Importance and implications of exosomes in nephrology and urology.

Exosomes are extracellular vesicles that are formed by two invaginations of the plasma membrane and can be released by all eukaryotic cells. Because of their bioactive contents, including nucleic acids and proteins, exosomes can activate a variety of functions in their recipient cells. Due to the plethora of physiological and pathophysiological functions, exosomes have received a lot of attention from researchers over the past few years. However, there is still no consensus regarding isolation and characterization protocols of exosomes and their subtypes. This heterogeneity poses a lot of methodical challenges but also offers new clinical opportunities simultaneously. So far, exosome-based research is still mostly limited to preclinical experiments and early-stage clinical trials since the translation of experimental findings remains difficult. Exosomes could potentially play an important role as future diagnostic and prognostic agents and might also be part of the development of new treatment strategies. Therefore, they have previously been investigated in a variety of nephrological and urological conditions such as acute kidney injury or prostate cancer.

IL-1ß-driven neutrophilia preserves antibacterial defense in the absence of the kinase IKKß.

Transcription factor NF-κB and its activating kinase IKKß are associated with inflammation and are believed to be critical for innate immunity. Despite the likelihood of immune suppression, pharmacological blockade of IKKß-NF-κB has been considered as a therapeutic strategy. However, we found neutrophilia in mice with inducible deletion of IKKß (Ikkß(Δ) mice). These mice had hyperproliferative granulocyte-macrophage progenitors and pregranulocytes and a prolonged lifespan of mature neutrophils that correlated with the induction of genes encoding prosurvival molecules. Deletion of interleukin 1 receptor 1 (IL-1R1) in Ikkß(Δ) mice normalized blood cellularity and prevented neutrophil-driven inflammation. However, Ikkß(Δ)Il1r1(-/-) mice, unlike Ikkß(Δ) mice, were highly susceptible to bacterial infection, which indicated that signaling via IKKß-NF-κB or IL-1R1 can maintain antimicrobial defenses in each other's absence, whereas inactivation of both pathways severely compromises innate immunity.

Modified Borggreve-Van Nes-Winkelmann rotationplasty for surgery in developing countries.

BACKGROUND: Amputation is still the most common therapy for patients suffering from osteosarcoma in Myanmar, despite the fact that limb salvage surgery e.g. Borggreve-Van Nes-Winkelmann rotationplasty for malignant tumors located within the distal femur or proximal tibia is the current state-of-the-art reconstructive procedure. A safe and reliable operation technique is crucial in order to perform a complex surgical procedure like the rotationplasty in lower-middle income economies with limited infrastructure and resources. The authors present seven cases of patients with osteosarcomas that received a Borggreve-Van Nes-Winkelmann rotationplasty with an evaluation of the procedures focusing on safety and sustainability. METHODS: From 2019 until 2020, seven young patients with osteosarcomas of the distal femur or proximal tibia were treated with Borggreve-Van Nes-Winkelmann rotationplasties in the Orthopaedic Hospital in Mandalay, Myanmar. As modification of the standard procedure the dissection and subsequent clamping of the femoral artery in order to minimize blood loss as well as the formation of an adipocutaneous flap that minimizes swelling and decreases the pressure on the vessels were successfully performed. This modified procedure resembles a safe and simplified surgical technique that is feasible under the circumstances of lower-middle income economies with good outcomes. RESULTS: All patients showed good functional and aesthetic results. One of the seven patients needed secondary wound closure due to wound dehiscence. CONCLUSIONS: A simplified and safe operation technique for the performance of the Van Nes-Borggreve rotationplasty was adapted to the given constraints in lower-middle income economies and proved to be successful. Trial registration All patients approved to participate in the study and have given consent to publication.

Laser speckle contrast analysis (LASCA) technology for the semiquantitative measurement of angiogenesis in in-ovo-tumor-model.

