Development of a cellular assay as a personalized model for testing chronic wound therapeutics.

Exudates of non-healing wounds contain drivers of pathogenicity. We utilized >800 exudates from non-healing and healing wounds of diverse etiologies, collected by three different methods, to develop a wound-specific, cell-based functional biomarker assay. Human dermal fibroblast proliferation served as readout to a) to differentiate between healing and non-healing wounds, b) follow the healing process of individual patients, and c) assess the effects of therapeutics for chronic wounds ex vivo. We observed a strong correlation between wound chronicity and inhibitory effects of individual exudates on fibroblast proliferation, with good diagnostic sensitivity (76-90%, depending on the sample collection method). Transition of a clinically non-healing to a healing phenotype restored fibroblast proliferation and extracellular matrix formation while reducing inflammatory cytokine production. Transcriptional analysis of fibroblasts exposed to ex vivo non-healing wound exudates revealed an induction of inflammatory cytokine- and chemokine pathways and the unfolded protein response, indicating that these changes may contribute to the pathology of non-healing wounds. Testing the wound therapeutics platelet derived growth factor and silver sulfadiazine yielded responses in line with clinical experience and indicate the usefulness of the assay to search for and profile new therapeutics.

Livedoid vasculopathy - A diagnostic and therapeutic challenge.

Livedoid vasculopathy is a rare, chronic-recurrent occlusive disorder in the microcirculation of dermal vessels. The clinical appearance is characterized by Livedo racemosa, painful ulceration, located in the distal parts of the lower extremities, followed by healing as porcelain-white, atrophic scars, the so-called Atrophie blanche. Different conditions that can promote a hypercoagulable state, such as inherited and acquired thrombophilias, autoimmune connective-tissue diseases and neoplasms, can be associated with livedoid vasculopathy. Therefore, livedoid vasculopathy is currently considered to be a coagulation disorder, clearly distinguished from inflammatory vasculitis. Although there are hints to hypercoaguability and secondary inflammation, pathophysiology is not completely understood. Diagnosis is made by synopsis of history, clinical and histopathological findings. Early and adequate therapy is essential to maintain life quality and avoid irreversible complications. Better understanding of molecular mechanisms is required to establish appropriate therapy regimens. This article presents the current state of knowledge about livedoid vasculopathy and proposes an algorithmic approach for diagnosis and therapy.

Quantitative analysis of the natural history of prolidase deficiency: description of 17 families and systematic review of published cases.

PURPOSE: Prolidase deficiency is a rare inborn error of metabolism causing ulcers and other skin disorders, splenomegaly, developmental delay, and recurrent infections. Most of the literature is constituted of isolated case reports. We aim to provide a quantitative description of the natural history of the condition by describing 19 affected individuals and reviewing the literature. METHODS: Nineteen patients were phenotyped per local institutional procedures. A systematic review following PRISMA criteria identified 132 articles describing 161 patients. Main outcome analyses were performed for manifestation frequency, diagnostic delay, overall survival, symptom-free survival, and ulcer-free survival. RESULTS: Our cohort presented a wide variability of severity. Autoimmune disorders were found in 6/19, including Crohn disease, systemic lupus erythematosus, and arthritis. Another immune finding was hemophagocytic lymphohistiocytosis (HLH). Half of published patients were symptomatic by age 4 and had a delayed diagnosis (mean delay 11.6 years). Ulcers were present initially in only 30% of cases, with a median age of onset at 12 years old. CONCLUSION: Prolidase deficiency has a broad range of manifestations. Symptoms at onset may be nonspecific, likely contributing to the diagnostic delay. Testing for this disorder should be considered in any child with unexplained autoimmunity, lower extremity ulcers, splenomegaly, or HLH.

Activated melanoma vessels: A sticky point for successful immunotherapy.

Metastatic melanoma is a devastating disease with a marginal-albeit increasing-hope for cure. Melanoma has a high mutation rate which correlates to the expression of numerous neo-antigens and thus is associated with the potential to induce and strengthen effective antitumoral immunity. However, the incomplete and potentially insufficient response to established immunotherapies (response rates usually do not markedly exceed 60%) already points to the need of further studies to improve treatment strategies. Multiple tumor escape mechanisms that allow melanoma to evade from antitumoral immune responses have been characterized and must be overcome to achieve a better clinical efficacy of immunotherapies. Recently, promising progress has been made in targeting tumor vasculature to control and increase the infiltration of tumors with effector lymphocytes. It has been hypothesized that amplified lymphocytic infiltrates in melanoma metastases result in a switch of the tumor microenvironment from a non-inflammatory to an inflammatory state. In this view point essay, we discuss the requirements for successful homing of lymphocytes to melanoma tissue and we present a mouse melanoma xenograft model that allows the investigation of human tumor vessels in vivo. Furthermore, current clinical studies dealing with the activation of melanoma vasculature for enhanced effectiveness of immunotherapy protocols are presented and open questions for routine clinical application are addressed.

