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Gastric cancer carries a poor prognosis even after curative resection (R0). Tumor progression in gastric cancer patients has been attributed to the persistence of disseminated tumor cells (DTC) in various body compartments as a sign of minimal residual disease, although the prognostic relevance of DTC is still unclear. In this study the prognostic relevance of DTC in the blood of gastric cancer patients was investigated. Material and methods Venous blood samples of 70 cancer patients were taken intraoperatively before surgical manipulation and examined by reverse transcription-polymerase chain reaction (RT-PCR) for expression of cytokeratin 20 (CK20) as a marker for DTC. Tumor-related survival was analyzed using univariate and multivariate models assessing occurrence of DTC, residual tumor classification, and tumor stage. Median follow-up was 20 months (range 1–57 months). Results Twenty-eight of the 70 patients (40%) were CK20 positive. The prevalence of DTC in patients following R0 resection (15/41, 37%) was similar to that in patients with residual tumor (13/29, 45%, NS). Furthermore, expression of CK20 was independent of TNM stage. Univariate analysis of R0-resected patients revealed CK20 to be a marker for significantly shorter tumor-related survival (p=0.0363). In a multivariate analysis, CK20 was an independent prognostic marker. Detection of CK20 had greatest impact for early tumor stages (T1 and T2, N0; p<0.0032). Conclusions Detection of DTC in venous blood of gastric cancer patients is an independent predictive marker of poor prognosis and thus could help to define patients for adjuvant therapy with this tumor entity. [ABSTRACT FROM AUTHOR]

Epidermolysis bullosa acquisita (EBA) is a sub epidermal blistering disorder associated with tissue-bound and circulating autoantibodies specific to type VII collagen, a major constituent of the dermal-epidermal junction. Previous attempts to transfer the disease by injection of patient autoantibodies into mice have been unsuccessful. To study the pathogenic relevance of antibodies specific to type WI collagen in vivo, we generated and characterized rabbit antibodies specific to a murine form of this antigen and passively transferred them into adult nude, BALB/c, and CS7BL/6 mice. Immune rabbit IgG bound to the lamina densa of murine skin and immunoblotted type WI collagen. Mice injected with purified IgG specific to type VII collagen, in contrast to control mice, developed subepidermal skin blisters, reproducing the human disease at the clinical, histological, electron microscopical, and immunopathological levels. Titers of rabbit IgG in the serum of mice correlated with the extent of the disease. F(ab')2 fragments of rabbit IgG specific to type WI collagen were not pathogenic. When injected into CS-deficient mice, antibodies specific to type WI collagen failed to induce the disease, where- as CS-sufficient mice were susceptible to blister induction. This animal model for EBA should facilitate further dissection of the pathogenesis of this disease and development of new therapeutic strategies. [ABSTRACT FROM AUTHOR]

Hepatic ischemia reperfusion injury (IRI) remains a major obstacle in liver resection and transplantation surgery, especially in diseased organs. Human mesenchymal stromal cells (MSCs) are reported to acutely alleviate hepatic IRI in mice by releasing bioactive membrane-enclosed extracellular vesicles (EVs), but the long-term effects of MSC-derived EV on hepatic IRI are unknown. Given the considerable differentiation capacity of fibroblasts (FBs) during wound healing and their morphological similarities with MSC, the present study aimed to investigate the potential of these two cell types and their cell-derived EV in attenuating liver damage after IRI. EVs were isolated and purified from the supernatant of MSC and FB cultures and, subsequently, characterized by electron microscopy, nanoparticle tracking analysis, and western blot. Liver injury and organ regeneration in a murine in vivo model of IRI were assessed by serum transaminase levels, histopathology, and immunohistochemistry. Changes in expression of inflammation-associated genes within liver tissue were evaluated by reverse transcriptase quantitative polymerase chain reaction. MSC, MSC-derived EV, FB, and FB-derived EV were systemically administered before hepatic IRI. We found that MSC and MSC-derived EV decreased serum transaminase levels, reduced hepatic necrosis, increased the amount of Ki67-positive hepatocytes, and repressed the transcription of inflammation-associated genes. Although they had no impact on organ damage, FB and FB-derived EV showed some regenerative potential in the late phase of hepatic IRI. Compared to FB, MSC and their derived EV had a stronger potential to attenuate liver damage and improve organ regeneration after hepatic IRI. These results suggest that the key therapeutic factors are located within EV. [ABSTRACT FROM AUTHOR]

