Whole stromal fibroblast signature is linked to specific chemokine and immune infiltration patterns and to improved survival in NSCLC.

Cancer associated fibroblasts (CAF) are known to orchestrate multiple components of the tumor microenvironment, whereas the influence of the whole stromal-fibroblast compartment is less understood. Here, an extended stromal fibroblast signature was investigated to define its impact on immune cell infiltration. The lung cancer adenocarcinoma (LUAD) data set of the cancer genome atlas (TCGA) was used to test whole stroma signatures and cancer-associated fibroblast signatures for their impact on prognosis. 3D cell cultures of the NSCLC cancer cell line A549 together with the fibroblast cell line SV80 were used in combination with infiltrating peripheral blood mononuclear cells (PBMC) for in-vitro investigations. Immune cell infiltration was assessed via flow cytometry, chemokines were analyzed by immunoassays and RNA microarrays. Results were confirmed in specimens from NSCLC patients by flow cytometry or immunohistochemistry as well as in the TCGA data set. The TCGA analyses correlated the whole stromal-fibroblast signature with an improved outcome, whereas no effect was found for the CAF signatures. In 3D microtumors, the presence of fibroblasts induced infiltration of B cells and CD69+CD4+ T cells, which was linked to an increased expression of CCL13 and CXCL16. The stroma/lymphocyte interaction was confirmed in NSCLC patients, as stroma-rich tumors displayed an elevated B cell count and survival in the local cohort and the TCGA data set. A whole stromal fibroblast signature was associated with an improved clinical outcome in lung adenocarcinoma and in vitro and in vivo experiments suggest that this signature increases B and T cell recruitment via induction of chemokines.

The circadian transcription factor ARNTL2 is regulated by weight-loss interventions in human white adipose tissue and inhibits adipogenesis.

Misalignment of physiological circadian rhythms promotes obesity which is characterized by white adipose tissue (WAT) expansion. Differentiation of Adipose stem/progenitor cells (ASCs) contributes to WAT increase but the importance of the cellular clock in this process is incompletely understood. In the present study, we reveal the role of the circadian transcription factor Aryl hydrocarbon receptor nuclear translocator-like 2 (ARNTL2) in human ASCs, isolated from subcutaneous (s)WAT samples of patients undergoing routine elective plastic abdominal surgery. We show that circadian synchronization by serum-shock or stimulation with adipogenic stimuli leads to a different expression pattern of ARNTL2 relative to its well-studied paralogue ARNTL1. We demonstrate that ARNTL2 mRNA is downregulated in ASCs upon weight-loss (WL) whereas ARNTL2 protein is rapidly induced in the course of adipogenic differentiation and highly abundant in adipocytes. ARNTL2 protein is maintained in ASCs cooperatively by mechanistic Target of Rapamycin (mTOR) and Mitogen-activated Protein Kinase (MAPK) signalling pathways while ARNTL2 functions as an inhibitor on both circuits, leading to a feedback mechanism. Consistently, ectopic overexpression of ARNTL2 repressed adipogenesis by facilitating the degradation of ARNTL1, inhibition of Kruppel-Like Factor 15 (KLF15) gene expression and down-regulation of the MAPK-CCAAT/enhancer-binding protein ß (C/EBPß) axis. Western blot analysis of sWAT samples from normal-weight, obese and WL donors revealed that ARNTL2 protein was solely elevated by WL compared to ARNTL1 which underscores unique functions of both transcription factors. In conclusion, our study reveals ARNTL2 to be a WL-regulated inhibitor of adipogenesis which might provide opportunities to develop strategies to ameliorate obesity.

The Great Auricular Nerve Trigger Site: Anatomy, Compression Point Topography, and Treatment Options for Headache Pain.