Evidence and impact of neutrophil extracellular traps in malignant melanoma.

Ulceration of melanoma is associated with neutrophil infiltrates and lower survival rates opposite to non-ulcerated melanoma. Neutrophils release neutrophil extracellular traps (NETs) that are chromatin structures loaded with antimicrobial proteins. Since NETs have been correlated with tumor progression, we investigated whether NETs appear in melanoma and affect melanoma cells. Indeed, human primary melanoma biopsies revealed neutrophils releasing NETs in all of 27 ulcerated melanomas, whereas NETs were absent in all of 7 non-ulcerated melanomas. However, the quantity of intratumoral NETs did not correlate with tumor progression of melanoma. Interestingly, in vitro assays showed that melanoma cells attach to NETs via integrin-mediated adhesion and that NETs inhibit tumor cell migration. Moreover, co-culturing of NETs and melanoma cells had a cytotoxic effect on melanoma cells resulting in necrosis. Hence, we discovered in vitro an antineoplastic role of NETs in melanoma.

Activation of human vascular endothelium in melanoma metastases induces ICAM-1 and E-selectin expression and results in increased infiltration with effector lymphocytes.

Lymphocytic infiltration into melanoma tissue is an important prerequisite for effective antitumoral immunity. However, analysis of human metastatic melanoma has shown that leucocyte adhesion receptor expression on melanoma blood vessels is very low or absent, thereby impairing the entry of cytotoxic lymphocytes into tumor tissue. We hypothesized that adhesion molecules can be induced on melanoma vasculature allowing better infiltration of cytotoxic lymphocytes. Quantitative real-time PCR and immunofluorescence staining indicated that the adhesion molecules ICAM-1 (CD54) and E-selectin (CD62E) can be significantly induced by intralesional application of TNF alpha in tissue from human melanoma metastases either in vitro or in vivo when grafted onto immunodeficient NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) mice that preserved human vessels. Furthermore, activated human autologous CD3+ lymphocytes were injected intravenously into mice bearing melanoma xenografts treated with TNF-α or PBS in addition to the leucocyte chemoattractant TARC (CCL17). Significantly increased numbers of CD8+ cells were detected in TNF-α-treated melanoma metastases compared with PBS-treated controls. In addition, tumor cell apoptosis was enhanced and melanoma cell proliferation reduced as shown by TUNEL assay and KI-67 staining. We conclude that adhesion molecules can be induced on human melanoma vasculature resulting in significantly improved homing of activated autologous cytotoxic T cells to melanoma tissue and inhibition of melanoma cell proliferation. These observations should be considered when designing protocols for immunotherapy of malignant melanoma.

Systemic therapies for leg ulcers.

Successful management of patients with leg ulcers requires identification of the underlying etiology, with subsequent initiation of causal treatment, if feasible. Supplementary measures of first choice include stage-adjusted wound treatment, usually combined with compression therapy. The significance of systemic drugs has been the subject of controversial debate, depending on the underlying cause of the condition. The present review article is therefore meant to highlight current aspects of systemic drug therapies for the treatment of leg ulcers associated with chronic venous insufficiency, peripheral arterial disease, livedoid vasculopathy, vasculitis, necrobiosis lipoidica, calciphylaxis and pyoderma gangrenosum. In summary, the majority of therapeutic options presented herein are used off-label. While systemic drugs are promising options for the more common types of wounds such as venous, mixed or arterial leg ulcers, they do not represent the current standard of treatment. By contrast, systemic agents play a key role in the management of many of the other disorders presented herein. These agents primarily include immunomodulatory and rheological drugs used to expedite wound healing.

Cellular stress induces erythrocyte assembly on intravascular von Willebrand factor strings and promotes microangiopathy.

