Deep learning classification for macrophage subtypes through cell migratory pattern analysis.

Macrophages can exhibit pro-inflammatory or pro-reparatory functions, contingent upon their specific activation state. This dynamic behavior empowers macrophages to engage in immune reactions and contribute to tissue homeostasis. Understanding the intricate interplay between macrophage motility and activation status provides valuable insights into the complex mechanisms that govern their diverse functions. In a recent study, we developed a classification method based on morphology, which demonstrated that movement characteristics, including speed and displacement, can serve as distinguishing factors for macrophage subtypes. In this study, we develop a deep learning model to explore the potential of classifying macrophage subtypes based solely on raw trajectory patterns. The classification model relies on the time series of x-y coordinates, as well as the distance traveled and net displacement. We begin by investigating the migratory patterns of macrophages to gain a deeper understanding of their behavior. Although this analysis does not directly inform the deep learning model, it serves to highlight the intricate and distinct dynamics exhibited by different macrophage subtypes, which cannot be easily captured by a finite set of motility metrics. Our study uses cell trajectories to classify three macrophage subtypes: M0, M1, and M2. This advancement holds promising implications for the future, as it suggests the possibility of identifying macrophage subtypes without relying on shape analysis. Consequently, it could potentially eliminate the necessity for high-quality imaging techniques and provide more robust methods for analyzing inherently blurry images.

Patterns of use of advanced wound matrices in the Veterans Administration clinics.

Chronic wounds are a common and costly health issue affecting millions of individuals in the United States, particularly those with underlying conditions such as diabetes, venous insufficiency, and peripheral artery disease. When standard treatments fail, advanced wound care therapies, such as skin substitutes, are often applied. However, the clinical effectiveness, indications, and comparative benefits of these therapies have not been well established. In this study, we report on the usage of both acellular and cellular, single and bilayer, natural and synthetic, dermal, and epidermal skin substitutes in a VA hospital system. We performed a retrospective chart review to understand the ordering and usage patterns of advanced wound therapies for patients with chronic wounds at the VA Northern California Health Care System. We examined types of products being recommended, categories of users recommending the products, indications for orders, and rate of repeated orders. Neuropathic, venous, or pressure ulcers were the main indications for using advanced wound matrices. Only 15.6% of patients for whom the matrices were ordered had supporting laboratory tests. Exactly 34.3% of the ordered matrices were not applied. And the use of wound matrices resulted in increased costs per patient visit of $1018-$3450. Our study sheds light on the usage patterns of these therapies in a VA healthcare facility and highlights the need for more robust evidence-based studies to determine the true benefits, efficacy, and cost-effectiveness of these innovative treatment options.

Cellular versus acellular matrix devices in the treatment of diabetic foot ulcers: Interim results of a comparative efficacy randomized controlled trial.

Healing of diabetic foot ulcers is a major challenge. Despite adhering to optimal standard of care (SOC), less than 30% of wounds heal after 20 weeks. Advanced cellular tissue-based products have shown better healing over SOC, albeit with great cost and modest improvement. We hypothesized no difference in healing effected by either cellular (Dermagraft), noncellular (Oasis) devices, relative to SOC in treating diabetic foot ulcer in a randomized controlled trial. The primary and secondary outcomes were the percentage of subjects that achieved complete wound closure by study endpoint (12 weeks of treatment) and study completion, respectively. During the 2-week screening phase with SOC, subjects with 40% change in ulcer size were excluded. After randomization, 56 patients entered an active treatment phase (8 weeks) followed by a maintenance phase (4-week SOC), with endpoint at visit 15, and 4 monthly follow-up visits. There was equal distribution of demographic data (p>.05) and no difference in initial wound characteristics (p>.05) between all groups. No differences were observed in complete wound closure by 12 and 28 weeks of treatment, nor were there any difference in percentage area reduction from treatment weeks 1 to 12 and from treatment weeks 1 to 28 between the groups. Each of the treatment arms showed statistically significant reduction in wound area from treatment weeks 1 to 28 (p<.05). This exploratory analysis suggests that the outcomes of treatment with either Dermagraft or Oasis matrix are comparable. We have completed enrollment, and the final data analysis is underway to make definitive conclusions.

Does class attendance matter? Results from a second-year medical school dermatology cohort study.

BACKGROUND: Little is known about the impacts of class attendance and learning preferences on academic performance in dermatology. OBJECTIVES: This study was designed to examine the effects of medical student class attendance and learning preferences on students' academic performance in an introductory dermatology course. METHODS: A total of 101 second-year medical students enrolled in a required introductory dermatology course were surveyed regarding their learning preferences. Records of class attendance and scores on the final examination were reviewed. RESULTS: The most frequently cited reason for attending classes was social expectation (96%), whereas the least cited was learning well in a classroom-type setting (65%). The top reasons cited by students for not attending classes were availability of lectures online (35%), preference for individual study outside the classroom setting (26%), and the inconvenience of traveling to class (24%). Multivariate analysis found no statistically significant relationship between class attendance and performance on the final examination (estimate -0.074, standard error 0.12; P = 0.54) after adjusting for sex, age, Medical College Admission Test (MCAT) score, having children at home, and reason for attending class. Those who prefer to learn by watching online videos scored significantly higher on the final examination (prefer online videos: 87 ± 5.5; neutral: 86 ± 5.9; do not prefer online videos: 82 ± 2.6 [P = 0.049]). CONCLUSIONS: Class attendance was not associated with improved academic performance in a dermatology course. Those who preferred to learn by watching online videos demonstrated a higher level of performance than those who did not prefer to learn this way.

Epithelialization in Wound Healing: A Comprehensive Review.

