Unique osteogenic profile of bone marrow stem cells stimulated in perfusion bioreactor is Rho-ROCK-mediated contractility dependent.

The fate determination of bone marrow mesenchymal stem/stromal cells (BMSC) is tightly regulated by mechanical cues, including fluid shear stress. Knowledge of mechanobiology in 2D culture has allowed researchers in bone tissue engineering to develop 3D dynamic culture systems with the potential for clinical translation in which the fate and growth of BMSC are mechanically controlled. However, due to the complexity of 3D dynamic cell culture compared to the 2D counterpart, the mechanisms of cell regulation in the dynamic environment remain relatively undescribed. In the present study, we analyzed the cytoskeletal modulation and osteogenic profiles of BMSC under fluid stimuli in a 3D culture condition using a perfusion bioreactor. BMSC subjected to fluid shear stress (mean 1.56 mPa) showed increased actomyosin contractility, accompanied by the upregulation of mechanoreceptors, focal adhesions, and Rho GTPase-mediated signaling molecules. Osteogenic gene expression profiling revealed that fluid shear stress promoted the expression of osteogenic markers differently from chemically induced osteogenesis. Osteogenic marker mRNA expression, type 1 collagen formation, ALP activity, and mineralization were promoted in the dynamic condition, even in the absence of chemical supplementation. The inhibition of cell contractility under flow by Rhosin chloride, Y27632, MLCK inhibitor peptide-18, or Blebbistatin revealed that actomyosin contractility was required for maintaining the proliferative status and mechanically induced osteogenic differentiation in the dynamic culture. The study highlights the cytoskeletal response and unique osteogenic profile of BMSC in this type of dynamic cell culture, stepping toward the clinical translation of mechanically stimulated BMCS for bone regeneration.

Rheumatoid neutrophilic dermatosis under treatment with the interleukin-6-receptor-antagonist sarilumab in a patient with seropositive rheumatoid arthritis.

Skin manifestations may arise as adverse events following the use of novel drugs. We report a case of a patient with seropositive rheumatoid arthritis who developed a rheumatoid neutrophilic dermatosis (RND) under treatment with the interleukin-6-receptor-antagonist sarilumab. The skin lesions developed 2-3 days after the first injection. RND presents with asymptomatic, symmetrical fixed urticarial-like papules, plaques, and nodules, localized typically on the extensor surfaces of the forearms and hands. After discontinuing the medication, the nodules in our patient disappeared within a month.

Virtual screening for small-molecule pathway regulators by image-profile matching.

Identifying the chemical regulators of biological pathways is a time-consuming bottleneck in developing therapeutics and research compounds. Typically, thousands to millions of candidate small molecules are tested in target-based biochemical screens or phenotypic cell-based screens, both expensive experiments customized to each disease. Here, our uncustomized, virtual, profile-based screening approach instead identifies compounds that match to pathways based on the phenotypic information in public cell image data, created using the Cell Painting assay. Our straightforward correlation-based computational strategy retrospectively uncovered the expected, known small-molecule regulators for 32% of positive-control gene queries. In prospective, discovery mode, we efficiently identified new compounds related to three query genes and validated them in subsequent gene-relevant assays, including compounds that phenocopy or pheno-oppose YAP1 overexpression and kill a Yap1-dependent sarcoma cell line. This image-profile-based approach could replace many customized labor- and resource-intensive screens and accelerate the discovery of biologically and therapeutically useful compounds.

Controllers of cutaneous regulatory T cells: ultraviolet radiation and the skin microbiome.