Microangiopathy with subsequent organ damage represents a major complication in several diseases. The mechanisms leading to microvascular occlusion include von Willebrand factor (VWF), notably the formation of ultra-large von Willebrand factor fibers (ULVWFs) and platelet aggregation. To date, the contribution of erythrocytes to vascular occlusion is incompletely clarified. We investigated the platelet-independent interaction between stressed erythrocytes and ULVWFs and its consequences for microcirculation and organ function under dynamic conditions. In response to shear stress, erythrocytes interacted strongly with VWF to initiate the formation of ULVWF/erythrocyte aggregates via the binding of Annexin V to the VWF A1 domain. VWF-erythrocyte adhesion was attenuated by heparin and the VWF-specific protease ADAMTS13. In an in vivo model of renal ischemia/reperfusion injury, erythrocytes adhered to capillaries of wild-type but not VWF-deficient mice and later resulted in less renal damage. In vivo imaging in mice confirmed the adhesion of stressed erythrocytes to the vessel wall. Moreover, enhanced eryptosis rates and increased VWF binding were detected in blood samples from patients with chronic renal failure. Our study demonstrates that stressed erythrocytes have a pronounced binding affinity to ULVWFs. The discovered mechanisms suggest that erythrocytes are essential for the pathogenesis of microangiopathies and renal damage by actively binding to ULVWFs.

VIPAR, a quantitative approach to 3D histopathology applied to lymphatic malformations.

BACKGROUND: Lack of investigatory and diagnostic tools has been a major contributing factor to the failure to mechanistically understand lymphedema and other lymphatic disorders in order to develop effective drug and surgical therapies. One difficulty has been understanding the true changes in lymph vessel pathology from standard 2D tissue sections. METHODS: VIPAR (volume information-based histopathological analysis by 3D reconstruction and data extraction), a light-sheet microscopy-based approach for the analysis of tissue biopsies, is based on digital reconstruction and visualization of microscopic image stacks. VIPAR allows semiautomated segmentation of the vasculature and subsequent nonbiased extraction of characteristic vessel shape and connectivity parameters. We applied VIPAR to analyze biopsies from healthy lymphedematous and lymphangiomatous skin. RESULTS: Digital 3D reconstruction provided a directly visually interpretable, comprehensive representation of the lymphatic and blood vessels in the analyzed tissue volumes. The most conspicuous features were disrupted lymphatic vessels in lymphedematous skin and a hyperplasia (4.36-fold lymphatic vessel volume increase) in the lymphangiomatous skin. Both abnormalities were detected by the connectivity analysis based on extracted vessel shape and structure data. The quantitative evaluation of extracted data revealed a significant reduction of lymphatic segment length (51.3% and 54.2%) and straightness (89.2% and 83.7%) for lymphedematous and lymphangiomatous skin, respectively. Blood vessel length was significantly increased in the lymphangiomatous sample (239.3%). CONCLUSION: VIPAR is a volume-based tissue reconstruction data extraction and analysis approach that successfully distinguished healthy from lymphedematous and lymphangiomatous skin. Its application is not limited to the vascular systems or skin. FUNDING: Max Planck Society, DFG (SFB 656), and Cells-in-Motion Cluster of Excellence EXC 1003.

Chronic venous insufficiency - a review of pathophysiology, diagnosis, and treatment.

Chronic venous disease is a common disorder associated with a variety of symptoms in later disease stages but also with complications such as venous leg ulcer. This, in turn, has substantial socioeconomic effects and significantly impacts patients' quality of life. While there are a number of diagnostic procedures available, color-flow duplex ultrasound has become the gold standard. As regards therapeutic options, major advances have been made in recent decades. Today, there are alternatives to saphenofemoral ligation and stripping of the great saphenous vein, including endovenous thermal ablation techniques. However, treatment selection continues to depend on many factors such as individual anatomical circumstances and disease stage. The following article provides an overview of the anatomy and pathophysiology as well as current diagnostic and therapeutic standards.

Die chronische venöse Insuffizienz - Eine Zusammenfassung der Pathophysiologie, Diagnostik und Therapie.

Die chronische Venenerkrankung ist eine weit verbreitete Krankheit, die in späteren Stadien mit einer Vielzahl an Symptomen, aber auch Komplikationen wie dem Ulcus cruris, einhergeht. Dies wiederum hat weitreichende Auswirkungen auf die Lebensqualität der Patienten wie auch auf das Gesundheitssystem. Für die Diagnostik der chronischen Venenerkrankungen steht eine Auswahl an Verfahren zur Verfügung, wobei sich die farbkodierte Duplexsonographie als Goldstandard etabliert hat. Im Bereich der Therapie kam es in den letzten Jahrzehnten zu großen Fortschritten, sodass heute auch Alternativen zum klassischen Stripping durch die endoluminalen Verfahren zur Verfügung stehen. Die Wahl der Therapieoption ist jedoch weiterhin stark abhängig von mehreren Faktoren, unter anderem von den anatomischen Gegebenheiten und dem Krankheitsstadium. Im folgenden Artikel werden die Anatomie und Pathophysiologie, sowie die aktuellen Standards der Diagnostik und Therapie zusammengefasst.