BACKGROUND: Peripheral nerve decompression surgery can effectively address headache pain caused by compression of peripheral nerves of the head and neck. Despite decompression of known trigger sites, there are a subset of patients with trigger sites centered over the postauricular area coursing. The authors hypothesize that these patients experience primary or residual pain caused by compression of the great auricular nerve. METHODS: Anatomical dissections were carried out on 16 formalin-fixed cadaveric heads. Possible points of compression along fascia, muscle, and parotid gland were identified. Ultrasound technology was used to confirm these anatomical findings in a living volunteer. RESULTS: The authors' findings demonstrate that the possible points of compression for the great auricular nerve are at Erb's point (point 1), at the anterior border of the sternocleidomastoid muscle in the dense connective tissue before entry into the parotid gland (point 2), and within its intraparotid course (point 3). The mean topographic measurements were as follows: Erb's point to the mastoid process at 7.32 cm/7.35 (right/left), Erb's point to the angle of the mandible at 6.04 cm/5.89 cm (right/left), and the posterior aspect of the sternocleidomastoid muscle to the mastoid process at 3.88 cm/4.43 cm (right/left). All three possible points of compression could be identified using ultrasound. CONCLUSIONS: This study identified three possible points of compression of the great auricular nerve that could be decompressed with peripheral nerve decompression surgery: Erb's point (point 1), at the anterior border of the sternocleidomastoid muscle (point 2), and within its intraparotid course (point 3).

Quiescence, Stemness and Adipogenic Differentiation Capacity in Human DLK1-/CD34+/CD24+ Adipose Stem/Progenitor Cells.

We explore the status of quiescence, stemness and adipogenic differentiation capacity in adipose stem/progenitor cells (ASCs) ex vivo, immediately after isolation from human subcutaneous white adipose tissue, by sorting the stromal vascular fraction into cell-surface DLK1+/CD34-, DLK1+/CD34dim and DLK1-/CD34+ cells. We demonstrate that DLK1-/CD34+ cells, the only population exhibiting proliferative and adipogenic capacity, express ex vivo the bonafide quiescence markers p21Cip1, p27Kip1 and p57Kip2 but neither proliferation markers nor the senescence marker p16Ink4a. The pluripotency markers NANOG, SOX2 and OCT4 are barely detectable in ex vivo ASCs while the somatic stemness factors, c-MYC and KLF4 and the early adipogenic factor C/EBPß are highly expressed. Further sorting of ASCs into DLK1-/CD34+/CD24- and DLK1-/CD34+/CD24+ fractions shows that KLF4 and c-MYC are higher expressed in DLK1-/CD34+/CD24+ cells correlating with higher colony formation capacity and considerably lower adipogenic activity. Proliferation capacity is similar in both populations. Next, we show that ASCs routinely isolated by plastic-adherence are DLK1-/CD34+/CD24+. Intriguingly, CD24 knock-down in these cells reduces proliferation and adipogenesis. In conclusion, DLK1-/CD34+ ASCs in human sWAT exist in a quiescent state, express high levels of somatic stemness factors and the early adipogenic transcription factor C/EBPß but senescence and pluripotency markers are barely detectable. Moreover, our data indicate that CD24 is necessary for adequate ASC proliferation and adipogenesis and that stemness is higher and adipogenic capacity lower in DLK1-/CD34+/CD24+ relative to DLK1-/CD34+/CD24- subpopulations.

Anatomical basis of a safe mini-invasive technique for lengthening of the anterior gastrocnemius aponeurosis.

BACKGROUND: The surgical procedure itself of lengthening the gastrocnemius muscle aponeurosis is performed to treat multiple musculoskeletal, neurological and metabolical pathologies related to a gastro-soleus unit contracture such as plantar fasciitis, Achilles tendinopathy, metatarsalgia, cerebral palsy, or diabetic foot ulcerations. Therefore, the aim of our research was to prove the effectiveness and safety of a new ultrasound-guided surgery-technique for the lengthening of the anterior gastrocnemius muscle aponeurosis, the "GIAR"- technique: the gastrocnemius-intramuscular aponeurosis release. METHODS AND RESULTS: An ultrasound-guided surgical GIAR on ten fresh-frozen specimens (10 donors, 8 male, 2 females, 5 left and 5 right) was performed. Exclusion criteria of the donated bodies to science were BMI above 35 (impaired ultrasound echogenicity), signs of traumas in the ankle and crural region, a history of ankle or foot ischemic vascular disorder, surgery or space-occupying mass lesions. The surgical procedures were performed by two podiatric surgeons with more than 6 years of experience in ultrasound-guided procedures. The anterior gastrocnemius muscle aponeurosis was entirely transected in 10 over 10 specimens, with a mean portal length of 2 mm (± 1 mm). The mean gain at the ankle joint ROM after the GIAR was 7.9° (± 1.1°). No damages of important anatomical structures could be found. CONCLUSION: Results of this study indicate that our novel ultrasound-guided surgery for the lengthening of the anterior gastrocnemius muscle aponeurosis (GIAR) might be an effective and safe procedure.

