Identifying Key Drivers in the Pathogenesis of Martorell Hypertensive Ischaemic Leg Ulcer: A Comparative Analysis with Chronic Venous Leg Ulcer.

Martorell hypertensive ischaemic leg ulcer (Martorell HYTILU) is a rare but significant cause of distal leg ulcers. Although hypertension and diabetes are known factors in its development, the precise pathogenesis of Martorell HYTILU remains elusive. To reach a better understanding of Martorell HYTILU, transcriptomic analysis was conducted through RNA sequencing and immunohistochemical comparison of Martorell HYTILU (n = 17) with chronic venous ulcers (n = 4) and healthy skin (n = 4). Gene expression analysis showed a marked activation of immune-related pathways in both Martorell HYTILU and chronic venous ulcers compared with healthy skin. Notably, neutrophil activity was substantially higher in Martorell HYTILU. While pathway analysis revealed a mild downregulation of several immune pathways in Martorell HYTILU compared with chronic venous ulcers, keratinization, cornification, and epidermis development were significantly upregulated in Martorell HYTILU. Additionally, STAC2, a gene encoding for a protein promoting the expression of the calcium channel Cav1.1, was significantly upregulated in Martorell HYTILU and was detected perivascularly in situ (Martorell HYTILU n = 24; chronic venous ulcers n = 9, healthy skin n = 11). The high expression of STAC2 in Martorell HYTILU suggests that increased calcium influx plays an important role in the pathogenesis of the disease. Consequently, calcium channel antagonists could be a promising treatment avenue for Martorell HYTILU.

NLRP3 activation induced by neutrophil extracellular traps sustains inflammatory response in the diabetic wound.

Persistent inflammatory response in the diabetic wound impairs the healing process, resulting in significant morbidity and mortality. Mounting evidence indicate that the activation of Nod-like receptor protein (NLRP) 3 inflammasome in macrophages (MΦ) contributes to the sustained inflammatory response and impaired wound healing associated with diabetes. However, the main trigger of NLRP3 inflammasome in the wounds is not known. Neutrophils, as sentinels of the innate immune system and key stimulators of MΦ, are immune cells that play the main role in the early phase of healing. Neutrophils release extracellular traps (NETs) as defense against pathogens. On the other hand, NETs induce tissue damage. NETs have been detected in the diabetic wound and implicated in the impaired healing process, but the mechanism of NETs suspend wound healing and its role in fostering inflammatory dysregulation are elusive. Here, we report that NLRP3 and NETs production are elevated in human and rat diabetic wounds. NETs overproduced in the diabetic wounds triggered NLRP3 inflammasome activation and IL-1ß release in MΦ. Furthermore, NETs up-regulated NLRP3 and pro-IL-1ß levels via the TLR-4/TLR-9/NF-κB signaling pathway. They also elicited the generation of reactive oxygen species, which facilitated the association between NLRP3 and thioredoxin-interacting protein, and activated the NLRP3 inflammasome. In addition, NET digestion by DNase I alleviated the activation of NLRP3 inflammasome, regulated the immune cell infiltration, and accelerated wound healing in diabetic rat model. These findings illustrate a new mechanism by which NETs contribute to the activation of NLRP3 inflammasome and sustained inflammatory response in the diabetic wound.

Rapidly progressing ulcer and a urine drainage bag.

Primary cutaneous mucormycosis is an opportunistic fungal infection caused by the order Mucorales, most frequently by the Rhizopus species. Both systemic factors, such as diabetes mellitus or malignancies and local factors disrupting the skin barrier are implicated in development of this entity. The initial manifestation is a red-to-black papule rapidly progressing to a necrotic and painful ulcer. Diagnosis is obtained by identification of fungal forms in a skin biopsy, typically showing branching and non-septate hyphae. The clinical course is highly variable and depends mostly on the fungal invasion of deep tissues. However, an early diagnosis is essential for implementation of prompt and optimal treatment, based upon antifungal therapy and aggressive surgical debridement.

Sickle Cell Anemia and Comorbid Leg Ulcer Treated With Curative Peripheral Blood Stem Cell Transplantation.

Allogeneic bone marrow transplantation or peripheral blood stem cell transplantation (PBSCT) are the only curative therapies for patients with sickle cell disease (SCD). Once the patients have successfully undergone transplantation and engrafted, the hallmark of hemolytic anemia resolves, and normal hemoglobin levels are achieved. Some transplant protocols exclude patients with open wounds, including leg ulcers, because of infection risks associated with transplantation and long-term immunosuppression required to prevent graft-versus-host disease. Recalcitrant and recurrent leg ulcers are a serious complication of SCD and a determinant of morbidity. Here, we report the case of a 37-year-old man with sickle cell anemia and a chronic leg ulcer, who underwent PBSCT, engrafted successfully, and then had complete healing of his ulcer 16 months posttransplant.