For the maintenance of homeostasis termination of immune reactions is as equally important as their induction. In this scenario regulatory T cells (Treg) play an important role. Accordingly a variety of inflammatory diseases are caused by an impairment of Treg. Hence, it is important to identify triggers by which Treg can be induced and activated, respectively. For quite a long time it is known that ultraviolet radiation can induce Treg which inhibit cutaneous immune reactions including contact hypersensitivity. Since these Treg inhibit in an antigen-specific fashion they may harbor therapeutic potential. However similar Treg can be induced also by other triggers which include vitamin D and antimicrobial peptides. Recently it was discovered that the gut microbiome controls the development of Treg in the intestine. The same may apply for the skin. Short chain fatty acids, microbiota-derived bacterial fermentation products, appear to induce and to activate Treg in the skin. Topical application of short chain fatty acids was shown to inhibit contact hypersensitivity and to reduce inflammation in the murine imiquimod-induced psoriasis-like skin inflammation model. Together, these data indicate that induction and activation of Treg may be a potential therapeutic strategy to treat inflammatory diseases in the future.

Induction of osteogenic differentiation of bone marrow stromal cells on 3D polyester-based scaffolds solely by subphysiological fluidic stimulation in a laminar flow bioreactor.

The fatal determination of bone marrow mesenchymal stem/stromal cells (BMSC) is closely associated with mechano-environmental factors in addition to biochemical clues. The aim of this study was to induce osteogenesis in the absence of chemical stimuli using a custom-designed laminar flow bioreactor. BMSC were seeded onto synthetic microporous scaffolds and subjected to the subphysiological level of fluid flow for up to 21 days. During the perfusion, cell proliferation was significantly inhibited. There were also morphological changes, with F-actin polymerisation and upregulation of ROCK1. Notably, in BMSC subjected to flow, mRNA expression of osteogenic markers was significantly upregulated and RUNX2 was localised in the nuclei. Further, under perfusion, there was greater deposition of collagen type 1 and calcium onto the scaffolds. The results confirm that an appropriate level of fluid stimuli preconditions BMSC towards the osteoblastic lineage on 3D scaffolds in the absence of chemical stimulation, which highlights the utility of flow bioreactors in bone tissue engineering.

Triple co-culture and perfusion bioreactor for studying the interaction between Neisseria gonorrhoeae and neutrophils: A novel 3D tissue model for bacterial infection and immunity.

Gonorrhea, a sexually transmitted disease caused by the bacteria Neisseria gonorrhoeae, is characterized by a large number of neutrophils recruited to the site of infection. Therefore, proper modeling of the N. gonorrhoeae interaction with neutrophils is very important for investigating and understanding the mechanisms that gonococci use to evade the immune response. We have used a combination of a unique human 3D tissue model together with a dynamic culture system to study neutrophil transmigration to the site of N. gonorrhoeae infection. The triple co-culture model consisted of epithelial cells (T84 human colorectal carcinoma cells), human primary dermal fibroblasts, and human umbilical vein endothelial cells on a biological scaffold (SIS). After the infection of the tissue model with N. gonorrhoeae, we introduced primary human neutrophils to the endothelial side of the model using a perfusion-based bioreactor system. By this approach, we were able to demonstrate the activation and transmigration of neutrophils across the 3D tissue model and their recruitment to the site of infection. In summary, the triple co-culture model supplemented by neutrophils represents a promising tool for investigating N. gonorrhoeae and other bacterial infections and interactions with the innate immunity cells under conditions closely resembling the native tissue environment.

QuoVadoPro, an Autonomous Tool for Measuring Intracellular Dynamics using Temporal Variance.

Trafficking of intracellular cargo is essential to cellular function and can be defective in pathological states including cancer and neurodegeneration. Tools to quantify intracellular traffic are thus necessary for understanding this fundamental cellular process, studying disease mechanisms, and testing the effects of therapeutic pharmaceuticals. In this article we introduce an algorithm called QuoVadoPro that autonomously quantifies the movement of fluorescently tagged intracellular cargo. QuoVadoPro infers the extent of intracellular motility based on the variance of pixel illumination in a series of time-lapse images. The algorithm is an unconventional approach to the automatic measurement of intracellular traffic and is suitable for quantifying movements of intracellular cargo under diverse experimental paradigms. QuoVadoPro is particularly useful to measure intracellular cargo movement in non-neuronal cells, where cargo trafficking occurs as short movements in mixed directions. The algorithm can be applied to images with low temporal or spatial resolutions and to intracellular cargo with varying shapes or sizes, like mitochondria or endoplasmic reticulum: situations in which conventional methods such as kymography and particle tracking cannot be applied. In this article we present a stepwise protocol for using the QuoVadoPro software, illustrate its methodology with common examples, discuss critical parameters for reliable data analysis, and demonstrate its use with a previously published example. © 2020 Wiley Periodicals LLC. Basic Protocol: QuoVadoPro, an autonomous tool for measuring intracellular dynamics using temporal variance.