Sprouty1 Prevents Cellular Senescence Maintaining Proliferation and Differentiation Capacity of Human Adipose Stem/Progenitor Cells.

The role of Ras-Mitogen-activated protein kinase (MAPK) signaling in cellular aging is not precisely understood. Recently, we identified Sprouty1 (SPRY1) as a weight-loss target gene in human adipose stem/progenitor cells (ASCs) and showed that Sprouty1 is important for proper regulation of adipogenesis. In the present study, we show that loss-of-function of Sprouty1 by CRISPR/Cas9-mediated genome editing in human ASCs leads to hyper-activation of MAPK signaling and a senescence phenotype. Sprouty1 knockout ASCs undergo an irreversible cell cycle arrest, become enlarged and stain positive for senescence-associated ß-galactosidase. Sprouty1 down-regulation leads to DNA double strand breaks, a considerably increased number of senescence-associated heterochromatin foci and induction of p53 and p21Cip1. In addition, we detect an increase of hypo-phosphorylated Retinoblastoma (Rb) protein in SPRY1 knockout ASCs. p16Ink4A is not induced. Moreover, we show that Sprouty1 knockout leads to induction of a senescence-associated secretory phenotype as indicated by the activation of the transcription factors NFκB and C/EBPß and a significant increase in mRNA expression and secretion of interleukin-8 (IL-8) and CXCL1/GROα. Finally, we demonstrate that adipogenesis is abrogated in senescent SPRY1 knockout ASCs. In conclusion, this study reveals a novel mechanism showing the importance of Sprouty1 for the prevention of senescence and the maintenance of the proliferation and differentiation capacity of human ASCs.

Postoperative Hypoparathyroidism in Thyroid Surgery: Anatomic-Surgical Mapping of the Parathyroids and Implications for Thyroid Surgery.

Hypoparathyroidism remains one of the most common complications in thyroid surgery. This study aims for an improved understanding of the complexity of the blood supply and the localisation of the parathyroids compared to the two most important intraoperative landmarks: the inferior laryngeal nerve (ILN) and Zuckerkandl's tubercle (ZT). We examined 103 laryngeal compounds to classify the blood supply and the localisation of the parathyroids. For intraoperative localisation we defined a Cartesian coordinate system with the ZT plane as x-axis and the course of the inferior laryngeal nerve as y-axis. The inferior thyroid artery (ITA) mainly supplies the parathyroids, whereas the superior thyroid artery provides a backup supply. It must be pointed out that 8.2% of parathyroids receive their blood directly from the thyroid gland. 73.5% of all parathyroids lie within 1 cm of the ILN and 1 cm cranial and 2.5 cm caudal to the ZT plane. Our described perimeters mark the most crucial areas during surgery and provide the surgeon with an anatomic mapping showing areas of special carefulness needed. One should keep bearing in mind all possible blood supply types of the parathyroids and therefore all branches should be handled with care.

Peripheral Nerve Anatomy Revisited: Modern Requirements for Neuroimaging and Microsurgery.

Detailed anatomic investigation of peripheral nerve topography underlies the correct application of intraoperative neuromonitoring (IONM) and ultrasonography, both well-established methods to prevent nerve palsy during surgical operations and to elucidate pathomechanisms in disease. In this study, we analyzed the anatomy of selected peripheral nerves in the head and neck regions to improve the outcome of endocrine and migraine surgeries. Anatomic dissections of 204 hemilarynges were performed to study the topography of the inferior laryngeal nerve (ILN). Measurements were taken from the lower rim of the cricoid and from the Zuckerkandl tubercle to the beginning of the furcation of the ILN. For the analysis of peripheral nerves contributing to migraine pathogenesis, 22 hemifaces were investigated by dissection and ultrasonography. The supratrochlear and supraorbital nerves and their relationship to the corrugator supercilii muscle are described. For identification of the ILN, the cricoid offers a suitable intraoperative landmark. A single branch existed in 5% of specimens on the left side and in 3% on the right side. Bifurcation was present in 72.5% and 62% and trifurcation in 18% and 29% of cases, respectively. IONM signals from the vagus nerve were positive if derived proximal to and negative if derived distal to the branching off of a nonrecurrent ILN (nrILN). By ultrasonographic identification of a brachiocephalic trunk, an nrILN could be excluded. For migraine surgery, possible compression points of the supratrochlear and supraorbital nerves were identified, and a workflow algorithm for ultrasound visualization of these nerves is provided. Anat Rec, 302:1325-1332, 2019. © 2019 Wiley Periodicals, Inc.