Extracorporal Shock Waves Activate Migration, Proliferation and Inflammatory Pathways in Fibroblasts and Keratinocytes, and Improve Wound Healing in an Open-Label, Single-Arm Study in Patients with Therapy-Refractory Chronic Leg Ulcers.

BACKGROUND/AIMS: Chronic leg ulcers (CLUs) are globally a major cause of morbidity and mortality with increasing prevalence. Their treatment is highly challenging, and many conservative, surgical or advanced therapies have been suggested, but with little overall efficacy. Since the 1980s extracorporal shock wave therapy (ESWT) has gained interest as treatment for specific indications. Here, we report that patients with CLU showed wound healing after ESWT and investigated the underlying molecular mechanisms. METHODS: We performed cell proliferation and migration assays, FACS- and Western blot analyses, RT-PCR, and Affymetrix gene expression analyses on human keratinocytes and fibroblasts, and a tube formation assay on human microvascular endothelial cells to assess the impact of shock waves in vitro. In vivo, chronic therapy-refractory leg ulcers were treated with ESWT, and wound healing was assessed. RESULTS: Upon ESWT, we observed morphological changes and increased cell migration of keratinocytes. Cell-cycle regulatory genes were upregulated, and proliferation induced in fibroblasts. This was accompanied by secretion of pro-inflammatory cytokines from keratinocytes, which are known to drive wound healing, and a pro-angiogenic activity of endothelial cells. These observations were transferred "from bench to bedside", and 60 consecutive patients with 75 CLUs with different pathophysiologies (e.g. venous, mixed arterial-venous, arterial) were treated with ESWT. In this setting, 41% of ESWT-treated CLUs showed complete healing, 16% significant improvement, 35% improvement, and 8% of the ulcers did not respond to ESWT. The induction of healing was independent of patient age, duration or size of the ulcer, and the underlying pathophysiology. CONCLUSIONS: The efficacy of ESWT needs to be confirmed in controlled trials to implement ESWT as an adjunct to standard therapy or as a stand-alone treatment. Our results suggest that EWST may advance the treatment of chronic, therapy-refractory ulcers.

Evaluation of CD4(+)CD25(+)FoxP3(+) T cell populations, IL-10 production, and their correlation with clinical and biochemical parameters in sickle cell anemia patients with leg ulcers.

Leg ulcers (LUs) are a debilitating complication of sickle cell anemia (SCA), with inflammation known to play a crucial role in their pathogenesis. Many studies have described the roles of T helper type 1 (Th1) and Th2 pathways in SCA; however, defects in anti-inflammatory responses are poorly understood. We evaluated interleukin (IL)-10 levels in serum and peripheral blood mononuclear cells (PBMCs) in SCA patients with leg ulcers (SCALU) and without leg ulcers (SCAWH) in addition to CD4(+) CD25(+)FoxP3(+) T cell populations and their its IL-10 expression. In stimulated and unstimulated PBMC cultures, SCALU patients produced higher levels of IL-10 than those in the SCAWH group. Higher levels of IL-10 in SCALU patients correlated with a history of osteonecrosis in stimulated and unstimulated cultures when compared with those in SCAWH. Immunophenotyping revealed that SCALU patients had a higher proportion of CD4(+)CD25(+)FoxP3(+), Tr1 and CD4(+)CD25(+)FoxP3(+)IL-10(+) T cells than other groups. Our findings revealed that IL-10 levels were increased in unstimulated cells from the SCALU group, and that this group also presented with a predominant CD4(+) CD25(+)FoxP3(+) cell population despite many of those cells being IL-10 negative.

Evaluation of Th17 related cytokines associated with clinical and laboratorial parameters in sickle cell anemia patients with leg ulcers.

Leg ulcers (LUs) represent one of the main causes of morbidity in sickle cell anemia (SCA). This manifestation has been related to hemolysis, infections predisposition and inflammation that leads cytokines secretion. In this context, our study aimed to evaluate Th17 related cytokines (IL-6, IL-17A, IL-22 and IL-23) in serum and peripheral mononuclear cells culture supernatants with and without lymphoproliferative stimulation (anti-human CD3 and anti-human CD28). The cytokines levels were also correlated to clinical, hematological and biochemical parameters in SCA patients with and without LUs history (SCALU and SCAWH) as well as in healthy controls. In SCALU patients, high levels of IL-17A were associated with absence of acute chest syndrome (ACS, p=0.0328). The other clinical parameters analyzed (osteonecrosis, stroke, priapism, splenectomy and blood transfusions history) were not significantly related with other cytokine levels. In SCALU patients was also observed that IL-17A increased levels were associated with high levels of LDH (p=0.0130), the same association pattern was found for IL-6 (0.0160) and IL-22 (p=0.0165) in the SCALU group. Interestingly, we did not find statistical correlations with these parameters in SCAWH group. The other hematological parameters (hemoglobin, leucocyte and reticulocyte count) and indirect bilirrubin did not show any correlation with analyzed cytokines in both groups. So, for the first time, we show that IL-17A present in SCALU patients may exert a preventive role in the ACS development. Furthermore, IL-6, IL-17A and IL-22 accompanied the LDH levels only in SCALU patients suggesting to serve as additional markers of hemolysis or to be related with immunity response against extracellular pathogens.

