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Background: Diabetes mellitus (DM) can cause severe complications, including diabetic foot ulcers (DFU). There is a significant gap in understanding the single-cell ecological atlas of DM and DFU tissues., Methods: Single-cell RNA sequencing data were used to create a detailed single-cell ecological landscape of DM and DFU. Enrichment analysis identified pathways involved in cellular subpopulations, and pseudo-time analysis inferred cell development processes. A gene regulatory network explored the role of transcription factors in DFU progression, and a potential herbal drug-target gene interaction network was constructed., Results: In the DFU group, immune cells were activated, with notable changes in several subpopulations. ATP5E was significantly overexpressed in Naive T cells, fibroblasts, endothelial cells, and CD8 + T cells in DM patients. Specific immune cell subsets, such as Naive T_RGCC, CTL_TYROBP_CL4, Mac_SLC40A1, and M1_CCL3L1, likely contribute to DFU formation through overactivation and proliferation, leading to tissue damage and ulcer exacerbation. Key genes TPP1, TLR4, and RIPK2 were identified, and 88 active ingredients in the herbal drug-target network showed strong correlations with these targets. Herbs like Angelica dahurica, Angelica sinensis, Boswellia carterii, liquorice, myrrh, and Semen armeniacae amarae were included., Conclusions: This study offers insights into DM and DFU cytology. T cells in DFU are activated, attacking normal tissues and worsening tissue damage. The ATP5E gene may be related to the ecological remodeling of DM, and TPP1, TLR4, and RIPK2 are potential targets for DFU treatment., Competing Interests: Declarations. Ethics approval and consent to participate: All samples were obtained with the consent of patients from the First Affiliated Hospital of Guangxi Medical University and approved by the Ethics Committee (No. 2022-KT-NSFC-260). Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Diabetic foot ulcers (DFUs) are serious skin injuries whereby the wound healing process is frequently stalled in the inflammatory phase. Currently, there is a lack of effective therapeutic strategies. MCC950, a highly selective nod-like receptor family pyrin domain containing 3 (NLRP3) inhibitor, has been reported to show strong anti-inflammation effects in many diseases. In this study, we unveiled the role of MCC950 in DFU mice model and its underlying molecular mechanisms. MCC950 could significantly accelerate diabetic wound healing, as shown by shortened healing time and better healing quality. Moreover, increased M2 phenotype macrophages and decreased pro-inflammatory genes were observed in MCC950-treated DFU mice. Additionally, myeloid-derived suppressor cells (MDSCs) were significantly increased in blood, spleen and wound tissues at different time courses. Specifically, MCC950 could recruit more MDSCs in an early phase in DFU mice, exerting an anti-inflammation effect. We identified the cell crosstalk between macrophages and MDSCs with MCC950 treatment process. Depleting MDSCs in vivo could eliminate the therapeutic effect of MCC950 on diabetic wound healing through inhibiting M2 macrophage polarization. Besides, MDSCs isolated from the wounds of MCC950 or saline treated mice were cocultured with bone marrow derived macrophage (BMDM) in a transwell system. Results confirmed that MDSCs sorted from MCC950 treated mice caused a significant increased percentage of M2 macrophages. Collectively, our findings suggest that the administration of MCC950 has the potential to accelerate diabetic wound healing by promoting M2 macrophage polarization in an MDSC-dependent manner. This study provides valuable insights into the utilization of pharmacological agents for DFU treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Diabetic foot ulcer (DFU) is a serious complication of diabetes mellitus, which causes great health damage and economic burden to patients. The pathogenesis of DFU is not fully understood. We screened wound healing-related genes using bioinformatics analysis, and full-thickness skin injury mice model and cellular assays were used to explore the role of target genes in diabetic wound healing. SFRP2 was identified as a wound healing-related gene, and the expression of SFRP2 is associated with immune cell infiltration in DFU. In vivo study showed that suppression of SFRP2 delayed the wound healing process of diabetic mice, impeded angiogenesis and matrix remodeling, but did not affect wound healing process of control mice. In addition, suppression of SFRP2 increased macrophage infiltration and impeded the transition of macrophages functional phenotypes during diabetic wound healing, and affected the transcriptome signatures-related to inflammatory response and energy metabolism at the early stage of wound healing. Extracellular flux analysis (EFA) showed that suppression of SFRP2 decreased mitochondrial energy metabolism and increased glycolysis in injury-related macrophages, but impeded both glycolysis and mitochondrial energy metabolism in inflammatory macrophages. In addition, suppression of SFRP2 inhibited wnt signaling-related genes in macrophages. Treatment of AAV-SFRP2 augmented wound healing in diabetic mice and demonstrated the therapeutic potential of SFRP2. In conclusions, SFRP2 may function as a wound healing-related gene in DFU by modulating functional phenotype transition of macrophages and the balance between mitochondrial energy metabolism and glycolysis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Yang, Xiong, He and Huang.)

In this study, we redefine the diagnostic landscape of diabetic ulcers (DUs), a major diabetes complication. Our research uncovers new biomarkers linked to immunogenic cell death (ICD) in DUs by utilizing RNA-sequencing data of Gene Expression Omnibus (GEO) analysis combined with a comprehensive database interrogation. Employing a random forest algorithm, we have developed a diagnostic model that demonstrates improved accuracy in distinguishing DUs from normal tissue, with satisfactory results from ROC analysis. Beyond mere diagnosis, our model categorizes DUs into novel molecular classifications, which may enhance our comprehension of their underlying pathophysiology. This study bridges the gap between molecular insights and clinical practice. It sets the stage for transformative strategies in DUs management, marking a significant step forward in personalized medicine for diabetic patients., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Diabetic foot ulcers (DFU), affecting a quarter of diabetic patients and leading to high rates of amputation and mortality, pose significant health and economic burdens. Wound healing in DFU is often compromised by chronic inflammation, underscoring the critical role of immune cells. However, the systematic investigation of immune-related genes (IRGs) in DFU pathogenesis remains elusive. To address this gap, our study aims to explore the association between IRGs and DFU., Methods: To explore biological changes in immune related gene expression in DFU, RNA-seq was performed on wound biopsies derived from 10 DFU patients and 11 healthy controls. Differentially expressed genes (DEGs) between DFU and normal samples were obtained by DEseq2. By intersecting the IRG list from the ImmPort database, the immune-related differentially expressed genes were identified. Function enrichment analysis and protein-protein interaction (PPI) analysis were applied by clusterProfiler and STRING database, and the hub genes of the PPI network were calculated by the cytoHubba plug-ins in Cytoscape. CIBERSORT algorithms was applied to analyze immune infiltration in DFU. And the correlation between immune cells infiltration and hub genes was explored by correlation analysis. Finally, to validate our findings, the transcriptional change of hub genes in DFU was confirmed using external scRNA-seq dataset and RT-qPCR., Results: RNA-seq analysis detected 8,800 DEGs in DFUs, with 2,351 upregulated and 6,449 downregulated.526 differential IRGs were obtained from intersection of DEGs and IRGs. 526 differential IRGs were obtained from intersection of DEGs and IRGs. Enrichment function analysis of DEGs showed that they played a significant role in immune response. The PPI network was constructed, and the most significant module containing 4 hub genes was identified. CIBERSORT analysis showing that there was a significant difference between DFU and normal controls in the infiltration of immune cells. Compared with normal tissue, DFU tissue contained a higher proportion of resting NK cell, M0 macrophages, and activated mast cell, while resting dendritic cell, activated mast cell, and activated NK cell contributed to a relatively lower portion. Additionally, the analysis for M1/M2 polarization of macrophage cells shown that DFU tissue contained a higher M1/M2 ratio than normal group. Finally, the expression levels of 4 hub genes were confirmed by external scRNA-seq dataset and RT-qPCR., Conclusions: The immune related hub genes and the difference in immune infiltration between DFU tissue and normal controls might provide new insight for understanding DFU healing., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Diabetic foot ulcer (DFU) is a foremost cause of amputation in diabetic patients. Consequences of DFU include infections, decline in limb function, hospitalization, amputation, and in severe cases, death. Immune cells including macrophages, regulatory T cells, fibroblasts and other damage repair cells work in sync for effective healing and in establishment of a healthy skin barrier post-injury. Immune dysregulation during the healing of wounds can result in wound chronicity. Hyperglycemic conditions in diabetic patients influence the pathophysiology of wounds by disrupting the immune system as well as promoting neuropathy and ischemic conditions, making them difficult to heal. Chronic wound microenvironment is characterized by increased expression of matrix metalloproteinases, reactive oxygen species as well as pro-inflammatory cytokines, resulting in persistent inflammation and delayed healing. Novel treatment modalities including growth factor therapies, nano formulations, microRNA based treatments and skin grafting approaches have significantly augmented treatment efficiency, demonstrating creditable efficacy in clinical practices. Advancements in local treatments as well as invasive methodologies, for instance formulated wound dressings, stem cell applications and immunomodulatory therapies have been successful in targeting the complex pathophysiology of chronic wounds. This review focuses on elucidating the intricacies of emerging physical and non-physical therapeutic interventions, delving into the realm of advanced wound care and comprehensively summarizing efficacy of evidence-based therapies for DFU currently available., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Significance: The incidence of diabetes continues to rise throughout the world in an alarming rate. Diabetic patients often develop diabetic foot ulcers (DFUs), many of which do not heal. Non-healing DFUs are a major cause of hospitalization, amputation, and increased morbidity. Understanding the underlying mechanisms of impaired healing in DFU is crucial for its management. Recent Advances: This review focuses on the recent advancements on macrophages and neutrophils in diabetic wounds and DFUs. In particular, we discuss diabetes-induced dysregulations and dysfunctions of macrophages and neutrophils. Critical Issues: It is well established that diabetic wounds are characterized by stalled inflammation that results in impaired healing. Recent findings in the field suggest that dysregulation of macrophages and neutrophils plays a critical role in impaired healing in DFUs. The delineation of mechanisms that restore macrophage and neutrophil function in diabetic wound healing is the focus of intense investigation. Future Directions: The breadth of recently generated knowledge on the activity of macrophages and neutrophils in diabetic wound healing is impressive. Experimental models have delineated pathways that hold promise for the treatment of diabetic wounds and DFUs. These pathways may be useful targets for further clinical investigation.

