epithelium from 15-week mice compared to 8-week mice. At 15 weeks, the epithelium of the control was significantly greater than that of the obese mice. There was a difference in the localization of Crb3 and PKCζ in the apical epithelium and a lack of lamellipodial extensions in the obese mouse. In the pre-Type 2 diabetic obese mouse there was a difference in the stiffness slope and after injury localization of fibronectin was negligible. These indicate that age and environmental changes incurred by diet alter the integrity of the tissue with age rendering it stiffer. The corneas from the pre-Type 2 diabetic obese mice were significantly softer and this may be a result of changes both in proteins on the apical surface indicating a lack of integrity and a decrease in fibronectin.

Corneal integrity is critical for vision. Corneal wounds frequently heal with scarring that impairs vision. Recently, human umbilical cord mesenchymal stem cells (cord stem cells) have been investigated for tissue engineering and therapy due to their availability and differentiation potential. In this study, we used cord stem cells in a 3-dimensional (3D) stroma-like model to observe extracellular matrix organization, with human corneal fibroblasts acting as a control. For 4 weeks, the cells were stimulated with a stable Vitamin C (VitC) derivative ±TGF-b1. After 4 weeks, the mean thickness of the constructs was ~30 mm; however, cord stem cell constructs had 50% less cells per unit volume, indicating the formation of a dense matrix. We found minimal change in decorin and lumican mRNA, and a significant increase in perlecan mRNA in the presence of TGF-b1. Keratocan on the other hand decreased with TGF-b1 in both cell lineages. With both cell types, the constructs possessed aligned collagen fibrils and associated glycosaminoglycans. Fibril diameters did not change with TGF-b1 stimulation or cell lineage; however, highly sulfated glycosaminoglycans associated with the collagen fibrils significantly increased with TGF-b1. Overall, we have shown that cord stem cells can secrete their own extracellular matrix and promote the deposition and sulfation of various proteoglycans. Furthermore, these cells are at least comparable to commonly used corneal fibroblasts and present an alternative for the 3D in vitro tissue engineered model.

Previously, we demonstrated that nucleotides released upon mechanical injury to corneal epithelium activate purinergic (P2) receptors resulting in mobilization of a Ca(2+) wave. However, the tissue is extensively innervated and communication between epithelium and neurons is critical and not well understood. Therefore, we developed a co-culture of primary trigeminal neurons and human corneal limbal epithelial cells. We demonstrated that trigeminal neurons expressed a repertoire of P2Yand P2X receptor transcripts and responded to P2 agonists in a concentration-dependent manner. Mechanical injuries to epithelia in the co-cultures elicited a Ca(2+) wave that mobilized to neurons and was attenuated by Apyrase, an ectonucleotidase. To elucidate the role of factors released from each cell type, epithelial and neuronal cells were cultured, injured, and the wound media from one cell type was collected and added to the other cell type. Epithelial wound media generated a rapid Ca(2+) mobilization in neuronal cells that was abrogated in the presence of Apyrase, while neuronal wound media elicited a complex response in epithelial cells. The rapid Ca(2+) mobilization was detected, which was abrogated with Apyrase, but it was followed by Ca(2+) waves that occurred in cell clusters. When neuronal wound media was preincubated with a cocktail of N-methyl-D-aspartate (NMDA) receptor inhibitors, the secondary response in epithelia was diminished. Glutamate was detected in the neuronal wound media and epithelial expression of NMDA receptor subunit transcripts was demonstrated. Our results indicate that corneal epithelia and neurons communicate via purinergic and NMDA receptors that mediate the wound response in a highly orchestrated manner.