BACKGROUND: The process of angiogenesis is a key element for tumor growth and proliferation and therefore one of the determining factors for aggressiveness and malignancy. A better understanding of the underlying processes of tumor induced angiogenesis is crucial for superior cancer treatment. Furthermore, the PeriCam perfusion speckle imager (PSI) system high resolution (HR) model by PERIMED presents a noninvasive method for semi-quantitative measurement of blood perfusion, based on laser speckle contrast analysis (LASCA). Aim of the present study was to utilize the chick chorioallantoic membrane (CAM) model as an in-ovo-tumor-model which enables rapid neovascularization of tumors while allowing real-time observation of the microcirculation via LASCA. METHODS: Fertilized chicken eggs were grafted with embryonal/alveolar rhabdomyosarcoma cells or primary sarcoma tumors. The blood perfusion was measured before and after tumor growth using LASCA. The procedure is accelerated and simplified through the integrated PIMSoft software which provides real-time graphs and color-coded images during the measurement. RESULTS: Sarcoma cells and primary sarcoma tumors exhibited satisfactory growth processes on the CAM. LASCA visualized microcirculation accurately and enabled an extensive investigation of the angiogenic potential of sarcoma cells on the CAM. We were able to show that sarcoma cells and primary sarcoma tumors induced larger quantities of neovasculature on the CAM than the controls. CONCLUSIONS: The utilization of LASCA for the investigation of tumor angiogenesis within the CAM model appears to be a highly beneficial, cost-efficient and easily practicable procedure. The proposed model can be used as a drug-screening model for individualized cancer therapy, especially with regards to anti-angiogenic agents.

How to Approach Secondary Breast Reduction: International Trends and a Systematic Review of the Literature.

BACKGROUND: Secondary breast reduction is complex and poses significant challenges to surgeons. Complication rates exceed those of primary reduction, commonly caused by impaired vascular supply of the nipple-areolar complex (NAC). Literature on the topic is scare and provides contradicting recommendations, especially with regard to pedicle choice in cases with unknown primary reduction technique. Aim of this study was to investigate international trends and to compare findings with literature. METHODS: A large-scale web-based questionnaire on international trends in mammaplasty (mastopexy and breast reduction) was designed and distributed to over five thousand surgeons in eight geographic regions. The presented manuscript evaluated information regarding pedicle choice in secondary breast reduction and compared data to literature identified in a systematic review. RESULTS: The survey was completed by 1431 participants. Overall, secondary procedures were performed in less than 5% or in 5 to 10% of cases. The preferred pedicle for secondary reductions differed significantly between geographic regions (p<0.001). The majority of respondents reported to use a superior or supero-medial pedicle (34.8% and 32.2%, respectively). Residual analysis revealed a strong association between the use of an inferior pedicle and procedures performed in North America. CONCLUSIONS: Secondary breast reduction is challenging and there remains international disparity with regard to pedicle choice for secondary procedures. Studies investigating outcome when the primary pedicle is unknown are scarce and provide incoherent recommendations. High-quality data is needed to provide evidence-based practice guidelines. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

pH sensing in skin tumors: Methods to study the involvement of GPCRs, acid-sensing ion channels and transient receptor potential vanilloid channels.

Solid tumors exhibit an inversed pH gradient with increased intracellular pH (pHi ) and decreased extracellular pH (pHe ). This inside-out pH gradient is generated via sodium/hydrogen antiporter 1, vacuolar-type H + ATPases, monocarboxylate transporters, (bi)carbonate (co)transporters and carboanhydrases. Our knowledge on how pHe -signals are sensed and what the respective receptors induce inside cells is scarce. Some pH-sensitive receptors (GPR4, GPR65/TDAG8, GPR68/OGR1, GPR132/G2A, possibly GPR31 and GPR151) and ion channels (acid-sensing ion channels ASICs, transient receptor potential vanilloid receptors TRPVs) transduce signals inside cells. As little is known on the expression and function of these pH sensors, we used immunostainings to study tissue samples from common and rare skin cancers. Our current and future work is directed towards investigating the impact of all the pH-sensing receptors in different skin tumors using cell culture techniques with selective knockdown/knockout (siRNA/CRISPR-Cas9). To study cell migration and proliferation, novel impedance-based wound healing assays have been developed and are used. The field of pH sensing in tumors and wounds holds great promise for the development of pH-targeting therapies, either against pH regulators or sensors to inhibit cell proliferation and migration.