Aim: The systemic immune inflammation index (SII) is a cost-effective biomarker calculated by lymphocyte, neutrophil and platelet counts and is currently being studied in various diseases. Since there is no study examining the relationship between SII and diabetic foot ulcers (DFU) in the literature, our aim was to investigate the relationship between SII and amputation rate in DFU., Methods: Type 2 DM 511 patients with DFU were screened from 2017 to 2021. Laboratory data obtained on the first day of hospitalization were considered. Platelet-to-lymphocyte ratio (PLR), neutrophil-to-lymphocyte ratio (NLR) and SII were calculated from routine blood count. Participants were divided into two groups as amputation (Group 1) and non-amputation (Group 2)., Results: Amputation rate was 18.8%. The A1c (8.80 (3.26) % vs. 9.52 (3.10) %, p = 0.007) and HGB (10.17 ± 2.16 g/dL vs. 12.05 ± 2.20 g/dL, p < 0.001) levels, and lymphocyte count (1.81 (1.16) vs. 2.05 (1.11), p = 0.015) were significantly lower in Group 1 than Group 2. The counts of WBC (14.01 (9.16) × 10 9 /L vs. 10.41 (5.82) × 10 9 /L), PLT (393.35 (196.98) × 10 9 /L vs. 312.05 (141.33) × 10 9 /L), neutrophil (11.52 (8.75) × 10 9 /L vs. 6.93 (5.96) × 10 9 /L), PLR (226.04 (159.24) × 10 9 /L vs. 153.12 (101.91) × 10 9 /L), NLR (6.64 (6.93) vs. 3.34 (3.99)) and SII (2505.86 (3957.47) × 10 9 /L vs. 1092.50 (1476.08) × 10 9 /L), and the levels of CRP (14.12 (12.66) mg/dL vs. 3.86 (12.63) mg/dL) and ESR (87.50 (50.50) mm/h vs. 63.00 (57.25) mm/h) were significantly higher in Group 1 than Group 2 (all p < 0.001). AUC of ROC analysis of PLR was 0.666 (95% CI, 0.604-0.728), NLR was 0.695 (95% CI, 0.638-0.752) and SII was 0.716 (95% CI, 0.661-0.772) for the predicting of amputation and the SII had the best AUC with 67.4% sensitivity and 63.3%specificty., Conclusion: SII is a cost-effective and readily available marker, but alone may not be sufficient to predict the risk of amputation in DFU. In our results, the predictive role of SII alone or with other markers for future DFU and its role in predicting other chronic diabetic complications will be evaluated in extensive studies., Competing Interests: Declaration of competing interest The authors have no conflict of interest to declare. This study was performed under the approval from the Ethics Committee of our Faculty of Medicine., (Copyright © 2023 The Japanese Orthopaedic Association. Published by Elsevier B.V. All rights reserved.)

Diabetic foot ulcers (DFUs) are one of the most common and challenging complications of diabetes, yet our understanding of their pathogenesis remains limited. We collected gene expression data of DFU patients from public databases. Bioinformatics tools were applied for systematic analysis, including the identification of differentially expressed genes (DEGs), weighted gene co-expression network analysis (WGCNA) and enrichment analysis. We further used single-cell RNA sequencing to identify the distribution of different cell populations in DFU. Finally, key results were validated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and flow cytometry. We identified 217 DEGs between ulcerated and healthy skin, and 37 DEGs between healing ulcers and ulcers. WGCNA revealed that the cyan module had the highest positive correlation with healthy skin and negative correlation with ulcers. The black module had the highest negative correlation with healthy skin and positive correlation with ulcers. Enrichment analysis showed that the genes in the cyan module were mainly associated with complement and coagulation cascades, while the genes in the black module were mainly associated with the IL-17 signalling pathway. In addition, CD8 T cells were significantly lower in ulcers than in healthy and healing ulcers. By comparing marker genes of CD8 T cells, we identified key genes in the cyan and black modules and validated their expression using RT-qPCR. The proportion of CD8 T cells was increased in healing ulcers. Flow cytometry detected increased levels of CD8 T, B and natural killer cells in healing ulcers. CD8 T cells and related key genes play an important role in the healing process of DFU. The results of this study provide a new perspective for understanding the pathogenesis and treatment of DFU., (© 2023 The Authors. International Wound Journal published by Medicalhelplines.com Inc and John Wiley & Sons Ltd.)

Impaired wound healing associated with recurrent Staphylococcus aureus infection and unresolved inflammation are hallmarks of nonhealing diabetic foot ulcers (DFUs). Perforin-2, an innate immunity molecule against intracellular bacteria, limits cutaneous infection and dissemination of S. aureus in mice. Here, we report the intracellular accumulation of S. aureus in the epidermis of DFUs with no clinical signs of infection due to marked suppression of perforin-2. S. aureus residing within the epidermis of DFUs triggers AIM2 inflammasome activation and pyroptosis. These findings were corroborated in mice lacking perforin-2. The effects of pyroptosis on DFU clinical outcomes were further elucidated in a 4-week longitudinal clinical study in patients with DFUs receiving standard care. Increased AIM2 inflammasome and ASC-pyroptosome coupled with induction of IL-1β were found in nonhealing DFUs compared with healing DFUs. Our findings revealed that perforin-2 suppression, intracellular S. aureus accumulation, and associated induction of pyroptosis contribute to healing inhibition and prolonged inflammation in patients with DFUs.