Aneurysm formation occurs most frequently as abdominal aortic aneurysm (AAA), but is also seen in other localizations like thoracic or peripheral aneurysm. While initial mechanisms for aneurysm induction remain elusive, observations from AAA samples show transmural inflammation with proteolytic imbalance and repair mechanisms triggered by the innate immune system. However, limited knowledge exists about aneurysm pathology, especially for others than AAA. We compared 42 AAA, 15 popliteal, 3 ascending aortic, five iliac, two femoral, two brachial, one visceral and two secondary aneurysms to non-aneurysmatic controls by histologic analysis, immunohistochemistry and cytokine expression. Muscular and elastic type arteries show a uniform way of aneurysm formation. All samples show similar morphology. The changes compared to controls are distinct and include matrix remodeling with smooth muscle cell phenotype switch and angiogenesis, adventitial lymphoid cell accumulation and M1 macrophage homing together with neutrophil inflammation. Inflammatory cytokines are up-regulated accordingly. Comparative analysis of different disease entities can identify characteristic pathomechanisms. The phenotype of human advanced aneurysm disease is observed for elastic and muscular type arteries, does not differ between disease localizations and might, thus, be a unique response of the vasculature to the still unknown trigger of aneurysm formation. [ABSTRACT FROM AUTHOR]

Colorectal carcinoma (CRC) is the most common cancer of the gastrointestinal tract with frequently dysregulated intracellular signaling pathways, including p53 signaling. The mainstay of chemotherapy treatment of CRC is 5-fluorouracil (5FU) and oxaliplatin. The two anticancer drugs mediate their therapeutic effect via DNA damage-triggered signaling. The small molecule reactivating p53 and inducing tumor apoptosis (RITA) is described as an activator of wildtype and reactivator of mutant p53 function, resulting in elevated levels of p53 protein, cell growth arrest, and cell death. Additionally, it has been shown that RITA can induce DNA damage signaling. It is expected that the therapeutic benefits of 5FU and oxaliplatin can be increased by enhancing DNA damage signaling pathways. Therefore, we highlighted the antiproliferative response of RITA alone and in combination with 5FU or oxaliplatin in human CRC cells. A panel of longterm established CRC cell lines (n = 9) including p53 wild-type, p53 mutant, and p53 null and primary patient-derived, low-passage cell lines (n = 5) with different p53 protein status were used for this study. A substantial number of CRC cells with pronounced sensitivity to RITA (IC50< 3.0 µmol/l) were identified within established (4/9) and primary patientderived (2/5) CRC cell lines harboring wild-type or mutant p53 protein. Sensitivity to RITA appeared independent of p53 status andwas associated with an increase in antiproliferative response to 5FUand oxaliplatin, a transcriptional increase of p53 targets p21 and NOXA, and a decrease in MYC mRNA. The effect of RITA as an inducer of DNA damage was shown by a strong elevation of phosphorylated histone variant H2A.X, which was restricted to RITA-sensitive cells. Our data underline the primary effect of RITA, inducingDNAdamage, and demonstrate the differential antiproliferative effect of RITA to CRC cells independent of p53 protein status. We found a substantial number of RITA-sensitive CRC cells within both panels of established CRC cell lines and primary patient-derived CRC cell lines (6/14) that provide a rationale for combining RITA with 5FU or oxaliplatin to enhance the antiproliferative response to both chemotherapeutic agents. [ABSTRACT FROM AUTHOR]

Background Evidence on bariatric surgery induced weight loss and its possible impact on cancer risk is limited, but also controversial. We used obese Zucker fa/fa and lean Zucker fa/+ to investigate the association between obesity, oxidative stress and genomic damage after weight loss induced either by Roux-en-Y gastric bypass surgery (RYGB) or caloric restriction. Methods Male Zucker fa/fa rats underwent RYGB ( n =15) or sham surgery ( n =17). Five shams were food restricted and body weight matched (BWM) to RYGB. Twelve Zucker fa/+ rats served as lean controls. Body weight and food intake were measured daily. An oral glucose tolerance test was performed on day 27. DHE staining and western blots of HSP70 and HO-1 were used to evaluate oxidative stress and anti-3-nitrotyrosine antibody staining for nitrative stress detection in colon and kidney. Lipid peroxidation products in urine were quantified by TBARS assay. LC/MS/MS was applied to measure urinary excretion of 8-oxoGua (oxidized DNA derived base), 8-oxodG (oxidized DNA derived nucleoside) and 8-oxoGuo (oxidized RNA derived nucleoside). DNA double strand breaks (DSBs) and cell proliferation (PCNA) were detected by immunohistochemistry. Results Sham-operated rats showed impaired glucose tolerance, elevated plasma insulin levels as well as elevated oxidative stress and nitrative stress markers, which were less severe after weight loss by RYGB or caloric restriction. Cell proliferation showed similar trends but no significant alteration. DNA DSBs were more frequent in sham-operated compared to all other groups. DNA damage in Zucker fa/fa rats positively correlated with basal plasma insulin values (Spearman's correlation coefficient for colon, 0.634 and for kidney, 0.525). Conclusions RYGB and caloric restriction were sufficient to significantly reduce elevated oxidative/nitrative stress and genomic damage in obese Zucker fa/fa rats. Further investigations are needed to elucidate the underlying mechanism of these genome protective effects. [ABSTRACT FROM AUTHOR]