Fibroblast feeder layer supports adipogenic differentiation of human adipose stromal/progenitor cells.

Adipose stromal/progenitor cells (ASCs) can differentiate into adipocytes in the course of adipogenesis. This process is governed by systemic factors and signals of the adipose stem cell niche. ASCs isolated from fat tissues and amplified in vitro provide an essential and reliable model system to study adipogenesis. However, current cell culture models routinely grow ASCs on plastic surfaces largely missing niche parameters. In the present communication, we employed human foreskin fibroblasts (HFFs) monolayers as feeder cells for ASCs, which were isolated from human subcutaneous white adipose tissue and amplified in vitro. We found that PPARγ2 and several adipocyte markers were significantly higher expressed in differentiated ASCs growing on feeder layers relative to plastic dishes. Moreover, a significant higher number of adipocytes was generated from ASCs cultured on feeder layer and these adipocytes contained larger fat droplets. Insulin strongly stimulated glucose uptake into adipocytes produced on feeder layer suggesting that these cells show characteristic metabolic features of fat cells.  Finally, we show that the HFF feeder layer allows adipogenic differentiation of low-density-seeded ASCs. In conclusion, we demonstrate that the HFF feeder layer increases adipocyte differentiation of ASCs and allows differentiation of low density seeded progenitor cells  into functional adipocytes.

Ultrasound-guided decompression surgery of the distal tarsal tunnel: a novel technique for the distal tarsal tunnel syndrome-part III.

BACKGROUND: The aim of this study was to provide a safe ultrasound-guided minimally invasive surgical approach for a distal tarsal tunnel release concerning nerve entrapments. METHODS AND RESULTS: The study was carried out on ten fresh-frozen feet. All of them have been examined by high-resolution ultrasound at the distal tarsal tunnel. The surgical approach has been marked throughout the course of the medial intermuscular septum (MIS, the lateral fascia of the abductor hallucis muscle). After the previous steps, nerve decompression was carried out through a MIS release through a 2.5 mm (± 0.5 mm) surgical portal. As a result, an effective release of the MIS has been obtained in all fresh-frozen feet. CONCLUSION: The results of our anatomic study indicate that this novel ultrasound-guided minimally invasive surgical approach for the release of the MIS might be an effective, safe and quick decompression technique treating selected patients with a distal tarsal tunnel syndrome.

Ultrasound-guided decompression surgery of the tarsal tunnel: a novel technique for the proximal tarsal tunnel syndrome-Part II.

BACKGROUND: The aim of this study is to provide a safe ultrasound-guided minimally invasive surgical approach for a proximal tarsal tunnel release concerning nerve entrapments. METHODS AND RESULTS: The study was carried out on ten fresh-frozen feet. All of them were examined by high resolution ultrasound at the medial ankle region. The surgical approach was marked throughout the course of the flexor retinaculum (laciniate ligament). Once the previous steps were done, the flexor retinaculum release technique was carried out with a 2-mm entry only. As a result, an effective and safe release of the flexor retinaculum was obtained in all fresh-frozen feet. CONCLUSION: The results of our anatomic study indicate that our novel ultrasound-guided minimally invasive surgical approach for the release of the flexor retinaculum might be an effective, safe and quick decompression technique treating selected patients with a proximal tarsal tunnel syndrome.

Clinical-anatomic mapping of the tarsal tunnel with regard to Baxter's neuropathy in recalcitrant heel pain syndrome: part I.