Delayed wound healing can cause significant issues for immobile and ageing individuals as well as those living with co-morbid conditions such as diabetes, cardiovascular disease, and cancer. These delays increase a patient's risk for infection and, in severe cases, can result in the formation of chronic, non-healing ulcers (e.g., diabetic foot ulcers, surgical site infections, pressure ulcers and venous leg ulcers). Chronic wounds are very difficult and expensive to treat and there is an urgent need to develop more effective therapeutics that restore healing processes. Sustained innate immune activation and inflammation are common features observed across most chronic wound types. However, the factors driving this activation remain incompletely understood. Emerging evidence suggests that the composition and structure of the wound microbiome may play a central role in driving this dysregulated activation but the cellular and molecular mechanisms underlying these processes require further investigation. In this review, we will discuss the current literature on: 1) how bacterial populations and biofilms contribute to chronic wound formation, 2) the role of bacteria and biofilms in driving dysfunctional innate immune responses in chronic wounds, and 3) therapeutics currently available (or underdevelopment) that target bacteria-innate immune interactions to improve healing. We will also discuss potential issues in studying the complexity of immune-biofilm interactions in chronic wounds and explore future areas of investigation for the field., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Versey, da Cruz Nizer, Russell, Zigic, DeZeeuw, Marek, Overhage and Cassol.)

Objective: To investigate the correlation between the platelet-to-lymphocyte ratio (PLR) and diabetic foot ulcer (DFU) in patients with type 2 diabetes mellitus (T2DM)., Method: From January 2018 to August 2019, 206 patients with T2DM admitted to the Central Hospital of Wuhan, China, were enrolled in this study, including 104 patients with DFU (DFU group) and 102 patients without DFU (T2DM group). During the same period, 90 healthy subjects were randomly screened as normal controls (NC group). The correlation between PLR and DFU in patients with T2DM was explored by comparing the PLR of the subjects in the three groups., Results: The PLRs of the DFU and T2DM groups were higher than that of the NC group, whereas the PLR of the DFU group was higher than that of the T2DM group (p < 0.05). PLR was positively correlated with the Wagner DFU grade (p < 0.001). Based on logistic regression analysis, PLR was found to be an independent risk factor for DFU (OR =1.029, 95% CI: 1.019 ~ 1.039, p < 0.001). The receiver operating characteristic curve analysis of the PLR showed that the area under the curve of the PLR for predicting diabetic foot ulcer was 0.776 (p < 0.001), and the analysis determined that the optimal critical value of the PLR for predicting DFU was 147.6., Conclusion: The PLR is significantly elevated in patients with DFU and positively correlated with the Wagner DFU grade, which might be a valuable marker for early diagnosis and assessment of severity of DFU., (© 2021 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Staphylococcus aureus is the most prevalent pathogen isolated from diabetic foot infections (DFIs). The purpose of this study was to evaluate its behavior in an in vitro model mimicking the conditions encountered in DFI. Four clinical S. aureus strains were cultivated for 16 weeks in a specific environment based on the wound-like medium biofilm model. The adaptation of isolates was evaluated as follows: by Caenorhabditis elegans model (to evaluate virulence); by quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) (to evaluate expression of the main virulence genes); and by Biofilm Ring test ® (to assess the biofilm formation). After 16 weeks, the four S. aureus had adapted their metabolism, with the development of small colony variants and the loss of β-hemolysin expression. The in vivo nematode model suggested a decrease of virulence, confirmed by qRT-PCRs, showing a significant decrease of expression of the main staphylococcal virulence genes tested, notably the toxin-encoding genes. An increased expression of genes involved in adhesion and biofilm was noted. Our data based on an in vitro model confirm the impact of environment on the adaptation switch of S. aureus to prolonged stress environmental conditions. These results contribute to explore and characterize the virulence of S. aureus in chronic wounds.

Persistent and impaired inflammation impedes tissue healing and is a characteristic of chronic wounds. A better understanding of the mechanisms controlling wound inflammation is needed. In this study, we show that in human wound-edge keratinocytes, the expressions of microRNA (miR)-17, miR-18a, miR-19a, miR-19b, and miR-20a, which all belong to the miR-17∼92 cluster, are upregulated during wound repair. However, their levels are lower in chronic ulcers than in acute wounds at the proliferative phase. Conditional knockout of miR-17∼92 in keratinocytes as well as injection of miR-19a/b and miR-20a antisense inhibitors into wound edges enhanced inflammation and delayed wound closure in mice. In contrast, conditional overexpression of the miR-17∼92 cluster or miR-19b alone in mice keratinocytes accelerated wound closure in vivo. Mechanistically, miR-19a/b and miR-20a decreased TLR3-mediated NF-κB activation by targeting SHCBP1 and SEMA7A, respectively, reducing the production of inflammatory chemokines and cytokines by keratinocytes. Thus, miR-19a/b and miR-20a being crucial regulators of wound inflammation, the lack thereof may contribute to sustained inflammation and impaired healing in chronic wounds. In line with this, we show that a combinatory treatment with miR-19b and miR-20a improved wound healing in a mouse model of type 2 diabetes., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Diabetic foot ulcers (DFUs) are a life-threatening disease that often result in lower limb amputations and a shortened lifespan. However, molecular mechanisms contributing to the pathogenesis of DFUs remain poorly understood. We use next-generation sequencing to generate a human dataset of pathogenic DFUs to compare to transcriptional profiles of human skin and oral acute wounds, oral as a model of "ideal" adult tissue repair due to accelerated closure without scarring. Here we identify major transcriptional networks deregulated in DFUs that result in decreased neutrophils and macrophages recruitment and overall poorly controlled inflammatory response. Transcription factors FOXM1 and STAT3, which function to activate and promote survival of immune cells, are inhibited in DFUs. Moreover, inhibition of FOXM1 in diabetic mouse models (STZ-induced and db/db) results in delayed wound healing and decreased neutrophil and macrophage recruitment in diabetic wounds in vivo. Our data underscore the role of a perturbed, ineffective inflammatory response as a major contributor to the pathogenesis of DFUs, which is facilitated by FOXM1-mediated deregulation of recruitment of neutrophils and macrophages, revealing a potential therapeutic strategy.

A central feature of diabetic (Db) wounds is the persistence of chronic inflammation, which is partly due to the prolonged presence of proinflammatory (M1) macrophages. Using in vivo and in vitro analyses, we have tested the hypothesis that long noncoding RNA GAS5 is dysregulated in Db wounds. We have assessed the contribution of GAS5 to the M1 macrophage phenotype, as well as the functional consequences of knocking down its expression. We found that expression of GAS5 is increased significantly in Db wounds and in cells isolated from Db wounds. Hyperglycemia induced GAS5 expression in macrophages in vitro. Overexpression of GAS5 in vitro promoted macrophage polarization toward an M1 phenotype by upregulating signal transducer and activator of transcription 1. Of most significance in our judgment, GAS5 loss-of-function enhanced Db wound healing. These data indicate that the relative level of long noncoding RNA GAS5 in wounds plays a key role in the wound healing response. Reductions in the levels of GAS5 in wounds appeared to enhance healing by promoting transition of M1 macrophages to M2 macrophages. Thus, our results suggest that targeting long noncoding RNA GAS5 may provide a therapeutic intervention for correcting impaired Db wound healing., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Diabetic foot ulcer (DFU) is one of the most common and severe complications of diabetes mellitus, which is becoming increasingly prevalent throughout the world, with high mortality and morbidity. Because of the complex pathophysiological processes involved, DFU is difficult to treat effectively with traditional therapies. Ozone therapy, an emerging method, has been reported as potentially beneficial for closure of DFUs and may gradually move to the forefront of clinical practice. Possible mechanisms of action include antioxidant capacity, pathogen inactivation, vascular and endogenous growth factor modulation, and immune system activation. However, some researchers are skeptical about its safety, and clinical trials are lacking. This article reviews the current research and application of ozone therapy for DFUs., (Copyright © 2020 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved.)

As the key defense molecules originally identified in Drosophila, Toll-like receptor (TLR) superfamily members play a fundamental role in detecting invading pathogens or damage and initiating the innate immune system of mammalian cells. The skin, the largest organ of the human body, protects the human body by providing a critical physical and immunological active multilayered barrier against invading pathogens and environmental factors. At the first line of defense, the skin is constantly exposed to pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), and TLRs, expressed in a cell type-specific manner by various skin cells, serve as key molecules to recognize PAMPs and DAMPs and to initiate downstream innate immune host responses. While TLR-initiated inflammatory responses are necessary for pathogen clearance and tissue repair, aberrant activation of TLRs will exaggerate T cell-mediated autoimmune activation, leading to unwanted inflammation, and the development of several skin diseases, including psoriasis, atopic dermatitis, systemic lupus erythematosus, diabetic foot ulcers, fibrotic skin diseases, and skin cancers. Together, TLRs are at the interface between innate immunity and adaptive immunity. In this review, we will describe current understanding of the role of TLRs in skin defense and in the pathogenesis of psoriasis and atopic dermatitis, and we will also discuss the development and therapeutic effect of TLR-targeted therapies., Competing Interests: The authors have nothing to disclose., (Copyright © 2019 Lixiang Sun et al.)