Ion channels in sarcoma: pathophysiology and treatment options.

Sarcomas are characterized by aggressive growth and a high metastasis potentially leading in most cases to a lethal outcome. These malignant tumors of the connective tissue have a high heterogeneity with numerous genetic mutations resulting in more than 100 types of sarcoma that can be grouped into two main kinds: soft tissue sarcoma and bone sarcoma. Sarcomas are often diagnosed at late disease stage, whereas a guaranteed diagnosis of the sarcoma type is fundamental for successful therapy. However, there is no appropriate therapy available. Therefore, the need for new therapies, which prolong survival and improve quality of life, is high. In the last two decades, the role of ion channels in cancer has emerged. Ion channels seem to be an ideal target for anti-tumor therapies. However, different cancer types have their own altered ion channel pattern, and the knowledge about the tumor-associated ion channel expression is fundamental. Here, we focus on the role of different ion channels in sarcoma, their pathophysiology, and possible treatment options.

Microsurgical reconstruction in patients greater than 80 years old.

BACKGROUND: Demographic change implies that the human population is getting older and the elderly are living longer. Consequently, achieving good functional and aesthetic outcomes in microvascular procedures, especially in very old patients with higher incidence of atherosclerosis and vessel calcifications, constitutes a microsurgical challenge. This study evaluates the feasibility of microsurgical procedures in a very old patient cohort. PATIENTS AND METHODS: Between 2009 and 2015, 754 patients underwent 838 free flap reconstructions. The patients were divided into two groups according to age in "middle-aged" (<80 years old; n = 711) or "very old" (≥80 years old; n = 43). The series was retrospectively analyzed regarding potential influence of medical comorbidities, surgical and medical complications and outcomes. RESULTS: Between the groups, there was a significant difference regarding comorbidities with a higher prevalence of hypertension (P < 0.0001) and peripheral artery disease (P < 0.0001) in the very old group. However, there was no significant difference regarding the rate of surgical or medical complications, flap failure (middle aged group 43/791 flaps (5.44%) versus very old group 4/47 flaps (8.51%); P = 0.328), and revision rate (117/791 flaps (14.79%) versus 6/47 flaps (12.77%); P = 0.834) between the patient groups during our 3-months follow-up period. CONCLUSION: Our findings suggest that despite higher rates of patient comorbidities, successful free tissue transfer can also be achieved in a very old population with acceptable risk for complications.

Populational equilibrium through exosome-mediated Wnt signaling in tumor progression of diffuse large B-cell lymphoma.

Tumors are composed of phenotypically heterogeneous cell populations. The nongenomic mechanisms underlying transitions and interactions between cell populations are largely unknown. Here, we show that diffuse large B-cell lymphomas possess a self-organized infrastructure comprising side population (SP) and non-SP cells, where transitions between clonogenic states are modulated by exosome-mediated Wnt signaling. DNA methylation modulated SP-non-SP transitions and was correlated with the reciprocal expressions of Wnt signaling pathway agonist Wnt3a in SP cells and the antagonist secreted frizzled-related protein 4 in non-SP cells. Lymphoma SP cells exhibited autonomous clonogenicity and exported Wnt3a via exosomes to neighboring cells, thus modulating population equilibrium in the tumor.

Exosomal evasion of humoral immunotherapy in aggressive B-cell lymphoma modulated by ATP-binding cassette transporter A3.

Targeting the surface of malignant cells has evolved into a cornerstone in cancer therapy, paradigmatically introduced by the success of humoral immunotherapy against CD20 in malignant lymphoma. However, tumor cell susceptibility to immunochemotherapy varies, with mostly a fatal outcome in cases of resistant disease. Here, we show that lymphoma exosomes shield target cells from antibody attack and that exosome biogenesis is modulated by the lysosome-related organelle-associated ATP-binding cassette (ABC) transporter A3 (ABCA3). B-cell lymphoma cells released exosomes that carried CD20, bound therapeutic anti-CD20 antibodies, consumed complement, and protected target cells from antibody attack. ABCA3, previously shown to mediate resistance to chemotherapy, was critical for the amounts of exosomes released, and both pharmacological blockade and the silencing of ABCA3 enhanced susceptibility of target cells to antibody-mediated lysis. Mechanisms of cancer cell resistance to drugs and antibodies are linked in an ABCA3-dependent pathway of exosome secretion.