Improper wound repair of the corneal epithelium can alter refraction of light resulting in impaired vision. We have shown that ATP is released after injury, activates purinergic receptor signaling pathways and plays a major role in wound closure. In many cells or tissues, ATP activates P2X(7) receptors leading to cation fluxes and cytotoxicity. The corneal epithelium is an excellent model to study the expression of both the full-length P2X(7) form (defined as the canonical receptor) and its truncated forms. When Ca(2+) mobilization is induced by BzATP, a P2X(7) agonist, it is attenuated in the presence of extracellular Mg(2+) or Zn(2+), negligible in the absence of extracellular Ca(2+), and inhibited by the competitive P2X7 receptor inhibitor, A438079. BzATP enhanced phosphorylation of ERK. Together these responses indicate the presence of a canonical or full-length P2X(7) receptor. In addition BzATP enhanced epithelial cell migration, and transfection with siRNA to the P2X(7) receptor reduced cell migration. Furthermore, sustained activation did not induce dye uptake indicating the presence of truncated or variant forms that lack the ability to form large pores. Reverse transcription-polymerase chain reaction and Northern blot analysis revealed a P2X(7) splice variant. Western blots identified a full-length and truncated form, and the expression pattern changed as cultures progressed from monolayer to stratified. Cross-linking gels demonstrated the presence of homo- and heterotrimers. We examined epithelium from age matched diabetic and non-diabetic corneas patients and detected a 4-fold increase in P2X(7) mRNA from diabetic corneal epithelium compared to non-diabetic controls and an increased trend in expression of P2X(7)variant mRNA. Taken together, these data indicate that corneal epithelial cells express full-length and truncated forms of P2X(7), which ultimately allows P2X(7) to function as a multifaceted receptor that can mediate cell proliferation and migration or cell death.

Background: Numerous retrospective analyses of prospective randomized clinical trials of patients treated with adjuvant tamoxifen and chemotherapy have demonstrated that the breast cancer intrinsic subtype Luminal A tumors generally have favorable early initial outcomes, while basal-like tumors are associated with a marked risk of early relapse. To determine the extended natural history of the intrinsic subtypes across two decades of follow up the PAM50 "non-commercial open source bioinformatics" qPCR assay was conducted on node negative tumors accrued through the Cooperative Breast Cancer Tissue Registry (CBCTR) from patients who did not receive systemic therapy. Methods: Intrinsic subtype calls were obtained from 331 CBCTR cases treated with local interventions only. Tumors were classified into Luminal A (LumA), Luminal B (LumB), HER2-enriched (HER2-E) and Basal-like (BLBC), and correlated relapse-free (RFS). Patient survival and hazard rate were estimated using Kaplan-Meier plots and log-rank test. Multivariable Cox regression analyses were used to determine the significance of the intrinsic subtypes, adjusted with standard clinicopathological variables including tumor size, age at diagnosis, grade, radiation therapy treatment, centralized reviewed estrogen receptor, progesterone receptor and human epidermal growth factor 2 status measured by immunohistochemistry. Patients were diagnosed from 1978 to 1992, with a mean follow-up time of 13 years (range 0.5-31). Results: Of the 331 tumors tested, 51% of cases were classified as LumA, 18% as LumB, 11 % as HER2-E and 20% as BLBC. Although LumA was associated with the best outcome for the first 10-year of follow-up, the final number of RFS events were eventually comparable with those observed for BLBC with prolonged follow up (Table 1). In the multivariable Cox model, only BLBC tumors were associated with worse prognosis than LumA with borderline significance (Hazard ratio: 2.0 (95% Cl 0.9-5), p = 0.07). BLBC had the highest hazard rates for the first 5 years (7% at first year to 5% at 5 yr), consistent with previous observations. Interestingly, in the absence of treatment, the slow growing LumA subtype had a gradual increase of hazard for an RFS event from 3% at 5 yrs to 4% at 10 yrs to 7% at 20 yrs. The hazard rates of LumA cross with those of BLBC at 10 years. Conclusions: Basal-like breast cancers are associated with an early risk of relapse that decreases over time. In contrast, Luminal A breast cancer has a low risk of relapse at the outset but the risk of relapse increases over time and is responsible of the majority of the RFS events after 20 years of follow up. Luminal A breast cancers are therefore not truly low risk, particularly if they do not receive endocrine therapy; and may experience the consequences of inadequate treatment decades after diagnosis. [ABSTRACT FROM AUTHOR]