Enhanced and prolonged expression of tumor necrosis factor alpha (TNF-α), a potent pro-inflammatory cytokine is evidenced during the chronic wound healing process of infected diabetic foot ulcer (IDFU). B-cell activating factor (BAFF) is the member of TNF-α family, which implicit in B-cell dysfunction. This study was aimed to evaluate the role of BAFF in diabetic foot ulcer (DFU) patients and to correlate its association with other family of inflammatory cytokines. Circulating levels of BAFF and other cytokines were measured in IDFU (n = 44) and non-IDFU patients (n = 40) using multiplexed bead-based cytokine immunoassay. A stepwise significant increase was observed in both circulatory BAFF and C-reactive protein (CRP) during the disease progression. The area under the receiver operating characteristic curve (AUC ROC ) for BAFF was found to be high (0.89; [95% CI: 0.73-1.0]), when compared to CRP (0.68; [95% CI: 0.61-0.76]). Optimum diagnostic cutoff level for BAFF was found to be ≥2.35 pg/mL with 62.0% sensitivity and 85.7% specificity. Further, BAFF levels showed a significant positive correlation with CRP among IDFU patients. With respect to other family cytokines, BAFF levels were positively correlated with TNF-α, interferon family cytokines such as IFN-α2, IL-28A/IFN-λ2, IFN-γ, and IL-10 family cytokines such as IL-19, IL-22, and IL-26 and negatively correlated with IL-6 receptor family such as gp130/sIL-6Rβ. Hence, our data suggest that devising therapeutic strategies to reduce the levels of BAFF may contribute in amelioration of IDFU., (© 2019 by the Wound Healing Society.)

Immune systems have evolved to recognize and eliminate pathogens and damaged cells. In humans, it is estimated to recognize 10 9 epitopes and natural selection ensures that clonally expanded cells replace unstimulated cells and overall immune cell numbers remain stationary. But, with age, it faces continuous repertoire restriction and concomitant accumulation of primed cells. Changes shaping the aging immune system have bitter consequences because, as inflammatory responses gain intensity and duration, tissue-damaging immunity and inflammatory disease arise. During inflammation, the glycolytic flux cannot cope with increasing ATP demands, limiting the immune response's extent. In diabetes, higher glucose availability stretches the glycolytic limit, dysregulating proteostasis and increasing T-cell expansion. Long-term hyperglycemia exerts an accumulating effect, leading to higher inflammatory cytokine levels and increased cytotoxic mediator secretion upon infection, a phenomenon known as diabetic chronic inflammation. Here we review the etiology of diabetic chronic inflammation and its consequences on wound healing., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Neutrophil to lymphocyte ratio (NLR) and platelet to lymphocyte ratio (PLR) are inflammatory markers used for prediction of chronic complications of diabetes. Lower extremity arterial disease (LEAD) is one of chronic complications of type 2 diabetes mellitus (T2DM). The correlation between NLR, PLR, and lower extremity vascular lesions was investigated in subjects with T2DM to determine the best predictive marker for LEAD., Methods: Three hundred thirty-five patients with T2DM (199 males and 136 females; age 54.12 ± 14.07 years) were enrolled. Blood differential counts and anklebrachial index (ABI) were assessed. Patients were divided into the LEAD group (ABI ≤ 0.9, n = 236) and non-LEAD group (ABI > 0.9, n = 99), and NLR and PLR were compared between the two groups. The independent risk factors for LEAD were analyzed using a logistic regression model. Receiver operating characteristic (ROC) curve analysis was used to assess the optimal cutoff values of PLR and NLR for prediction of LEAD., Results: NLR and PLR in the LEAD group were significantly increased compared to non-LEAD group patients. Univariate analyses identified that NLR was positively correlated with age, glycosylated hemoglobin (HbA1c), triglycerides (TG), total cholesterol (TC), low-density lipoprotein (LDL), and 2-hours postprandial glucose levels. PLR was positively correlated with age, duration of T2DM, HbA1c, TG, TC, and LDL, but negatively correlated with diastolic blood pressure and fasting C-peptide levels. Binary logistic regression analysis identified that age, total number of white blood cells, PLR, and TC were significant and independent factors of diabetic LEAD. Moreover, ROC curve analysis showed that NLR and PLR were both predictive markers of LEAD in diabetes, and that the area under the PLR curve was larger than NLR., Conclusions: NLR and PLR are positively correlated with LEAD in diabetes. PLR was superior to NLR as a predictor of LEAD in diabetes.

The present study aimed to investigate the existence of immunoregulation‑associated long non‑coding (lnc)RNAs mediated by T lymphocytes in the wound surfaces of diabetic foot ulcers (DFUs). The wound skin tissues of patients receiving debridement for trauma or DFUs associated with infection were obtained. Dermatological histological changes were observed by pathological staining, and T lymphocyte subsets and inflammation‑associated cytokines were identified. Gene chip technology was used to screen for lncRNAs regulated by immune cells. The wound skin structure in the control group was revealed to be complete, and the inflammatory response was not marked. However, the wound skin structure in the ulcer group was disordered and exhibited a notable inflammatory response. Compared with the control group, expression levels of cluster of differentiation (CD)3 and CD8 in the wound surface tissues of the ulcer group were significantly increased, while the expression levels of interleukin (IL)‑1β, IL‑2, IL‑10, interferon‑γ and tumor necrosis factor‑α were significantly upregulated. A target regulatory association was identified between downregulated lncRNA‑ENST00000411554 and upregulated mitogen‑activated protein kinase (MAPK)1 in DFU tissues, and a negative correlation was detected between this RNA and protein. The present results suggested that an immune functional disorder of T lymphocytes may be closely associated with the development of DFUs. Furthermore, activation of the MAPK signal transduction pathway mediated by the lncRNA‑ENST00000411554/MAPK1 axis may affect the DFU immune regulatory imbalance.

A major burden of the healthcare system resides in providing proper medical treatment for all types of chronic wounds, which are usually treated with dressings to induce a faster regeneration. Hence, to reduce healing time and improve the patient's quality of life, it is extremely important to select the most appropriate constituent material for a specific wound dressing. A wide range of wound dressings exist but their mechanisms of action are poorly explored, especially concerning the immunomodulatory effects that occur from the interactions between immune cells and the biomaterial. Tissue-resident and monocyte-derived recruited macrophages are key regulators of wound repair. These phagocytic immune cells exert specific functions during the different stages of wound healing. The recognition of the substantial role of macrophages in the outcome of the wound healing process requires specific understanding of the immunomodulatory effects of commercially available or newly developed wound dressings. For a precise intervention, it is necessary to obtain more knowledge on macrophage polarization in different phases of wound healing in the presence of the dressings. The main purpose of this review is to collect clinical cases in which macrophage immunomodulation was taken into consideration as an indicator of the performances of novel or mainstream wound dressing materials, including those provided with antimicrobial properties.