Livedoid vasculopathy: clinical features and treatment in 24 Chinese patients.

Livedo vasculopathy (LV) is a chronic cutaneous disorder characterised by recurrent, painful ulceration ending in stellate scars. We have conducted a retrospective study of clinical features and treatment response of LV in 24 Chinese patients. LV occurred more frequently in women (male:female ratio 1:3). The peak age at onset of disease ranged from 14 to 20 years, younger than previously published data. 87.5% of the patients (21/24) showed significant summer exacerbation with ulcer formation. Out of 24 patients tested, 14 (58.3%) had positive antiphospholipid antibodies. Ten out of 14 patients (71.4%) were tested to be hypersensitive to multivalent insect antigens. Combinative anti-inflammatory therapy with steroids, tetracycline and Tripterygium glycosides plus antiplatelet/profibrinolytic drugs promoted quick healing of ulcer and reduce recurrence. The younger age of disease presentation and significant summer exacerbation are 2 novel clinical features observed in this study. These findings suggest that apart from procoagulation other risk factors may contribute significantly to the pathogenesis of LV. Although antiplatelet/profibrinolytic drugs are deemed as a first line therapy for LV, anti-inflammatory medications such as steroids, tetracycline and Tripterygium glycosides, from our experiences, are indispensable, especially for acute, ulcerative stage of disease.

Clinical and immunohistopathological aspects of venous ulcers treatment by Low-Intensity Pulsed Ultrasound (LIPUS).

The immunological mechanisms that are triggered by Low-Intensity Pulsed Ultrasound (LIPUS) in wound healing are unknown. In the present study, experimental groups were used to assess the treatment of chronic venous ulcers with 30mW/cm(2) SATA peripheral LIPUS three times per week compared to a daily treatment of 1% silver sulfadiazine (SDZ). The ulcers of the SDZ group (n=7) (G1) and LIPUS group (n=9) (G2) were photographed five times three months, and the images were analyzed using ImageJ software to quantify the total area (S), fibrin/sphacel area (yellow) and granulation area (red). The healing process was evaluated by the wound healing rate (WHR), granulation tissue rate (GTR) and fibrin/sphacel tissue rate (FTR). The ulcers were biopsied on days 1 and 45 and stained for collagen fiber quantification (picrosirius) and CD68(+) protein and VEGF (vascular endothelial growth factor) expression using HRP-streptavidin (horseradish peroxidase-streptavidin). On day 90, G2 had a mean 41% decrease in the ulcer area, while no decrease was observed in G1 (p<0.05). An increased tendency toward positive labeling of collagen fibers and VEGF (p>0.05) was observed in G2 compared to G1, and the number of CD68(+) cells was greater in G2 than in G1 (p<0.05). LIPUS presents superior activity compared to SDZ in stimulating the inflammatory and proliferative (angiogenesis and collagenesis, respectively) phases of chronic venous wound healing.

Prevalence of immune disease in patients with wounds presenting to a tertiary wound healing centre.

Chronic leg ulcers are a significant cause of morbidity and mortality and account for considerable healthcare and socioeconomic costs. Leg ulcers are a recognised complication of immune disease, and the purpose of this study was to establish the prevalence of immune disease in a cohort of patients with chronic wounds, and to compare wound outcomes in the subjects with and without immune disease. Retrospective chart review was completed on consecutive patients scheduled with the plastic surgeon in the Georgetown University Center for Wound Healing between 1 January 2009 and 31 March 2009. Of the 520 patients scheduled for appointments, 340 were eligible for inclusion. The prevalence of immune disease was higher than expected with 78 of 340 patients (23%) having associated immune disease. At presentation, wounds in patients with immune disease had a significantly larger mean surface area [33·4 cm(2) (69·05) compared to 22·5 cm(2) (63·65), P = 0·02]. Split thickness skin graft (STSG) and bioengineered alternative tissue (BAT) graft data was available on 177 grafts from 55 subjects. There was a significantly lower response rate to STSG in subjects with immune disease (50% compared to 97%, P = 0·0002), but response rates to BAT were not different. The association between immune diseases and chronic wounds may provide unique insights into pathways of wound healing, and warrants further study.