Ultra high frequency ultrasound enables real-time visualization of blood supply from chorioallantoic membrane to human autosomal dominant polycystic kidney tissue.

Ultra high frequency (UHF) ultrasound enables the visualization of very small structures that cannot be detected by conventional ultrasound. The utilization of UHF imaging as a new imaging technique for the 3D-in-vivo chorioallantoic membrane (CAM) model can facilitate new insights into tissue perfusion and survival. Therefore, human renal cystic tissue was grafted onto the CAM and examined using UHF ultrasound imaging. Due to the unprecedented resolution of UHF ultrasound, it was possible to visualize microvessels, their development, and the formation of anastomoses. This enabled the observation of anastomoses between human and chicken vessels only 12 h after transplantation. These observations were validated by 3D reconstructions from a light sheet microscopy image stack, indocyanine green angiography, and histological analysis. Contrary to the assumption that the nutrient supply of the human cystic tissue and the gas exchange happens through diffusion from CAM vessels, this study shows that the vasculature of the human cystic tissue is directly connected to the blood vessels of the CAM and perfusion is established within a short period. Therefore, this in-vivo model combined with UHF imaging appears to be the ideal platform for studying the effects of intravenously applied therapeutics to inhibit renal cyst growth.

Tyrosinekinase inhibition facilitates cooperation of transcription factor SALL4 and ABC transporter A3 towards intrinsic CML cell drug resistance.

Although BCR-ABL1 tyrosine kinase inhibitors reliably induce disease remission for patients with chronic myeloid leukaemia (CML), unlimited extension of therapy is necessary to prevent relapse from persistent leukaemic cells. Here, we analysed model cell lines and primary CML cells for the expression and functions of the ABC transporter A3 (ABCA3) as well as the embryonic stem cell-associated transcription factor SALL4. ABCA3 protected leukaemic cells from the cytotoxic effects of the tyrosine kinase inhibitors imatinib, dasatinib, and nilotinib. In the surviving cells, exposure to tyrosine kinase inhibitors significantly enhanced ABCA3 expression in vivo and in vitro, and was associated with increased expression of SALL4, which binds the ABCA3 promoter. Inhibition of ABCA3 or SALL4 by genetic silencing or indomethacin, but not interferon gamma, interrupted SALL4-dependent regulation of ABCA3 and restored susceptibility of leukaemic cells to tyrosine kinase inhibition. Tyrosine kinase inhibitor exposure facilitates a protective loop of SALL4 and ABCA3 cooperation in persistent leukaemic cells.

Intracellular ATP-binding cassette transporter A3 is expressed in lung cancer cells and modulates susceptibility to cisplatin and paclitaxel.

Patients with advanced-stage bronchial cancer benefit from systemic cytostatic therapy, in particular from regimens integrating cisplatin and taxanes. However, eventual disease progression leads to a fatal outcome in most cases, originating from tumor cells resisting chemotherapy. We here show that the intracellular ATP-binding cassette transporter A3 (ABCA3), previously recognized as critical for the secretion of surfactant components from type 2 pneumocytes, is expressed in non-small-cell lung cancer (NSCLC) cells. With some heterogeneity in a given specimen, expression levels detected immunohistochemically in primary cancer tissue were highest in adenocarcinomas and lowest in small cell lung cancers. Genetic silencing of ABCA3 in the NSCLC cell line models A549, NCI-H1650 and NCI-H1975 significantly increased tumor cell susceptibility to the cytostatic effects of both cisplatin (in all cell lines) and paclitaxel (in two of three cell lines). Taken together, ABCA3 emerges as a modulator of NSCLC cell susceptibility to cytostatic therapy.

Immunostimulatory activity of murine keratinocyte-derived exosomes.