Objectives: As a complication of diabetes mellitus (DM), one of the leading causes for death and disability for DM patients is diabetic foot ulcers (DFUs). Epithelial to mesenchymal transition (EMT) plays a critical role in wound healing of DFUs. miR-203 is specifically enriched in keratinocytes and has been shown to target interleukin 8 (IL-8), which acts as an activator for the EMT process. In this study, we explored the interaction between miR-203 and IL-8 in DFU rat models and human keratinocyte cells, underlying the mechanism of miR-203's function in DFUs progression., Methods: DFU rat models were used to test gene expression in DFU progression. Diabetic keratinocyte cell lines were used to validate in vitro. Wound healing and Transwell assays were applied to evaluate cell migration and invasion abilities. The EMT process was estimated by testing expression of E-cadherin, Vimentin and Slug. The interaction between miR-203 and IL-8 was determined by Luciferase assay., Results: Our results demonstrated that the wound-healing process had been slowed in DFUs, and the advanced glycation end products (AGEs) and the receptor for advanced glycation end products (RAGEs) in wound tissue were of a higher expression than those in normal rat. miR-203 was increased in skin tissues from DFU rat models, while IL-8 was decreased. Through knock-down of miR-203 in AGE-treated keratinocyte cells, it had been shown that the downregulation of miR-203 could promote cell proliferation and migration, and facilitate the EMT process. Meanwhile, Luciferase assay proved that miR-203 could directly target and inhibit IL-8. The repression of IL-8 could rescue the outcomes brought about by miR-203 inhibition., Conclusions: The upregulation of miR-203 in DFU tissues impaired wound healing by the repress EMT process. Specific knock-down of miR-203 could promote wound healing through the reactivation of its target gene IL-8 and the downstream IL-8/AKT pathway., (© 2019 S. Karger AG, Basel.)

Management of foot salvage therapy (FST) for diabetic foot infections (DFI) is challenging due to the absence of reliable diagnostics to identify the etiologic agent and prognostics to justify aggressive treatments. As Staphylococcus aureus is the most common pathogen associated with DFI, we aimed to develop a multiplex immunoassay of IgG in serum and medium enriched for newly synthesized anti- S. aureus antibodies (MENSA) generated from cultured peripheral blood mononuclear cells of DFI patients undergoing FST. Wound samples were collected from 26 DFI patients to identify the infecting bacterial species via 16S rRNA sequencing. Blood was obtained over 12 weeks of FST to assess anti- S. aureus IgG levels in sera and MENSA. The results showed that 17 out of 26 infections were polymicrobial and 12 were positive for S. aureus While antibody titers in serum and MENSA displayed similar diagnostic potentials to detect S. aureus infection, MENSA showed a 2-fold-greater signal-to-background ratio. Multivariate analyses revealed increases in predictive power of diagnosing S. aureus infections (area under the receiver operating characteristic curve [AUC] > 0.85) only when combining titers against different classes of antigens, suggesting cross-functional antigenic diversity. Anti- S. aureus IgG levels in MENSA decreased with successful FST and rose with reinfection. In contrast, IgG levels in serum remained unchanged throughout the 12-week FST. Collectively, these results demonstrate the applicability of serum and MENSA for diagnosis of S. aureus DFI with increased power by combining functionally distinct titers. We also found that tracking MENSA has prognostic potential to guide clinical decisions during FST., (Copyright © 2018 American Society for Microbiology.)

Objective: Diabetic foot ulcer (DFU), the major complication associated with diabetes mellitus, has been shown to precede amputation in up to 90% of cases. Recent data reveal that procalcitonin (PCT) is a valid marker for the diagnosis of bacterial infections compared with traditional markers like white blood cell count, C-reactive protein levels, and erythrocyte sedimentation rate in DFU patients. Furthermore, cytokines are proposed to act as modulators and mediators for the expression and release of PCT into the circulation. Hence, this preliminary study was conducted to evaluate the diagnostic accuracy of PCT compared with other traditional markers and to predict the association of PCT plasma levels with inflammatory cytokines and clinical parameters of incident diabetes among South Indian DFU subjects., Methods: There were 185 subjects with type 2 diabetes mellitus (T2DM) selected in this cross-sectional study, subdivided into three groups: group I, control/T2DM subjects free from DFU (n = 75; male, 43; female, 32); group II, T2DM subjects with noninfected DFU (n = 34; male, 19; female, 15); and group III, T2DM subjects with infected DFU (IDFU; n = 76; male, 46; female, 30). Patients with IDFU were further divided into three subgroups as per the Infectious Diseases Society of America-International Working Group on the Diabetic Foot classification criteria: grade 2 (n = 27), grade 3 (n = 38), and grade 4 (n = 11). Subjects with type 1 diabetes, gestational diabetes, pneumonia, sepsis, inflammatory bowel disease, meningitis, or hematologic diseases and those who underwent surgery in the past 2 to 3 weeks were excluded from this study. For investigation of clinical parameters, blood samples were drawn from all the study subjects; plasma samples were used for estimating PCT by the enzyme-linked immunosorbent assay method. The profiling of plasma cytokines was carried out using a multiplex bead-based assay. Data are presented as mean ± standard deviation for clinical and biochemical variables and as geometric mean with 95% confidence interval (CI) for cytokines. All analysis was done using the Statistical Package for the Social Sciences (version 20.0; IBM Corp, Armonk, NY); P < .05 was considered to be statistically significant., Results: We found PCT to be a valid diagnostic marker for IDFU with higher sensitivity and specificity than other traditional markers. For PCT, the area under the receiver operating characteristic curve was found to be high (0.99; 95% CI, 0.96-1.0), followed by C-reactive protein levels (0.78; 95% CI, 0.65-0.81), white blood cell count (0.76; 95% CI, 0.67-0.86), and erythrocyte sedimentation rate (0.74; 95% CI, 0.68-0.80) in IDFU subjects. We found the cutoff value of ≥0.5 ng/mL to have 54% sensitivity and 100% specificity for PCT with a positive predictive value of 100% and a negative predictive value of 12% for IDFU diagnosis. Moreover, PCT circulatory levels showed a positive correlation with helper T-cell subtype 1 cytokines, such as interferon γ (r = 0.21; P = .03) and interleukin 28A (r = 0.31; P = .003), and subtype 17 cytokines, such as interleukin 29/interferon λ1 (r = 0.20; P = .037)., Conclusions: PCT could be a valuable marker for diagnosis of T2DM patients with IDFU., (Copyright © 2017 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Inflammation is a critical phase in the healing of skin wounds. Excessive inflammation and inflammatory macrophages are known to cause impaired wound closure and outcome. This prompted us to test the role of IL-23 in IL-17 expression and in modulating wound inflammation and macrophage polarization. Full-thickness wounds (4 × 6 mm) were created on the dorsal surface of multiple genetically modified mouse models. Obese diabetic mouse wounds were treated with anti-IL-17A, anti-IL-23, or isotype-matched antibodies. We found IL-23- but not IL-12-deficient mice displayed significantly reduced IL-17 expression in wounds. This was rescued by delivery of recombinant IL-23. IL-23- and IL-17-deficient mice showed a significant increase in noninflammatory macrophages. Obese diabetic mice treated with anti-IL-17A and anti-IL-23p19 blocking antibodies had significantly improved wound reepithelialization. Similarly, IL-17 -/- obese mice had accelerated wound closure, resulting in reduced iNOS expression and inflammatory macrophages while maintaining prohealing CD206 and lymphatic vessel endothelial hyaluronic acid receptor 1 (LYVE1)-expressing macrophages. This study highlights the importance of the IL-17 pathway in wound closure offering new possibilities of therapeutic intervention in chronic wounds.-Lee, J., Rodero, M. P., Patel, J., Moi, D., Mazzieri, R., Khosrotehrani, K. Interleukin-23 regulates interleukin-17 expression in wounds, and its inhibition accelerates diabetic wound healing through the alteration of macrophage polarization.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief due to an author reporting that they were listed without their permission., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Type 2 diabetes mellitus (T2DM) is a growing health problem in Egypt. T2DM is recognized as chronic inflammatory disease with involvement of immune cells including B cells. We aimed to determine the frequency of antibody secreting B1a and B2 B cells in T2DM patients, their correlation with diabetes metabolic parameters and whether they play a role in diabetic foot infection (DFI) development. This study included 56 participants, recruited from Al-Zahraa hospital, Al-Azhar University, Egypt. Of these, 36 patients were diagnosed with T2DM, divided to two groups; (1) DM group (n=19) recently diagnosed, without foot complication; (2) DFI group (n=17); in addition to a Control group (n=20). The study assessed the frequency of circulating B1a (CD19+CD23-CD5+), and B2 (CD19+CD23+CD5-) cells by flow cytometry in diabetic patients. Comparison of the 3 studied groups revealed significant differences in frequency of studied total B cells (P=0.011), B1a (P < 0.001) and B2 subsets (P < 0.001). Comparison of B cell subsets between DFI, DM groups showed significant decrease in B1a in DFI group (P < 0.001). B1a cells % showed inverse correlation with HgA1c (r=-0.47, P < 0.001), LDL (r=-0.64, P < 0.001), and TG (r=-0.67, P < 0.001) but showed positive correlation with HDL (r=0.61, P < 0.001), while B2 cells showed opposite correlations. We concluded that imbalance of B cell subsets is seen in T2DM subjects. Beneficial role of B1a cells was spotted as they correlated inversely with glycemia and lipidemia in contrary to B2 cells. Decrease in B1a cells may predispose to DFI development., (Copyright© by the Egyptian Association of Immunologists.)