Background: Intrinsic breast cancer subtypes determined by the PAM50 assay are reported to be prognostic independent of standard clinicopathological variables. The CALGB (Alliance) 9741 adjuvant trial randomized treatment in a 2x2 factorial design to (i) 2-wk (dose dense; DD) vs 3-wk therapy and (ii) sequential vs concurrent doxorubicin, cyclophosphamide, paclitaxel (A>T>C vs AC>T). DD therapy improved disease-free survival and overall survival (OS) (Citron et al. JCO 2003.). A significant interaction between intrinsic breast cancer subtypes and the use of DD therapy was hypothesized. Methods: C9741 was conducted in collaboration with ECOG, NCCTG, and SWOG. Biospecimens were collected from 1652 of 2005 subjects. Multiplexed gene expression profiling (NanoString Technologies, Inc) generated PAM50 subtype calls (luminal A, luminal B, HER2-enriched, basal-like), risk of relapse (ROR) score, and proliferation score for 1321 cases (80%). Excluded samples were due to insufficient tumor (n = 181) or RNA (n = 150). The primary endpoint was relapse-free survival (RFS). The primary analysis to determine if the clinical benefit of DD therapy is dependent on intrinsic subtype was conducted as a test of interaction in a Cox proportional hazards model (3 degrees of freedom, df; alpha=0.05). Similar analyses were performed for ROR score and proliferation score as continuous measures of risk (1 df). PAM50 subtype, ROR score, and proliferation score were evaluated in separate multivariate models adjusting for number of positive nodes, menopausal status, dose density, and sequence of therapy. Results: Improved outcomes for DD therapy in the evaluable subset mirrored results from the complete dataset (HR = 1.20; 95%CI 0.99-1.44) with a median follow-up of 12.3 yrs. Subtype analysis identified 32% luminal A (n = 417), 26% luminal B (n = 341), 20% HER2-enriched (n = 267), and 22% basal-like (n = 296). Intrinsic subtypes were prognostic of RFS (p < 0.0001) irrespective of treatment assignment with HRs relative to the luminal A subgroup of 1.50 (luminal B), 1.70 (HER2-enriched) and 1.66 (basal-like). No subtype specific treatment effect on RFS was identified (p = 0.44). ROR scores (range 0-100, median 60) were also prognostic of RFS (HR = 1.12 for a 10-unit change; 95% CI 1.07-1.18; p < 0.0001), but no association with DD therapy benefit was seen (p = 0.58). Similar results were obtained for the proliferation score, for OS as a secondary endpoint, and from multivariate models with clinical covariates. Conclusion: The prognostic value of PAM50 subtype, ROR score, and proliferation score remains highly significant in patients treated with contemporary adjuvant anthracycline and taxane based chemotherapy. Intrinsic subtype did not predict for improved survival with the 2- wk DD vs 3-wk treatment schedule; we hypothesize that this is because the prognostic differences by subtype outweighed the modest but significant clinical benefit of DD chemotherapy administration for the overall population. Planned clinical validation studies will assess the ability of PAM50 and other gene signatures to stratify patients and individualize systemic therapy regimens based on the expected risk of distant recurrence. [ABSTRACT FROM AUTHOR]