Neonatal lupus erythematosus in identical twins, showing transient bullous lesions.

One of identical twin girls was born with ulcers on her leg, and shortly after birth developed a flaccid blister on the leg. Subepidermal blister with vacuolar degeneration of basal cell layer and the heavy infiltration of mononuclear cells in the upper dermis were observed in the blister lesion. She also had generalized livedo. Her identical twin sister did not exhibit ulcers or blisters, but was born with milia on her limbs. Their mother was found to have lupus erythematosus with positive anti-Ro/SSA antibodies and developed Sjögren syndrome. We emphasize neonatal blistering and congenital milia unique manifestations of neonatal lupus erythematosus.

Contact sensitization in the elderly.

Contact dermatitis from irritant and allergic sources is the reason for 6% to 10% of all dermatologic visits with considerable morbidity and economic impact. Allergic contact dermatitis is a T-cell-mediated inflammatory reaction and develops in predisposed individuals as a consequence of environmental exposure to allergens. Aging is correlated with the rate and type of contact sensitization because of "immunosenescence." The number of old people is growing around the world. This contribution reviews the main findings from published epidemiologic studies on contact allergy in elderly populations. In all examined studies, patch testing was performed in patients with cutaneous manifestations possibly related to contact dermatitis; the prevalence of contact dermatitis in the elderly was from 33% to 64%. Establishing the most frequent allergens responsible for allergic contact dermatitis in the elderly is a hard task. The commonest allergens reported were nickel sulfate, fragrance mix, diamino diphenylmethane, lanolin alcohols, paraben mix, Euxyl K400, quinoline mix, and balsam of Peru. We emphasize that allergens surveillance is needed to realize an "elderly series" for having a useful adjunct to contact allergy that may help the treatment of each patient.

Interleukin-8 production by polymorphonuclear leukocytes from patients with chronic infected leg ulcers treated with Lactobacillus plantarum.

Bacterial infection impairs the healing process, promoting the chronicity of inflammation and wounds. Because antibiotics fail to eradicate bacteria, especially in biofilm form, new therapeutic modalities may be required. In the present study, the effectiveness of bacteriotherapy with Lactobacillus plantarum on infected chronic venous ulcers was investigated and its effects on interleukin (IL)-8 production by cells from the ulcer bed and neutrophils isolated from peripheral blood that were previously challenged in vitro with Pseudomonas aeruginosa and L. plantarum were studied. Topical application of L. plantarum culture to lesions (25-60 cm(2)) of 14 diabetic and 20 non-diabetic patients induced debridement, granulation tissue formation and total healing after 30 days in 43% diabetics and in 50% non-diabetics. No significant differences between the groups were observed. The cells from ulcer beds collected after treatment with L. plantarum for 10 days showed a decrease in the percentage of polymorphonuclear, apoptotic and necrotic cells and an enhancement of IL-8 production. IL-8 production by isolated neutrophils from these patients was compared with that in diabetics without ulcers, as well as normal subjects under basal conditions, and after infection of polymorphonuclear cells with P. aeruginosa preincubated either with or without L. plantarum. The basal values in diabetic and ulcer patients were higher than normal (p <0.001) and were increased by P. aeruginosa infection in normal, diabetics (p <0.001) and non-diabetics with ulcers (p <0.01). Preincubation with L. plantarum decreased IL-8 production in patients with ulcers non-diabetic and diabetic (p <0.001). Lactobacillus plantarum treatment reduced wound bacterial load, neutrophils, apoptotic and necrotic cells, modified IL-8 production and induced wound healing.

Contact allergy to glucocorticosteroids in patients with chronic venous leg ulcers, atopic dermatitis and contact allergy.

The aim of the study was to assess the prevalence of contact allergy to glucocorticosteroids in patients with chronic venous leg ulcers (CVLU), atopic dermatitis (AD) and contact dermatitis (CD), and in a group of healthy individuals; and to estimate differences among these patient groups. Patch tests with the European standard series, antibiotics, glucocorticosteroid contact allergy screening markers and ointment vehicles were performed in a population of 140 patients. Positive patch tests results were recorded in 80% and contact allergy to glucocorticosteroids in 40% of CVLU patients. In the group of AD patients, the respective figures were 30% and 3%. In the group of CD patients, allergic type of disease was detected in 80% and positive patch tests for glucocorticosteroids in 20% of patients. In healthy individuals, allergic contact reaction was observed in 17% of cases. Statistically significant differences among patient groups were found according to the prevalence of contact allergy, polyvalent allergy and contact allergy to glucocorticosteroids. We suggest that glucocorticosteroid contact allergy should be considered as a crucial clinical problem in patients with inflammatory dermatoses like CVLU, AD and CD.