Localization of IL-1α Responsive Dermal Populations in an Ex Vivo Human Model of Wound Healing

While playing a critical role in skin wound healing, the inflammatory phase of this process is poorly understood. To gain a better understanding of the inflammatory phase of wound healing, we developed an ex vivo skin culture model of skin injury-induced inflammation. Previous work in our laboratory showed ex vivo culture of human skin induces an interleukin 1 alpha (IL-1α)-dependent response characterized by increased transcript and protein levels for the inflammatory cytokines/chemokines, IL-6, CXCL1, and CSF3. However, the cellular sources of these factors in ex vivo cultured human skin have not been determined. Prior work with ex vivo cultured mouse skin and single cell RNA sequencing suggested fibroblasts and endothelial cells were potential cellular sources for these inflammatory mediators. The current studies used spatial transcriptomics analysis of ex vivo cultured human skin to localize the IL-1α target cell populations/skin tissue regions that produce IL-6, CXCL1 and CSF3. The Visium Gene Expression Solution platform (10x Genomics Inc.) was used to generate spatial transcriptomics data from skin specimens preserved immediately after biopsy or after skin culture for 24 hours. Loupe Browser version 5.1.0 (10x Genomics Inc) was used for data analysis to identify and characterize cell populations/regions expressing IL6, CXCL1, and CSF3 and associated differentially expressed genes (including cell type-specific transcripts). Notably, these IL-1α-induced transcripts were localized to the parent dermis region cluster. Analysis of subclusters in the dermal region showed differential expression of these inflammatory transcripts in regions enriched with either or both fibroblast and endothelial cell specific-type markers. Potential novel markers of this inflammatory response, like SOD2, were identified and warrant future investigation. Subsequent studies in identifying the targets of IL-1α in skin inflammation is called for, as they may lead to better understanding of this processes in wound healing and better clinical outcomes.

Cutaneous Anthrax on the Upper Eyelid

A 46-year-old female patient, who presented with a black, crusty lesion on the upper eyelid, was diagnosed with cutaneous anthrax after the detection of <i>Bacillus anthracis</i> in the skin culture. It was determined that the symptoms started after she cooked the meat she bought from a butcher. Anthrax is a disease that should be kept in mind in cutaneous infections even in isolated lesions, especially in endemic areas.

Reduction of bacterial colonization at the exit site of peripherally inserted central catheters: A comparison between chlorhexidine-releasing sponge dressings and cyano-acrylate

Introduction: A serious complication associated with Central Venous Access Device (CVAD) is infection because of bacterial contamination, either by the extra-luminal or by the intra-luminal route. We evaluated the efficacy, the safety, and the cost-effectiveness of two strategies for non-inferiority in controlling bacterial colonization of the exit-site of Peripherally-Inserted Central Catheters (PICC). Methods: After PICC placement, a skin swab of the exit site was taken and cultured. In group A the exit site was sealed with N-butyl-cyanoacrylate glue, while in group B a chlorhexidine-releasing sponge dressing was applied. A second skin culture was taken at day 7. Results: A total of 51 patients were enrolled in each group. In 42 patients the second skin culture was not performed because of 20 patients were lost at follow-up or deceased and in 22 patients the dressing needed to be changed early, because of local bleeding (13 cases, in group B) or because of dressing detachment (four in group A and five in group B). The microbiological study was completed in 36 patients in group A and 24 in group B. No microorganisms were isolated in any patient. Conclusions: Both strategies were effective in controlling bacterial colonization. Glue was effective in reducing local bleeding, and it was more cost-effective than sponge dressing. During the first week, when local bleeding and bacterial colonization must be prevented, glue might be more appropriate than chlorhexidine-releasing dressing; after the first week chlorhexidine-releasing dressing might be preferable, considering that the safety of glue application on the skin for prolonged periods is still questionable.

Impact of diabetic (diabetes mellitus) patients immune factors on the skin cell viability in vitro

At the moment, a lot of scientific research focused on the role of immune mechanisms in diabetic foot ulcers development and impaired healing. A 3D skin culture system as a relevant skin model may prove valuable in investigating these mechanisms and may be a useful tool to study interactions between different cell types such as keratinocytes, fibroblasts, and immune cells. The aim of our research was to study keratinocytes and fibroblasts viability in co-culture with immune factors of patients with diabetes mellitus type 2 (DM2) and patients with diabetes and chronic foot ulcers in a 3D skin culture system. In this study, the multilayer 3D immunocompetent model of human skin comprising keratinocytes, fibroblasts, and mononuclears in an agarose-fibronectin gel was used. The human immortalized keratinocyte cell line, HaCaT, and primary fibroblast cell culture isolated from skin samples of healthy man in abdominal surgery were used for the 3D system. For the experiment 20 % serum of 9 patients with chronic diabetic foot ulcers (without active inflammation signs), 9 diabetic type 2 patients and 9 healthy people, and mononuclears of the same groups of patients were used. 9 experimental series with 3 repeats were carried out. Mononuclears of patients with DM2 and DM2 and diabetic foot syndrome (DFS) had a greater inhibitory effect on fibroblasts, significantly inhibiting their proliferation to a level of 83.78 [79.03; 89.53] % vs 70.18 [66.38; 72.10] % vs 95.40 [91.75; 99.05] %, H = 21.259, p <0.001 – DM2, DFS, and the control group, respectively. There was no significant difference in the cytoinhibitory effect of mononuclears on keratinocytes between different groups: 96.40 [92.82; 100.50] % vs 93.61 [86.80; 97.10] % vs 92.87 [85.15; 95.25] %, H = 4.459, p = 0.108 – control, DM2 and DFS group, respectively. Adding serum to the culture system influenced significantly the viability of neither keratynocytes – 99.40 [95.35; 102.05] % vs 98.60 [90.55; 100.40] % vs 94.79 [91.65; 98.16] %, H = 3.030, p = 0.220 nor of fibroblasts – 95.61 [92.39; 100.19] % vs 95.80 [88.99; 102.15] % vs 96.20 [99.69; 88.70] %, H = 0.353, p = 0.838, control, DM2 and DFS group, respectively. It was determined that the fibroblasts vialability significantly decreases after introducing mononuclears of patients with DM and patients with DM and chronic diabetic foot ulcers to the co-culture system. Adding serum of these patient groups to the culture system doesn’t influence significantly the viability of skin cells.