Foot ulceration is one of the most debilitating complications associated with diabetes, but its cause remains poorly understood. Several studies have been undertaken to understand healing kinetics or find possible therapies to enhance healing. However, few studies have been directed at understanding the immunological alterations that could influence wound healing in diabetes. In this study, we analysed the T-cell receptor (TCR) repertoire diversity in TCR-αβ + T cells. We also analysed the distribution and phenotype of T cells obtained from the peripheral blood of healthy controls and diabetic individuals with or without foot ulcers. Our results showed that diabetic individuals, especially those with foot ulcers, have a significantly lower naive T-cell number and a poorer TCR-Vβ repertoire diversity. We also showed that the reduced TCR-Vβ repertoire diversity in diabetic individuals was mainly owing to the accumulation of effector T cells, the major source of tumour necrosis factor-α production, which was even more pronounced in patients with acute foot ulceration. Moreover, the expression of several inflammatory chemokine receptors was significantly reduced in diabetic patients. In conclusion, effector T-cell accumulation and TCR repertoire diversity reduction appear to precede the development of foot ulcers. This finding may open new immunological therapeutic possibilities and provide a new prognostic tool in diabetic wound care.

Objective: The neutrophil-to-lymphocyte ratio (NLR) has been used as a surrogate marker of systemic inflammation. We sought to investigate the association between NLR and wound healing in diabetic wounds., Methods: The outcomes of 120 diabetic foot ulcers in 101 patients referred from August 2011 to December 2014 were examined retrospectively. Demographic, patient-specific, and wound-specific variables as well as NLR at baseline visit were assessed. Outcomes were classified as ulcer healing, minor amputation, major amputation, and chronic ulcer., Results: The subjects' mean age was 59.4 ± 13.0 years, and 67 (66%) were male. Final outcome was complete healing in 24 ulcers (20%), minor amputation in 58 (48%) and major amputation in 16 (13%), and 22 chronic ulcers (18%) at the last follow-up (median follow-up time, 6.8 months). In multivariate analysis, higher NLR (odds ratio, 13.61; P = .01) was associated with higher odds of nonhealing., Conclusions: NLR can predict odds of complete healing in diabetic foot ulcers independent of wound infection and other factors., (Copyright © 2016 Society for Vascular Surgery. All rights reserved.)

Chronic wounds and in particular diabetic foot ulcers (DFUs) are a growing clinical challenge, but the underlying molecular pathophysiological mechanisms are unclear. Recently, we reported reduced levels of the immunomodulating and antimicrobial S100A8/A9 in non-healing venous leg ulcers (VLUs), while another study found increased S100A8/A9 in DFUs. To clarify these apparently contradictory findings, we compared S100A8/A9 as well as an inducer, lipopolysaccharide (LPS) and selected innate immune response mediators in wound fluids from non-healing DFUs and VLUs with healing wounds. Wound fluids were collected from neuropathic DFUs (n = 6) and VLUs (n = 9) of median 2-year duration, and split-thickness skin graft donor site wounds (n = 10) by standardized method. None of the patients had ischaemic extremities or clinically infected wounds. LPS was determined by limulus amoebocyte lysate test, and S100A8/A9, granulocyte colony-stimulating factor (G-CSF), interleukin (IL)-10 and vascular endothelial growth factor (VEGF) by immunospecific quantitative assays. LPS levels were median 8.7 (interquartile range 5.4-21.2) ng/ml in DFUs compared with 121 (22-2000) ng/ml in VLUs. S100A8/A9 was higher (p = 0.020) in DFUs [718 (634-811) µg/ml] than in VLUs [303 (252-533) µg/ml]. Neither G-CSF nor IL-10 wound fluid levels differed significantly between the chronic wound groups. VEGF levels correlated with LPS (r = 0.758, p = 0.011, n = 10) and were higher (p = 0.024) in VLU wound fluids. LPS (p < 0.0001), S100A8/A9 (p = 0.005), G-CSF (p = 0.003), IL-10 (p = 0.003) and VEGF (p = 0.005) were increased in chronic wound fluids combined compared with the sterile donor site wound fluids. The protein alterations in the wounds were not reflected in the patients' sera. Low S100A8/A9 levels may contribute to poor wound healing in colonized chronic wounds with striking difference between DFUs and VLUs.

Upon activation, neutrophils undergo histone citrullination by protein arginine deiminase (PAD)4, exocytosis of chromatin and enzymes as neutrophil extracellular traps (NETs), and death. In diabetes, neutrophils are primed to release NETs and die by NETosis. Although this process is a defense against infection, NETosis can damage tissue. Therefore, we examined the effect of NETosis on the healing of diabetic foot ulcers (DFUs). Using proteomics, we found that NET components were enriched in nonhealing human DFUs. In an independent validation cohort, a high concentration of neutrophil elastase in the wound was associated with infection and a subsequent worsening of the ulcer. NET components (elastase, histones, neutrophil gelatinase-associated lipocalin, and proteinase-3) were elevated in the blood of patients with DFUs. Circulating elastase and proteinase-3 were associated with infection, and serum elastase predicted delayed healing. Neutrophils isolated from the blood of DFU patients showed an increased spontaneous NETosis but an impaired inducible NETosis. In mice, skin PAD4 activity was increased by diabetes, and FACS detection of histone citrullination, together with intravital microscopy, showed that NETosis occurred in the bed of excisional wounds. PAD4 inhibition by Cl-amidine reduced NETting neutrophils and rescued wound healing in diabetic mice. Cumulatively, these data suggest that NETosis delays DFU healing., (© 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Objective: The objective of this work is to describe the microbiology of diabetic foot infections (DFIs)., Materials and Methods: The authors included all samples of infected diabetic foot ulcers between January 2009 and June 2014 at the Mohammed Vth Military Teaching Hospital of Rabat, Morocco., Results: The researchers collected 199 samples corresponding to 157 patients. The mean age of the patients was 59 years ± 12 years. Of the collected samples, deep samples represented 41% and swab samples 59%. Direct examination indicated anaerobic infection in 32% of the cases. There were 307 bacteria isolates from both deep and swab samples. There was no statistically significant association between the sampling method and isolate species (P = 0.237). Enterobacteriaceae, Staphylococcus aureus, Streptococcus sp, nonfermenting gram-negative bacilli (NFGNB), and Enterococcus sp represented 31.8%, 12.6 %, 12.3%, 11.7%, and 8.7% of the isolates, respectively. Methicillin-resistant S. aureus represented 4.7% of S. aureus isolates. Enterobacteriaceae and NFGNB-producing extended spectrum β-lactamases represented 14.1% and 5.1%, respectively, with isolates producing carabapenemase representing 3.8% and 38.5%. Piperacillin-tazobactam, imipenem, and ciprofloxacin resistance concerned 7.5%, 4.7%, and 25.5%, respectively, of isolated Enterobacteriaceae, and 35.9%, 30.7%, and 35.9% of NFGNB. Low susceptibility to β-lactams was found in 4.9% of Streptococcus sp isolates and 4.9% of Streptococcus sp isolates were resistant to moxifloxacin., Conclusion: Gram-negative bacilli are responsible for 43% of DFIs, and multidrug-resistant GNB is a challenging issue in DFI management. The sampling method doesn't seem to impact the bacteriological profile; however, this finding must be confirmed with further study.