Lymphoscintigraphy for Sentinel Lymph Node Biopsy in Breast Cancer: Do We Need a Delayed Image?

BACKGROUND: Sentinel lymph node biopsy has become a standard of care in the treatment of patients with early breast cancer, but clinical guidelines continue to be vague on details of the procedure. We were interested in the results of our 2-day protocol, which includes delayed lymphoscintigraphy at 18 h. METHODS: We reviewed the results of preoperative lymphoscintigrams in patients undergoing surgery for breast cancer. Lymphoscintigraphy was performed 2 h after periareolar injection of 4 × 37 MBq 99mTc nanocolloid (early lymphoscintigraphy) and 18 h following injection (delayed lymphoscintigraphy). The early results were compared with the late results. RESULTS: A total of 238 lymphoscintigraphies were performed in 232 patients (6 bilateral). At 2 h, ≥1 sentinel nodes were visualized in 154/238 (65%) cases; in 84 (35%), no sentinel node was visualized. Delayed lymphoscintigraphy visualized a sentinel node in 40 of 76 (53%) cases with no visualization at 2 h and failed to show a sentinel node in 36 (47%) of these cases (in 8 cases, no delayed lymphoscintigram was obtained). CONCLUSIONS: Delayed lymphoscintigraphy was useful in about 50% of the breast cancer patients in whom immediate scintigraphy failed to demonstrate a sentinel lymph node.

A novel high-dimensional NMR experiment for resolving protein backbone dihedral angle ambiguities.

Intrinsically disordered proteins (IDPs) are challenging established structural biology perception and urge a reassessment of the conventional understanding of the subtle interplay between protein structure and dynamics. Due to their importance in eukaryotic life and central role in protein interaction networks, IDP research is a fascinating and highly relevant research area in which NMR spectroscopy is destined to be a key player. The flexible nature of IDPs, as a result of the sampling of a vast conformational space, however, poses a tremendous scientific challenge, both technically and theoretically. Pronounced signal averaging results in narrow signal dispersion and requires higher dimensionality NMR techniques. Moreover, a fundamental problem in the structural characterization of IDPs is the definition of the conformational ensemble sampled by the polypeptide chain in solution, where often the interpretation relies on the concept of 'residual structure' or 'conformational preference'. An important source of structural information is information-rich NMR experiments that probe protein backbone dihedral angles in a unique manner. Cross-correlated relaxation experiments have proven to fulfil this task as they provide unique information about protein backbones, particularly in IDPs. Here we present a novel cross-correlation experiment that utilizes non-uniform sampling detection schemes to resolve protein backbone dihedral ambiguities in IDPs. The sensitivity of this novel technique is illustrated with an application to the prototypical IDP [Formula: see text]-Synculein for which unexpected deviations from random-coil-like behaviour could be observed.

ROR1-CAR T cells are effective against lung and breast cancer in advanced microphysiologic 3D tumor models.