PURPOSE: Neuropathy of the Baxter nerve (BN) seems to be the first cause of the heel pain syndrome (HPS) of neurological origin. METHODS: 41 alcohol-glycerol embalmed feet were dissected. We documented the pattern of the branches of the tibial nerve (TN) and describe all relevant osteofibrous structures. Measurements for the TN branches were related to the Dellon-McKinnon malleolar-calcaneal line also called DM line (DML) for the proximal TT and the Heimkes Triangle for the distal TT. Additionally, we performed an ultrasound-guided injection procedure of the BN and provide an algorithm for clinical usage. RESULTS: The division of the TN was 16.4 mm proximal to the DML. The BN branches off 20 mm above the DML center or 30 mm distally to it. In most of the cases, the medial calcaneal branch (MCB) originated from the TN proximal to the bifurcation. Possible entrapment spots for the medial and lateral plantar nerve (MPN, LPN), the BN and the MCB are found within a circle of 5 mm radius with a probability of 80%, 83%, and 84%, respectively. In ten out of ten feet, the US-guided injection was precisely allocated around the BN. CONCLUSIONS: Our detailed mapping of the TN branches and their osteofibrous tubes at the TT might be of importance for foot and ankle surgeons during minimally invasive procedures in HPS such as ultrasound-guided ankle and foot decompression surgery (UGAFDS).

The influence of stromal cells and tumor-microenvironment-derived cytokines and chemokines on CD3+CD8+ tumor infiltrating lymphocyte subpopulations.

The tumor microenvironment has been identified as a major mediator of immunological processes in solid tumors. In particular, tumor-associated fibroblasts are known to interact with tumor infiltrating immune cells. We describe the influence of fibroblasts and tumor-microenvironment-derived cytokines on the infiltration capacity of CD3+CD8+ cytotoxic T lymphocyte subpopulations using a multicellular 3D co-culture system. 3D tumor microtissues were cultivated using a hanging drop system. Human A549 and Calu-6 cancer cell lines were incubated alone or together with the human fibroblast cell line SV80 for 10 d to form microtissues. On day 10, peripheral blood mononuclear cells (PBMC) were added with or without cytokine stimulation for 24 h. Infiltrating PBMC subpopulations were investigated by flow cytometry. Aggregation of the microtissues and the infiltration of the PBMCs were analyzed by immunohistochemistry, and endogenous cytokine and chemokine expression was analyzed with a multi-cytokine immunoassay. Secretion of chemokines is increased in microtissues consisting of cancer cells and fibroblasts. PBMC infiltrate the whole spheroid in cancer cell monocultures, whereas in co-cultures of cancer cells and fibroblasts, PBMCs are rather localized at the margin. Activated CD69+ and CD49d+ T lymphocytes show an increased microtissue infiltration in the presence of fibroblasts. We demonstrate that the stromal component of cancer microtissues significantly influences immune cell infiltration. The presence of fibroblasts in cancer microtissues induces a shift of T lymphocyte infiltration toward activated T lymphocytes.

Development of a 3D angiogenesis model to study tumour - endothelial cell interactions and the effects of anti-angiogenic drugs.

The tumour microenvironment and tumour angiogenesis play a critical role in the development and therapy of many cancers, but in vitro models reflecting these circumstances are rare. In this study, we describe the development of a novel tri-culture model, using non-small cell lung cancer (NSCLC) cell lines (A549 and Colo699) in combination with a fibroblast cell line (SV 80) and two different endothelial cell lines in a hanging drop technology. Endothelial cells aggregated either in small colonies in Colo699 containing microtissues or in tube like structures mainly in the stromal compartment of microtissues containing A549. An up-regulation of hypoxia and vimentin, ASMA and a downregulation of E-cadherin were observed in co- and tri-cultures compared to monocultures. Furthermore, a morphological alteration of A549 tumour cells resembling "signet ring cells" was observed in tri-cultures. The secretion of proangiogenic growth factors like vascular endothelial growth factor (VEGF) was measured in supernatants. Inhibition of these proangiogenic factors by using antiangiogenic drugs (bevacizumab and nindetanib) led to a significant decrease in migration of endothelial cells into microtissues. We demonstrate that our method is a promising tool for the generation of multicellular tumour microtissues and reflects in vivo conditions closer than 2D cell culture.

CD146 (MCAM) in human cs-DLK1-/cs-CD34+ adipose stromal/progenitor cells.