Background: Type 2 diabetes mellitus (T2DM) is characterized by persistent hyperglycemia which causes a chain of abrupt biochemical and physiological changes. Immune dys-regulation is the hallmark of T2DM that could contribute to prolonged inflammation causing transformation of wounds into non-healing chronic ulcers. Toll like receptor -9 (TLR9) is a major receptor involved in innate immune regulation. TLR9 activation induces release of pro-inflammatory molecules like S100A8 and interleukin-8 (IL-8) by myeloid cells causing migration of myeloid cells to the site of inflammation. We hypothesized that pro-inflammatory S100A8 and IL-8 proteins could cause persistent inflammation in chronic wounds like diabetic foot ulcer (DFU) and may contribute to impaired wound healing in T2DM patients., Materials and Methods: Expression of TLR9 and its downstream effector molecules S100A8, and IL-8 were analyzed in chronic diabetic wound and non-diabetic control wound tissue samples by semiquantitative reverse transcriptase - polymerase chain reaction (RT-PCR), quantitative RT-PCR, western blot and immunofluorescence. CD11b(+)CD33(+) myeloid cells were analyzed by flow cytometry., Results: TLR9 message and protein were higher in diabetic wounds compared to control wounds (p=0.03, t=2.21 for TLR9 mRNA; p=<0.001, t=4.21 for TLR9 protein). TLR9 down-stream effector molecules S100A8 and IL-8 were also increased in diabetic wounds (p=0.003, t=3.1 for S100A8 mRNA; p=0.04, t=2.04 for IL-8). CD11b(+) CD33(+) myeloid cells were decreased in T2DM as compared to non-diabetic controls (p=0.001, t=3.6). DFU subjects had higher levels of CD11b(+) CD33(+) myeloid cells as compared to non-DFU T2DM control (p=0.003, t=2.8). Infection in the wound microenvironment could be the cause of increase in CD11b(+)CD33(+) myeloid cells in DFU (p=0.03, t=2.5)., Conclusion: The up-regulation of myeloid cell-derived pro-inflammatory molecules S100A8 and IL-8 in combination with lower levels of CD11b(+) CD33(+) myeloid cells may cause the impairment of wound healing in T2DM subjects leading to chronic ulcers., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The aim of our study was to analyse immune abnormalities in patients with chronic infected diabetic foot ulcers (DFUs) especially those infected by resistant microorganisms. Methods . 68 patients treated in our foot clinic for infected chronic DFUs with 34 matched diabetic controls were studied. Patients with infected DFUs were subdivided into two subgroups according to the antibiotic sensitivity of causal pathogen: subgroup S infected by sensitive ( n = 50) and subgroup R by resistant pathogens ( n = 18). Selected immunological markers were compared between the study groups and subgroups. Results . Patients with infected chronic DFUs had, in comparison with diabetic controls, significantly reduced percentages ( p < 0.01) and total numbers of lymphocytes ( p < 0.001) involving B lymphocytes ( p < 0.01), CD4+ ( p < 0.01), and CD8+ T cells ( p < 0.01) and their naive and memory effector cells. Higher levels of IgG ( p < 0.05) including IgG1 ( p < 0.001) and IgG3 ( p < 0.05) were found in patients with DFUs compared to diabetic controls. Serum levels of immunoglobulin subclasses IgG2 and IgG3 correlated negatively with metabolic control ( p < 0.05). A trend towards an increased frequency of IgG2 deficiency was found in patients with DFUs compared to diabetic controls (22% versus 15%; NS). Subgroup R revealed lower levels of immunoglobulins, especially of IgG4 ( p < 0.01) in contrast to patients infected by sensitive bacteria. The innate immunity did not differ significantly between the study groups. Conclusion . Our study showed changes mainly in the adaptive immune system represented by low levels of lymphocyte subpopulations and their memory effector cells, and also changes in humoral immunity in patients with DFUs, even those infected by resistant pathogens, in comparison with diabetic controls., Competing Interests: All participated authors of this study have no conflict of interests.

The analysis of changes in the specific immunity in patients with purulent—necrotic complications of diabetic foot syndrome (DFS) and sepsis was presented. Complicated DFS was diagnosed in 436 patients, of whom 29 (6.6%) — weighed down by sepsis. The concentration of the major classes of immunoglobulins IgA, IgM, IgG were determined by enzyme—linked immunosorbent assay (ELISA). To evaluate the immune state was determined lymphocyte subpopulations (CD4+, CD3+, CD8+, CD25+) with monoclonal antibodies to antigens use by flow cytometry. It was found that in patients with DFS indicators specific cellular immunity were in a state of suppression, which deepened at complication of sepsis. Based on the derived formula with DFS marked disorders of the immune system I degree, in sepsis — II degree. Thus, the cellular and humoral immune system in patients with both DFS and sepsis qualified as a general immune depression.

Aim: Persistent hyperglycemic microenvironment in type 2 diabetes mellitus (T2DM) leads to the development of secondary complications like wound healing impairment. Proper co-ordination of innate immune system plays an integral role in wound healing. Toll like receptors (TLRs) are prominent contributors for the induction of the innate immune and inflammation response. TLR2 is an important extracellular member in mammalian TLR family and has been shown to be a potent player in the wound healing mechanism., Methods: Expressional status of TLR2 was seen in wounds of T2DM cases with respect to the severity of wounds in 110 human lower extremity wounds. The methylation status of TLR2 promoter was also examined., Results: Although TLR2 transcripts were downregulated in T2DM wounds compared to control, their levels tend to increase with the severity of T2DM wounds. The methylation status of TLR2 gene promoter was not significantly different among different grades of wounds in T2DM subjects. The CpG sites investigated were totally or partially methylated in majority of DFU cases., Conclusion: TLR2 down regulation in wounds of T2DM patients compared to non diabetic patients may lead to development of non healing chronic ulcers in them., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Despite extensive research, the pathophysiology of Charcot foot is still not fully understood. In the case of late diagnosis and insufficient therapy, severe deformity may occur., Objectives: Different theories on the pathophysiology of the Charcot foot are presented and discussed., Materials and Methods: This article presents different theories on the pathophysiology of the Charcot foot and presents studies on the cellular, immunological, and bone metabolism level., Results: One theory is based on repetitive microtraumas in the sensoric deficient foot which lead to development of Charcot foot. Another theory is based on pathologic neurovascular innervation leading to morphologic changes. Probably both mechanisms are responsible in combination as etiological factors inducing neuropathy, Conclusion: A combination of mechanical, vascular, and biologic factors induce the neuropathic foot and result in severe deformity if diagnosis is too late.

Aims: Diabetic foot osteomyelitis is an important risk factor of lower limb amputation. Antibiotic therapy is often effective in preventing surgery. However, the duration of antibiotic therapy is difficult to define in the absence of a marker to diagnose osteomyelitis remission at the end of the treatment. In this study, we assessed the diagnostic performance of white blood cell SPECT/CT imaging for evaluating osteomyelitis remission., Patients and Methods: Twenty-nine out of 42 episodes of diabetic foot osteomyelitis seen between December 2009 and April 2012 had radiographs, a three-phase bone scintigraphy and a white blood cell SPECT/CT at the end of antibiotic therapy. They were treated with antibiotics alone and considered in clinical remission. White blood cell SPECT/CT results were considered positive when abnormal uptake in the osteomyelitis location was identified. Osteomyelitis remission was defined by the absence of an osteomyelitis relapse after 12 months' follow-up., Results: A negative white blood cell SPECT/CT was seen for 22 episodes of osteomyelitis. All of them were in remission. A positive white blood cell SPECT/CT was observed for seven episodes. A relapse occurred in five episodes (71.5%) after a median duration of 4 months (2-7 months). Sensitivity, specificity, positive predictive value and predictive negative value in predicting osteomyelitis relapse after the discontinuation of antibiotic treatment were, respectively, for radiographs 80%, 33%, 20% and 89%; for three-phase bone scintigraphy 100%, 12.5%, 15.5% and 100%; and for the white blood cell SPECT/CT 100%, 91.5%, 71.5% and 100%., Conclusion: Negative uptake on white blood cell SPECT/CT is a good marker for diagnosis of diabetic foot osteomyelitis remission and might be very useful in guiding antibiotic therapy., (© 2014 The Authors. Diabetic Medicine © 2014 Diabetes UK.)