Background: Neoantigen vaccines can induce or enhance highly specific antitumor immune responses with minimal risk of autoimmunity. We have developed a neoantigen DNA vaccine platform capable of efficiently presenting both HLA class I and II epitopes and performed a phase 1 clinical trial in triple-negative breast cancer patients with persistent disease on surgical pathology following neoadjuvant chemotherapy, a patient population at high risk of disease recurrence., Methods: Expressed somatic mutations were identified by tumor/normal exome sequencing and tumor RNA sequencing. The pVACtools software suite of neoantigen prediction algorithms was used to identify and prioritize cancer neoantigens and facilitate vaccine design for manufacture in an academic GMP facility. Neoantigen DNA vaccines were administered via electroporation in the adjuvant setting (i.e., following surgical removal of the primary tumor and completion of standard of care therapy). Vaccines were monitored for safety and immune responses via ELISpot, intracellular cytokine production via flow cytometry, and TCR sequencing., Results: Eighteen subjects received three doses of a neoantigen DNA vaccine encoding on average 11 neoantigens per patient (range 4-20). The vaccinations were well tolerated with relatively few adverse events. Neoantigen-specific T cell responses were induced in 14/18 patients as measured by ELISpot and flow cytometry. At a median follow-up of 36 months, recurrence-free survival was 87.5% (95% CI: 72.7-100%) in the cohort of vaccinated patients., Conclusion: Our study demonstrates neoantigen DNA vaccines are safe, feasible, and capable of inducing neoantigen-specific immune responses., Clinical Trial Registration Number: NCT02348320., Competing Interests: Declarations Ethics approval and consent to participate The clinical protocol was reviewed and approved by the Institutional Review Board (ID# 201505074) at Washington University School of Medicine. The research conformed to the principles of the Helsinki/Tokyo/Venice Declaration on experimentation in humans. Consent for publication Written consent for publication was obtained from all participating patients. Competing interests K.S., M.G., and O.G. are consultants for the Jaime Leandro Foundation. All other authors declare that they have no competing interests., (© 2024. The Author(s).)

Questions: What are the current barriers and facilitators to the role of ED physiotherapists? How do ED physiotherapists believe their role may be optimised within the context of the ED?, Design: Mixed methods study using a cross sectional survey., Participants: Australian physiotherapists currently providing services to patients within an Australian emergency department., Intervention: N/A., Outcome Measures: The survey included questions related to the level of integration of ED physiotherapy into emergency department teams and wider health system, and open answer questions to identify the factors which impact and influence ED physiotherapy practice, and the future of ED physiotherapy., Results: 1 - Organisational culture, 2 - training and credentialling, 3 - governance, legislation and policies, 4 - funding, and 5 - advocacy and research were the five major themes generated from participant comments on existing facilitators and barriers to their role and the role of ED physiotherapists nationally. Six themes were generated from participant comments regarding the strategies to overcome barriers and facilitate an increased contribution by ED physiotherapists in the future: 1 - Training opportunities and specialisation pathways, 2 - Organisational culture, 3 - Governance, legislation and policies, 4 - Funding, 5 - Advocacy, 6 - Medicolegal Risks., Conclusion: Australian emergency department physiotherapists perceive their roles and emergency physiotherapy service provision to be impacted by complex and multi-factorial influences. The overall contribution of ED physiotherapy is susceptible to influence from non-linear interactions of various agents and factors which span all levels of the health system., Trial Registration: N/A., (© 2024. The Author(s).)

Questions: To provide workforce data on the status of Australian Emergency Department (ED) physiotherapy practice, including physiotherapist demographic data, staffing levels and funding sources. Evaluate the scope of practice currently undertaken by ED physiotherapists and alignment of ED physiotherapy service provision with demand levels., Design: Cross sectional survey., Participants: Australian physiotherapists working within an Australian ED., Outcome Measures: Workforce data, scope of practice and alignment of physiotherapy service provision to ED presentations and demand., Results: 94 Australian ED physiotherapists completed the survey, 76.9% were working as primary contact clinicians. They had a diverse scope of practice, 100% perform mobility assessments, 89.9% provide care for paediatric patients and 10.1% were involved in administration of medications. 86.2% of participants (75/87) reported working within a service model that provided seven-day per week physiotherapy coverage to ED., Conclusion: The sample of ED physiotherapists were found to be experienced and highly trained clinicians with a diverse scope of practice, who are well placed to meet the unpredictable and highly variable patient caseload of Australian EDs., (© 2024. The Author(s).)