Solid tumors impose immunologic and physical barriers to the efficacy of chimeric antigen receptor (CAR) T cell therapy that are not reflected in conventional preclinical testing against singularized tumor cells in 2-dimensional culture. Here, we established microphysiologic three-dimensional (3D) lung and breast cancer models that resemble architectural and phenotypical features of primary tumors and evaluated the antitumor function of receptor tyrosine kinase-like orphan receptor 1-specific (ROR1-specific) CAR T cells. 3D tumors were established from A549 (non-small cell lung cancer) and MDA-MB-231 (triple-negative breast cancer) cell lines on a biological scaffold with intact basement membrane (BM) under static and dynamic culture conditions, which resulted in progressively increasing cell mass and invasive growth phenotype (dynamic > static; MDA-MB-231 > A549). Treatment with ROR1-CAR T cells conferred potent antitumor effects. In dynamic culture, CAR T cells actively entered arterial medium flow and adhered to and infiltrated the tumor mass. ROR1-CAR T cells penetrated deep into tumor tissue and eliminated multiple layers of tumor cells located above and below the BM. The microphysiologic 3D tumor models developed in this study are standardized, scalable test systems that can be used either in conjunction with or in lieu of animal testing to interrogate the antitumor function of CAR T cells and to obtain proof of concept for their safety and efficacy before clinical application.

[Endovenous Treatment of Varicose Veins and Chronic Venous Insufficiency]. / Endovenöse Behandlungsverfahren der Varikosis und der chronisch-venösen Insuffizienz.

ENDOVENOUS LASER ABLATION: Treatment with longer wavelengths and radial fibers should be used. RADIOFREQUENCY ABLATION: Results after 5 years show a very good efficacy and safety. FURTHER ENDOVENOUS TREATMENTS: Data is limited. The short-term studies show a good efficacy of nonthermal ablation methods. FOLLOW-UP STUDIES: There is no data about a 10 year follow-up until now. Data with comparison of surgical and endovenous therapy show after 5 years show more sonographical reflux in patients treated with first generation endothermal treatment without any differences in the clinical outcome.

The EANM practical guidelines for sentinel lymph node localisation in oral cavity squamous cell carcinoma.

PURPOSE: Sentinel lymph node biopsy is an essential staging tool in patients with clinically localized oral cavity squamous cell carcinoma. The harvesting of a sentinel lymph node entails a sequence of procedures with participation of specialists in nuclear medicine, radiology, surgery, and pathology. The aim of this document is to provide guidelines for nuclear medicine physicians performing lymphoscintigraphy for sentinel lymph node detection in patients with early N0 oral cavity squamous cell carcinoma. METHODS: These practice guidelines were written and have been approved by the European Association of Nuclear Medicine (EANM) and the International Atomic Energy Agency (IAEA) to promote high-quality lymphoscintigraphy. The final result has been discussed by distinguished experts from the EANM Oncology Committee, and national nuclear medicine societies. The document has been endorsed by the Society of Nuclear Medicine and Molecular Imaging (SNMMI). These guidelines, together with another two focused on Surgery and Pathology (and published in specialised journals), are part of the synergistic efforts developed in preparation for the "2018 Sentinel Node Biopsy in Head and Neck Consensus Conference". CONCLUSION: The present practice guidelines will help nuclear medicine practitioners play their essential role in providing high-quality lymphatic mapping for the care of early N0 oral cavity squamous cell carcinoma patients.

Decreased expression of G-protein-coupled receptors GPR43 and GPR109a in psoriatic skin can be restored by topical application of sodium butyrate.

The G-protein-coupled receptors GPR43 and GPR109a are known to play an important role in mediating anti-inflammatory and anti-cancer functions in the gut. Short-chain fatty acids, such as sodium butyrate (SB), are activators of GPR43 and GPR109a and thereby promote anti-inflammatory effects. The present study aimed to examine the expression of these receptors and their reaction to SB in psoriasis. Lesional and non-lesional biopsies of 6 psoriasis patients and of 4 controls were obtained and stained for GPR109a and GPR43. Ex vivo stimulation with SB was performed on fresh biopsy material. Lesional and non-lesional psoriatic skin showed a decreased expression of GPR109a and GPR43 on keratinocytes in comparison with control skin. Topical application of SB was able to increase the low-level expression of both receptors. The data suggest that SB by restoring the impaired expression of GPR109a and GPR43 might exert anti-inflammatory effects and may be utilized as a topical tool for the treatment of psoriasis, which has to be proven in future clinical trials.