To precisely characterize CD146 in adipose stromal/progenitor cells (ASCs) we sorted the stromal vascular faction (SVF) of human abdominal subcutaneous white adipose tissue (sWAT) according to cell surface (cs) expression of CD146, DLK1 and CD34. This test identified three main SVF cell populations: ~50% cs-DLK1-/cs-CD34+/cs-CD146- ASCs, ~7.5% cs-DLK1+/cs-CD34dim/+/cs-CD146+ and ~7.5% cs-DLK1+/cs-CD34dim/+/cs-CD146- cells. All cells contained intracellular CD146. Whole mount fluorescent IHC staining of small vessels detected CD146+ endothelial cells (CD31+/CD34+/CD146+) and pericytes (CD31-/CD34-/CD146+ ASCs). The cells in the outer adventitial layer showed the typical ASC morphology, were strongly CD34+ and contained low amounts of intracellular CD146 protein (CD31-/CD34+/CD146+). Additionally, we detected wavy CD34-/CD146+ and CD34dim/CD146+ cells. CD34dim/CD146+ cells were slightly more bulky than CD34-/CD146+ cells. Both CD34-/CD146+ and CD34dim/CD146+ cells were detached from the inner pericyte layer and protruded into the outer adventitial layer. Cultured early passage ASCs contained low levels of CD146 mRNA, which was expressed in two different splicing variants, at a relatively high amount of the CD146-long form and at a relatively low amount of the CD146-short form. ASCs contained low levels of CD146 protein, which consisted predominantly long form and a small amount of short form. The CD146 protein was highly stable, and the majority of the protein was localized in the Golgi apparatus. In conclusion, the present study contributes to a better understanding of the spatial localization of CD34+/CD146+ and CD34-/CD146+ cells in the adipose niche of sWAT and identifies CD146 as intracellular protein in cs-DLK1-/cs-CD34+/cs-CD146- ASCs.

L1CAM in the Early Enteric and Urogenital System.

L1 cell adhesion molecule (L1CAM) is a transmembrane molecule belonging to the L1 protein family. It has shown to be a key player in axonal guidance in the course of neuronal development. Furthermore, L1CAM is also crucial for the establishment of the enteric and urogenital organs and is aberrantly expressed in cancer originating in these organs. Carcinogenesis and embryogenesis follow a lot of similar molecular pathways, but unfortunately, comprehensive data on L1CAM expression and localization in human developing organs are lacking so far. In the present study we, therefore, examined the spatiotemporal distribution of L1CAM in the early human fetal period (weeks 8-12 of gestation) by means of immunohistochemistry and in situ hybridization (ISH). In the epithelia of the gastrointestinal organs, L1CAM localization cannot be observed in the examined stages most likely due to their advanced polarization and differentiation. Despite these results, our ISH data indicate weak L1CAM expression, but only in few epithelial cells. The genital tracts, however, are distinctly L1CAM positive throughout the entire fetal period. We, therefore, conclude that in embryogenesis L1CAM is crucial for further differentiation of epithelia.

Case report: a common trunk of the coronary arteries.

We describe the heart from a 79-year-old woman with no medical history of cardiac complaints. Her heart shows a regular right coronary artery (RCA) and a variant left coronary artery (LCA) arising from the right sinus of Valsalva. The common stem of the RCA and the LCA is extremely short. The LCA depicts a preinfundibular course with a cranial-anterior loop and reaches the intersection of the anterior interventricular sulcus and the left coronary sulcus, where it divides into the regular branches, the anterior interventricular branch (left anterior descending, LAD) and the circumflex branch (left circumflex, LCx). All further branching resembles a normal distribution with the posterior interventricular branch coming for the RCA. Such a variant LCA is extremely rare with a reported incidence of 0.17 %. However, recognition and angiographic demonstration of such a variation assume the highest priority in a patient undergoing, for instance, direct coronary artery surgery or prosthetic valve replacement.

Weight Loss Upregulates the Small GTPase DIRAS3 in Human White Adipose Progenitor Cells, Which Negatively Regulates Adipogenesis and Activates Autophagy via Akt-mTOR Inhibition.