It has long been known that keratinocytes influence cutaneous immunity through secretion of soluble factors. Exosomes, small membrane vesicles of endocytotic origin, have been implicated in intercellular communication processes such as the transfer of tumor cell antigens and the activation of recipient dendritic cells (DC). However, little is known about immunomodulatory functions of keratinocyte-derived exosomes. To address this question, we analysed exosome secretion of the murine keratinocyte cell line MPEK under steady state as well as inflammatory conditions (+/- IFNγ). These exosomes were readily taken up by bone marrow-derived DC (BMDC) in vitro resulting in a matured phenotype, as evidenced by increased CD40 expression as well as by the production of large amounts of IL-6, IL-10 and IL-12. When the transfer of antigen-specific information through exosomes was investigated, it was found that keratinocytes took up antigen (ovalbumin) and transferred it to their exosomes. However, these antigen-harbouring exosomes failed to induce antigen-specific T cell responses via BMDC. Together, this novel biological function suggests that keratinocytes are able to direct unspecific immune processes but do not elicit specific immune responses.

Application of Laser Speckle Contrast Imaging (LSCI) for the Angiogenesis Measurement of Tumors in the Chorioallantoic Membrane (CAM) Model.

Tumor angiogenesis is one essential aspect for the growth and metastasis of cancer cells, which means that adequate in vivo angiogenesis models are of utmost importance for the investigation of such diseases. The chick chorioallantoic membrane (CAM) model is one established method for this purpose and has already been used for research on multiple cancer types. One important part of the evaluation of tumors grafted onto the CAM is the measurement of tumor-induced angiogenesis. In order to address this central aspect, we utilized the novel PeriCam perfusion speckle imager (PSI) system high resolution (HR) model (Perimed AB, Järfälla, Sweden), which is based on laser speckle contrast imaging (LSCI) for the semiquantitative measurement of blood flow in the CAM model. This method enables a fast and accurate analysis of the angiogenesis of cell line tumors and primary tumors that are grafted onto the CAM. The proposed model can be regarded as a precursor model for personalized cancer therapy.

The 3D in vivo chorioallantoic membrane model and its role in breast cancer research.

PURPOSE: We aimed to evaluate the role of the chorioallantoic membrane model (CAM) in breast cancer research. METHODS: The following is an overview of the use of the CAM in the field of breast cancer research based on a PubMed literature query. RESULTS: The CAM is a 3D in vivo model that can be used for the analysis of tumor growth, biology and angiogenesis of primary tumor tissue or tumor cell lines. The CAM model has been used in breast cancer research for drug testing, migration assays and the evaluation of vascularization, amongst others. The CAM model is a valuable method that offers a better imitation of the physiological phenomena compared to 2D or 3D in vitro models. CONCLUSION: The CAM model has primarily and successfully been utilized for the assessment of the tumor biology of established breast cancer cell lines. Further, the CAM model is a promising method to analyze patient derived primary tumor material and could be used as a "patient-specific 3D-tumor-therapy-model" for the cost-efficient evaluation of anti-cancer drugs to find the optimal treatment for breast cancer patients.

New, Innovative, Three-Dimensional In Vivo Model for High-Level Microsurgical and Supermicrosurgical Training: A Replacement for Animal Models.

SUMMARY: Microsurgery and supermicrosurgery are surgical subdomains necessary for a large variety of surgical disciplines. So far, there is no training model for lymphatic surgery or perforator flap surgery, and the most commonly used microsurgical training models are living animals. However, the ethical principles of replacement, refinement, and reduction (the three Rs) of living animals for training purposes were implemented, highlighting the necessity of an animal-sparing microsurgical training model. Formed during embryogenesis, the chick chorioallantoic membrane resembles a highly vascularized, noninnervated membrane within fertilized chicken eggs. The aim of this study was to utilize the chorioallantoic membrane model as an innovative and versatile training model for supermicrosurgery and microsurgery that can reduce the number of animals used for these purposes. The variety of different sized vessels for the implementation of an anastomosis proved the chorioallantoic membrane model as a well-functioning supermicrosurgical and microsurgical training model. The circulatory system is resilient enough to withstand the mechanical stress applied to the tissue, and the patency of the implemented anastomosis can be tested for the verification of the procedures. In summary, the integration of the chorioallantoic membrane model into a surgical training program can benefit its quality by representing a realistic anatomical and physiological model with a high variety of vascular structures. Moreover, the chorioallantoic membrane model satisfies the principles of replacement, refinement, and reduction as an animal-sparing model, indicating the potential of this model as an innovative microsurgical training model for the improvement of surgical skills.