Diagnostic and prognostic value of additional SPECT/CT in sentinel lymph node mapping in breast cancer patients.

THE AIM: of the study was to demonstrate the diagnostic and prognostic value of SPECT/CT in sentinel lymph node mapping (SLNM) in patients with invasive breast cancer. METHODS: 114 patients with invasive breast cancer with clinically negative lymph nodes were included in this retrospective study as they were referred for SLNM with 99mTc-nanocolloid. Planar image acquisition was accomplished in a one-day or two-day protocol depending on the schedule of the surgical procedure. Low dose SPECT/CT was performed after the planar images. The sentinel lymph node biopsy (SLNB) was considered false negative if a primary recurrence developed within 12 months after SLNB in the axilla from which a tumor-free SLN had been removed. RESULTS: Between December 2009 and December 2011, 114 patients (pts.) underwent SLNM with additional SPECT/CT. Planar imaging identified in 109 pts. 139 SLNs, which were tumor-positive in 42 nodes (n = 41 pts.). SPECT/CT identified in 81 pts. 151 additional SLNs, of which 19 were tumor-positive and led to therapy change (axillary lymph node dissection) in 11 pts. (9.6 %). Of overall 61 tumor-positive SLNs (n = 52 pts.) SPECT/CT detected all, whereas planar imaging detected only 42 of 61 (P < 0.0001). No patient had lymph node metastasis within 12 months after SLNB in the axilla from which a tumor-free SLN had been removed resulting in a false-negative rate of 0 %. The local relapse rate was 1.8 % leading to a 4-year disease-free survival rate of 90 %. CONCLUSION: Among patients with breast cancer, the use of SPECT/CT-aided SLNM correlated due to a better anatomical localization and identification of planar not visible SLNs with a higher detection rate of SLNs. This led to therapeutic consequences and an excellent false-negative and 4-year disease-free survival rate.

Ultraviolet radiation induces Melan-A-expressing cells in interfollicular epidermis in wild-type mice.

Adult wild-type mice are not supposed to be proper models for ultraviolet radiation (UVR)-induced melanoma since melanocytes are confined to hair follicles and cannot be sufficiently reached by UVR. On the other hand, in mutated mouse models used for melanoma research limitations, including an altered immune system and selection of affected pathways, lead to tumors phenotypically quite different from naturally occurring melanomas. We compared the distribution of epidermal melanocytes in UVR and not-UVR-exposed wild-type C57BL/6 mice. Starting at the age of 8 weeks, mice were exposed to physiologic doses of UVR three times weekly over 16 weeks. Back skin biopsies were taken 4, 8, 12 and 16 weeks after initiation of exposure, and stained for Melan-A, representing a highly selective marker for melanocytes. Surprisingly, after exposure to UVR, Melan-A positive cells were detected also in the interfollicular epidermis of C57BL/6 mice. We conclude that UVR is capable of inducing interfollicular epidermal melanocytes in wild-type mice.

Clinically defined subgroups of mycosis fungoides display differing histopathological features at initial biopsy.

Most patients with mycosis fungoides (MF) remain in early disease stages but some progress to tumor stage. The individual course of the disease cannot be predicted. We wanted to assess the clinical and histological characteristics of the first available biopsy. An end-of-spectrum approach with two groups was used, comparing MF remaining long-term stable in T1a ('MF stable') and MF with later tumor development or present T3 stage ('MF tumor'). The clinical and histomorphological features of the initial skin biopsy were compared. Patients in the 'MF tumor' group presented initially with higher disease stages. The first biopsies of 'MF tumor' patients showed significantly higher infiltrate density and depth, more large cells and a higher proliferative index. In summary, long-term stable MF seems to differ in clinical and histopathological parameters from MF with T3 evolution/presence already at the time point of the initial biopsy. Our findings might indicate a predetermined biologic behavior.