This paper describes important aspects of the diabetic foot which the vascular surgeon needs to understand to efficiently manage the diabetic foot. Firstly, it emphasises the three main pathologies which come together in the diabetic foot, namely neuropathy, ischemia and immunopathy, the latter predisposing to infection. As a result of neuropathy, the signs and symptoms of tissue breakdown, infection and ischemia may be minimal. Nevertheless the pathology emanating from such clinical events proceeds rapidly without the body being aware of it and the end stage of tissue death and necrosis is quickly reached. It is important to have a prompt system of evaluation and intervention to prevent the rapid progression to necrosis. Thus, secondly, the paper describes a simple rapid assessment of the diabetic foot, which comprises inspection, palpation and sensory testing and leads on to a modern classification and staging of the diabetic foot. This classifies six subdivisions of the diabetic foot: foot with neuropathic ulceration, Charcot foot, neuroischemic foot, critically ischemic foot, acutely ischemic foot and renal ischemic foot and six stages in the natural history of each of these subdivisions: normal foot, high risk foot, ulcerated foot, infected foot, necrotic foot and unsalvageable foot. Thirdly, it describes modern management of the diabetic foot, emphazising wound care and revascularization within the context of a multidisciplinary care team that provides integrated care focused in a diabetic foot clinic, to which patients with diabetes should have easy and rapid access. Members of the team include podiatrist, nurse, orthotist, physician, radiologist and surgeons.

Context: Lipoic acid (LA) and hyperbaric oxygenation therapy (HBOT) improve chronic wound healing., Objective: We compared the effects of LA or its enantiomer R-(+)-lipoic acid (RLA) on wound healing., Materials and Methods: Groups LA + HBOT (L), RLA + HBOT (R) and placebo + HBOT (P). Lesion areas measured before treatment and on 20th and 40th day. The biopsies and plasma were harvested before treatment and on 7th and 14th (measurements of VEGF, vascular endothelial growth factor; EGF, epidermal growth factor, TNF-α and IL-6)., Results: Ulcers improved more on RLA. In both L and R groups, EGF and VEFG increased in time. RLA decreased IL-6 on T7 and T14, which did not happen with LA. TNF-α levels decreased on T14 in both LA and RLA., Discussion: The improved wound healing is associated with increased EGF and VEGF and reduced plasma TNF-α and IL-6., Conclusion: RLA may be more effective than LA in improving chronic wound healing in patients undergoing HBO therapy.

Background: Diabetic foot (DF) can be defined as an infection and/or an ulceration with or without destruction of deep tissues associated with neurological abnormalities and varying degrees of peripheral vascular disease of the lower limb in patients with diabetes. Both neuropathy and vascular disease, along with the well-known impairment of immune function in patients with diabetes, contribute to polymicrobial foot infections, which further aggravate the already severe clinical manifestations of diabetes., Discussion: The immunocompromised district (ICD) is a novel pathogenic concept referring to a site in which there is an obstacle to the normal trafficking of immunocompetent cells through lymphatic channels, and/or interference with the signals that neuropeptides and neurotransmitters, released by peripheral nerves, send to cell membrane receptors of immunocompetent cells. These loci minoris resistentiae have the propensity to develop a secondary disease, which may occur after an extremely variable length of time., Conclusions: In this work, we provide an overview of etiopathogenetic mechanisms of DF and propose a unifying view of this topic based on the concept of the ICD., (© 2013 The International Society of Dermatology.)

Objective: CTX0E03 (CTX) is a clinical-grade human neural stem cell (hNSC) line that promotes angiogenesis and neurogenesis in a preclinical model of stroke and is now under clinical development for stroke disability. We evaluated the therapeutic activity of intramuscular CTX hNSC implantation in murine models of hindlimb ischemia for potential translation to clinical studies in critical limb ischemia., Approach and Results: Immunodeficient (CD-1 Fox(nu/nu)) mice acutely treated with hNSCs had overall significantly increased rates and magnitude of recovery of surface blood flow (laser Doppler), limb muscle perfusion (fluorescent microspheres, P<0.001), and capillary and small arteriole densities in the ischemic limb (fluorescence immunohistochemistry, both P<0.001) when compared with the vehicle-treated group. Hemodynamic and anatomic improvements were dose related and optimal at a minimum dose of 3×10(5) cells. Dose-dependent improvements in blood flow and increased vessel densities by hNSC administration early after ischemia were confirmed in immunocompetent CD-1 and streptozotocin-induced diabetic mice, together with marked reductions in the incidence of necrotic toes (P<0.05). Delayed administration of hNSCs, 7 days after occlusion, produced restorative effects when comparable with acute treatment of 35 days after hindlimb ischemia. Histological studies in hindlimb ischemia immunocompetent mice for the first 7 days after treatment revealed short-term hNSC survival, transient elevation of early host muscle inflammatory, and angiogenic responses and acceleration of myogenesis., Conclusions: hNSC therapy represents a promising treatment option for critical limb ischemia.

Within chronic wounds, the relationship between the clinical diagnosis of infection and bacterial/immuno-inflammatory responses is imprecise. This study prospectively examined the interrelationship between clinical, microbiological, and proinflammatory biomarker levels between chronic venous leg ulcers (CVLUs) and diabetic foot ulcers (DFUs). Wound swabs and fluids were collected from CVLUs (n = 18) and DFUs (n = 15) and diagnosed clinically as noninfected or infected; and qualitative/quantitative microbiology was performed. CVLU and DFU fluids were also analyzed for cytokine, growth factor, receptor, proteinase/proteinase inhibitor; and oxidative stress biomarker (protein carbonyl, malondialdehyde, and antioxidant capacity) levels. While no correlations existed between clinical diagnosis, microbiology, or biomarker profiles, increasing bacterial bioburden (≥10(7) colony-forming unit/mL) was associated with significant alterations in cytokine, growth factor, and receptor levels. These responses contrasted between ulcer type, with elevated and decreased cytokine, growth factor, and receptor levels in CVLUs and DFUs with increasing bioburden, respectively. Despite proteinase biomarkers exhibiting few differences between CVLUs and DFUs, significant elevations in antioxidant capacities correlated with increased bioburden in CVLU fluids, but not in DFUs. Furthermore, oxidative stress biomarker levels were significantly elevated in all DFU fluids compared with CVLUs. This study provides further insight into the contrasting disease-specific host responses to bacterial challenge within infected CVLUs and DFUs., (© 2013 by the Wound Healing Society.)

Background: The different grading systems for diabetic foot disease pose a challenge in clinical decision making because each system fails to accurately reflect the individual course of disease progression. This study attempts to identify laboratory measurements for classifying diabetic foot disease to guide clinical treatment., Methods: The sera of 111 clinically graded diabetic foot patients were measured for several laboratory parameters including serum amyloid A (SAA), C-reactive protein (CRP), and apo A-I. By using the molar sum of CRP and SAA and then dividing by the molarity of apo A-I, an acute phase index was introduced to assess the inflammatory status of the patients., Results: Based on a newly defined acute phase index (API), diabetic foot patients were classified into 3 distinct groups that provide a diagnostic tool complementing the established Texas grading system for clinical decision making., Conclusions: The integration of the serum concentrations of SAA, CRP and apo A-I into an acute phase index (API) offers an opportunity to triage diabetic foot patients who may benefit from personalized medicine.

Treatment of chronic conditions like diabetic foot ulcer (DFU) is challenging due to increased susceptibility for infection and delayed wound healing. Complexity of existing therapy, adverse effects and microbial resistance emphasizes the need of an alternative approach for the management of DFU. The increasing body of evidence associated with probiotic application in diverse disease states merits its use in wound healing and infection too. Different probiotic strains have shown their efficacy in various infections like gut infections, oral infections and urogenital infections. Experimental studies have demonstrated probiotics' ability for gastric ulcer healing. Underlying mechanism of the above therapeutic effects of probiotics involves modulation of local and systemic immunity. The hypothesis is based on the concept that mechanism of anti-infective and ulcer healing action of probiotics will be similar in peripheral wounds and ulcers as on any other part of the body. This paper focuses on the hypothesis that topical applications/formulation of probiotics may be effective for the treatment of diabetic foot ulcers., (Copyright © 2013 Elsevier Ltd. All rights reserved.)