The Role of Citrate Homeostasis in Merkel Cell Carcinoma Pathogenesis.

Merkel cell carcinoma (MCC) is a rare but highly aggressive tumor of the skin with a poor prognosis. The factors driving this cancer must be better understood in order to discover novel targets for more effective therapies. In the search for targets, we followed our interest in citrate as a central and critical metabolite linked to fatty acid synthesis in cancer development. A key to citrate uptake in cancer cells is the high expression of the plasma membrane citrate transporter (pmCiC), which is upregulated in the different adenocarcinoma types tested so far. In this study, we show that the pmCiC is also highly expressed in Merkel cell carcinoma cell lines by western blot and human tissues by immunohistochemistry staining. In the presence of extracellular citrate, MCC cells show an increased proliferation rate in vitro; a specific pmCiC inhibitor (Na+-gluconate) blocks this citrate-induced proliferation. Furthermore, the 3D in vivo Chick Chorioallantoic Membrane (CAM) model showed that the application of Na+-gluconate also decreases Merkel cell carcinoma growth. Based on our results, we conclude that pmCiC and extracellular citrate uptake should be considered further as a potential novel target for the treatment of Merkel cell carcinoma.

An Innovative Simulation Model for Microvascular Training.

SUMMARY: Preclinical/clinical microsurgical training is essential for clinical practice. Therefore, various training models have been established, such as synthetic and cadaveric models. The most common limitation of these models is the lack of circulation, which limits the simulation of real intraoperative circumstances. Thus, the authors aimed to create a novel model that provides blood circulation with an extracorporeal perfusion device that they attached to rat cadavers for the reestablishment of a circulatory system. Patent blue and heparin were added to the perfusion fluid to visualize circulation and to dissolve thrombosis, and indocyanine green fluorescent imaging was applied to show the perfusion of the entire body. The femoral and brachial vessels were dissected, and an end-to-end anastomosis was performed on the femoral artery. The patency of the operated vessel was visualized with indocyanine green fluorescent imaging. Indocyanine green fluorescent imaging showed appropriate vessel patency and extremity perfusion through the anastomosis. The use of this novel rat model enables a solution for ethical problems encountered when using rats for surgical training courses. By practicing on these animal-sparing models with intact circulation, microsurgical skills can be improved. Future studies on further microsurgical techniques and vascular perfusion of organs or tumors may benefit from our model.

Deep Learning-Based Image Analysis for the Quantification of Tumor-Induced Angiogenesis in the 3D In Vivo Tumor Model-Establishment and Addition to Laser Speckle Contrast Imaging (LSCI).

(1) Background: angiogenesis plays an important role in the growth and metastasis of tumors. We established the CAM assay application, an image analysis software of the IKOSA platform by KML Vision, for the quantification of blood vessels with the in ovo chorioallantoic membrane (CAM) model. We added this proprietary deep learning algorithm to the already established laser speckle contrast imaging (LSCI). (2) Methods: angiosarcoma cell line tumors were grafted onto the CAM. Angiogenesis was measured at the beginning and at the end of tumor growth with both measurement methods. The CAM assay application was trained to enable the recognition of in ovo CAM vessels. Histological stains of the tissue were performed and gluconate, an anti-angiogenic substance, was applied to the tumors. (3) Results: the angiosarcoma cells formed tumors on the CAM that appeared to stay vital and proliferated. An increase in perfusion was observed using both methods. The CAM assay application was successfully established in the in ovo CAM model and anti-angiogenic effects of gluconate were observed. (4) Conclusions: the CAM assay application appears to be a useful method for the quantification of angiogenesis in the CAM model and gluconate could be a potential treatment of angiosarcomas. Both aspects should be evaluated in further research.