Significance: Keratinocytes, a major cellular component of the epidermis, are responsible for restoring the epidermis after injury through a process termed epithelialization. This review will focus on the pivotal role of keratinocytes in epithelialization, including cellular processes and mechanisms of their regulation during re-epithelialization, and their cross talk with other cell types participating in wound healing. Recent Advances: Discoveries in epidermal stem cells, keratinocyte immune function, and the role of the epidermis as an independent neuroendocrine organ will be reviewed. Novel mechanisms of gene expression regulation important for re-epithelialization, including microRNAs and histone modifications, will also be discussed. Critical Issues: Epithelialization is an essential component of wound healing used as a defining parameter of a successful wound closure. A wound cannot be considered healed in the absence of re-epithelialization. The epithelialization process is impaired in all types of chronic wounds. Future Directions: A comprehensive understanding of the epithelialization process will ultimately lead to the development of novel therapeutic approaches to promote wound closure.

Catecholamine stress alters neutrophil trafficking and impairs wound healing by ß2-adrenergic receptor-mediated upregulation of IL-6.

Stress-induced hormones can alter the inflammatory response to tissue injury; however, the precise mechanism by which epinephrine influences inflammatory response and wound healing is not well defined. Here we demonstrate that epinephrine alters the neutrophil (polymorphonuclear leukocyte (PMN))-dependent inflammatory response to a cutaneous wound. Using noninvasive real-time imaging of genetically tagged PMNs in a murine skin wound, chronic, epinephrine-mediated stress was modeled by sustained delivery of epinephrine. Prolonged systemic exposure of epinephrine resulted in persistent PMN trafficking to the wound site via an IL-6-mediated mechanism, and this in turn impaired wound repair. Further, we demonstrate that ß2-adrenergic receptor-dependent activation of proinflammatory macrophages is critical for epinephrine-mediated IL-6 production. This study expands our current understanding of stress hormone-mediated impairment of wound healing and provides an important mechanistic link to explain how epinephrine stress exacerbates inflammation via increased number and lifetime of PMNs.

TLR2 expression and signaling-dependent inflammation impair wound healing in diabetic mice.

Toll-like receptor-2 (TLR2) is a pivotal pathogen recognition receptor that has a key role in inflammation, diabetes, and injury. Hyperglycemia, inflammation, and oxidative stress induce TLR2-myeloid differentiation factor-88 (MyD88) expression and signaling, and are major pathophysiological mechanisms in the impaired diabetic wound-healing process. The aim of the study was to examine the contribution of TLR2-MyD88 expression and signaling to the prolonged inflammation observed in diabetic wounds. Diabetes was induced in male C57BL/6J and TLR2(-/-) mice using streptozotocin (STZ) with matching nondiabetic mice as control. In addition, nonobese diabetic (NOD) mice were used to represent the spontaneous type 1 diabetes condition. After 2 weeks of persistent hyperglycemia in the mice, full-thickness excision wounds were made on the backs aseptically. Total RNA and protein were subjected to real-time PCR and western blot analyses. Wound sizes were measured using digital planimetry. TLR2 mRNA and protein expression increased significantly in wounds of C57BL/6J+STZ and NOD mice (P<0.05) compared with nondiabetic C57BL/6J mice. MyD88 expression, interleukin receptor-associated kinase-1 phosphorylation, and nuclear factor-κ B (NF-κB) activation were increased in diabetic wounds compared with nondiabetic wounds. Wounds of TLR2(-/-)+STZ mice showed less oxidative stress, decreased MyD88 signaling, NF-κB activation, and cytokine secretion. The wound closure was significant in TLR2(-/-)+ STZ mice compared with C57BL/6J+STZ mice. Collectively, our findings show that increased TLR2 mRNA and protein expression, signaling, and activation contribute to the prolonged inflammation in the diabetic wounds and that absence of TLR2 may result in decreased inflammation and improved wound healing.

Factors modulating p63 expression in cultured limbal epithelial cells.

PURPOSE: The expression pattern of p63, a homologue of the transcription factor p53, and whether it can be used as a corneal epithelial stem cell specific marker remain controversial. We investigated the p63 expression pattern in cultured limbal epithelial cells at different time points in culture, in sparse and confluent cultures, after growth factor starvation, and in single-cell-derived colonies. METHODS: Harvested limbal epithelial cells were plated at 2.5 (sparse) or 5 (dense) x 10 cells/cm and evaluated for p63 expression at day 1, day 4, day 7, after starvation for 72 hours, or in colonies derived from single cells. Expression of corneal lineage specific differentiation marker keratin 3 (K3) was correlated with p63 expression. Results were compared by 1-way ANOVA. RESULTS: More than 85% (85%-90%) of cells expressed p63 on day 1 regardless of cell plating density. On day 4, sparsely plated cultures were subconfluent and demonstrated high p63 expression (87.4%), whereas densely plated cells were confluent and had markedly reduced p63 expression (16.9%). Starvation of subconfluent cultures arrested cell division but did not decrease p63 expression. High-p63-expressing cultures expressed low levels of K3, and this trend was reversed in confluent cultures. Most cells in all colonies derived from single cells expressed p63. CONCLUSIONS: The majority of corneal limbal epithelial cells express p63 in colonies derived from single cells and in subconfluent cultures regardless of time in culture or continuance of cell division. This suggests that p63 expression in culture cannot be used as a marker for stem cells. Significantly reduced number of cells express p63 in confluent cultures, associated with increased cell-cell contact. It is notable that these cells continue to express p63 amid areas of increased cell-cell contact several days after cultures have attained full confluency. This may represent a unique subpopulation of cells that retain proliferative potential in a confluent culture and may be analogous to a subpopulation of stem cells present in vivo.