Long-term weight-loss (WL) interventions reduce insulin serum levels, protect from obesity, and postpone age-associated diseases. The impact of long-term WL on adipose-derived stromal/progenitor cells (ASCs) is unknown. We identified DIRAS3 and IGF-1 as long-term WL target genes up-regulated in ASCs in subcutaneous white adipose tissue of formerly obese donors (WLDs). We show that DIRAS3 negatively regulates Akt, mTOR and ERK1/2 signaling in ASCs undergoing adipogenesis and acts as a negative regulator of this pathway and an activator of autophagy. Studying the IGF-1-DIRAS3 interaction in ASCs of WLDs, we demonstrate that IGF-1, although strongly up-regulated in these cells, hardly activates Akt, while ERK1/2 and S6K1 phosphorylation is activated by IGF-1. Overexpression of DIRAS3 in WLD ASCs completely inhibits Akt phosphorylation also in the presence of IGF-1. Phosphorylation of ERK1/2 and S6K1 is lesser reduced under these conditions. In conclusion, our key findings are that DIRAS3 down-regulates Akt-mTOR signaling in ASCs of WLDs. Moreover, DIRAS3 inhibits adipogenesis and activates autophagy in these cells.

The nonrecurrent laryngeal nerve: A clinical anatomic mapping with regard to intraoperative neuromonitoring.

BACKGROUND: We investigated the nonrecurrent inferior laryngeal nerve (nrILN), an important variant in the course of the inferior laryngeal nerve (ILN; 0.5-6.0%). Its importance was demonstrated in a clinical case as well as in cadaver specimens, and the pattern was identified with intraoperative neuromonitoring (IONM). METHODS: The ILN and the presence of an nrILN were investigated in 36 formaldehyde-embalmed specimens. Our anatomic findings showed differences in the anatomic course of the ILN and thus produced possible explanations for different IONM signals that would correlate with differences in the anatomic course of the ILN. Preoperative ultrasonographic evaluation of the brachiocephalic trunk and the recurrent laryngeal nerve were used for the exclusion or identification of an nrILN, respectively. RESULTS: We found 2 nrILNs (ascending, horizontal; 6%) in the anatomic specimens. These 2 specimens each showed an aberrant right subclavian artery (lusorial artery) and were, therefore, associated with the absence of a brachiocephalic trunk. The intraoperative case displayed a descending nrILN. Signals derived from the vagus nerve were positive if derived proximal to and negative if derived distal to the branching of an nrILN. By ultrasonographic identification of a normal brachiocephalic trunk, an nrILN could be excluded. CONCLUSION: Surgeons need a working knowledge about nrILNs to avoid recurrent nerve palsy and should be familiar with all the possible course variations in the ILN when IONM signals are absent with vagal stimulation. Moreover, endocrine surgeons need to be able to interpret correctly negative as well as positive signals. Preoperative ultrasonography should ideally be performed, because the presence of a normal brachiocephalic trunk is a quick method to exclude or identify a nrILN.

Infiltration of lymphocyte subpopulations into cancer microtissues as a tool for the exploration of immunomodulatory agents and biomarkers.

INTRODUCTION: The interaction between the immune system and malignant diseases is a proven key target for cancer therapy. We describe an innovative 3D cell culture system comprising both immune and cancer cells to evaluate their interaction and immune cell infiltration to provide an innovative in vitro screening of immunomodulatory agents and biomarkers. METHODS: 3D tumor microtissues were cultivated using a hanging drops system. Human non-small-cell lung cancer cell lines were incubated for 7 days to form microtissues. On day 5, peripheral blood mononuclear cells (PBMC) were added with or without interleukin-2 (IL-2) for 24 or 48h. Viability of cancer cells and the infiltrating PBMC subpopulations were investigated by flow cytometry. Aggregation of tumor cells and PBMC and the infiltration of the PBMC into the tumor microtissues were analyzed by immunohistochemistry. Quantification of infiltration was measured by applying the TissueFAXS system. RESULTS: Immunohistochemistry revealed PBMC infiltration in all cell lines which increased under IL-2 stimulation. Analysis of infiltrating populations showed both lymphocyte subpopulations and monocytes within the tumor microtissues. In all three co-cultures, CD3+CD8+ and CD3+CD8+CD45R0+CD28+ lymphocytes were increased with IL-2, whereas CD3+CD8+CD45R0-CD28+ PBMCs were decreased with and without IL-2 stimulation. CONCLUSION: In summary, we present a novel cell culture system to study the interaction between cancer cells and immune cells in 3-dimensional microtissues. In addition, we report for the first time an in vitro infiltration assay based on 3D microtissues. This model has the potential to provide a tool for ex-vivo drug testing and biomarker screening of immunomodulatory agents.