Donor-derived immunity against tumor-associated antigens (TAAs) may exert selective antileukemic activity reprieving the allogeneic recipient from graft-versus-host disease. As TAAs are highly expressed in placental tissues we hypothesized that pregnancy could drive respective immunity in healthy individuals. Thus, we investigated the frequency and level of immune responses against clinically relevant TAAs in 114 blood donors and 44 women during their first pregnancy. Quantitative reverse-transcription polymerase chain reaction was employed to detect low levels of interferon-γ after primary peptide stimulation of CD8+ T lymphocytes. In blood donors, primary immune responses of low and/or high avidity were found against WT1 (15%), MUC1 (14%), PRAME (7%), and HER2/neu (5%) and exerted killing functions against leukemic cells. Men had higher responses than women, likely due to gonadal cancer-testis-antigen expression. Interestingly, a history of prior delivery was not associated with increased responses, whereas the strongest responses during pregnancy were found in early trimesters to disappear after delivery. This boost and loss of TAA-specific immunity suggests that virtually every donor harbors the potential to mount antileukemic immune responses in a recipient. However, in the absence of the driving target and a permissive environment, they are short-lived and thus require supplemental strategies such as vaccination or immunomodulation to facilitate their persistence. [ABSTRACT FROM AUTHOR]

Clinical prognosis of metastasized colorectal carcinoma (CRC) is still not at desired levels and novel drugs are needed. Here, we focused on the multi-tyrosine kinase inhibitor E7080 (Lenvatinib) and assessed its therapeutic efficacy against human CRC cell lines in vitro and human CRC xenografts in vivo. The effect of E7080 on cell viability was examined on 10 human CRC cell lines and humanendothelial cells (HUVEC). The inhibitory effect of E7080 on VEGF-induced angiogenesis was studied in an ex vivo mouse aortic ring angiogenesis assay. In addition, the efficacy of E7080 against xenografts derived from CRC cell lines and CRC patient resection specimens with mutated KRAS was investigated in vivo. A relatively low cytotoxic effect of E7080 on CRC cell viability was observed in vitro. Endothelial cells (HUVEC) were more susceptible to the incubation with E7080. This is in line with the observation that E7080 demonstrated an anti-angiogenic effect in a three-dimensional ex vivo mouse aortic ring angiogenesis assay. E7080 effectively disrupted CRC cell-mediated VEGF-stimulated growth of HUVEC in vitro. Daily in vivo treatment with E7080 (5 mg/kg) significantly delayed the growth of KRAS mutated CRC xenografts with decreased density of tumor-associated vessel formations and without tumor regression. This observation is in line with results that E7080 did not significantly reduce the number of Ki67-positive cells in CRC xenografts. The results suggest antiangiogenic activity of E7080 at a dosage that was well tolerated by nudemice. E7080 may provide therapeutic benefits in the treatment of CRC with mutated KRAS. [ABSTRACT FROM AUTHOR]

R2323 --> 826.12 --> Primordial germ cells (PGCs) are precursors of eggs and spermatozoa and serve as an effective means for the transgenic chickens production, including using CRISPR / Cas9 technology. The aim study is selection of PGCs culture media suitable for long‐term cultivation. All experiments on chickens were conducted with ethical approval of the Russian Research Institute of Farm Animal Genetics and Breeding ‐ Branch of the L.K. Ernst Federal Research Center for Animal Husbandry (Protocol Number: 2020‐3). For the work, eggs of the Pushkin breed chickens (n = 21) were selected from the Bioresource collection RRIFAGB (Pushkin, St. Petersburg). PGCs were selected from the embryos dorsal aorta for 3‐4 days of incubation (stage Hamburger‐Hamilton 14) using a microinjector (Narishige IM‐11‐2, Japan). Cells were cultured for 21 days at 37 ° C with 5% CO2. Three variants of the culture medium were used. The first basic medium was Opti‐MEM (Reduced Serum Medium, GlutaMAX Supplement) (Gibco, Thermo Fisher) with the addition of sodium pyruvate 1M, nucleosides 100X ‐ 2.5 ml, Chicken Serum (Gibco, Thermo Fisher) ‐ 2%, 2‐mercaptoethanol (NF, VWR) ‐ 3.9 μl. On day 21, the concentration of cells in the first culture reached 1,2х103, and some of the wells with cells were overgrown. The second base medium was similar to the first base medium, but with the addition of antimycotic antibiotic (Thermo Fisher) up to 1X. The medium with the use of the antibiotic‐antimycotic was less susceptible to overgrowth, on day 21, the concentration of cells in the culture reached 3.5x104. The third basic medium was KnockOut DMEM / F‐12, without L‐glutamine (Gibco, ThermoFisher) with the addition sodium pyruvate 1M, nucleosides 100X ‐ 2.5 ml, Human Activin A Recombinant Protein (Gibco, Thermo Fisher) ‐ 25 ng / μl, Human FGF‐basic (FGF‐2 / bFGF) Recombinant Protein (Gibco, Thermo Fisher) ‐ 10 ng / μl, Chicken Serum (Gibco, Thermo Fisher) ‐ 2%, 2‐mercaptoethanol (NF, VWR) ‐ 3.9 μl), antimycotic antibiotic (Thermo Fisher) up to 1X. The third medium with the addition of Activin A and FGF‐2 / bFGF supported the cell culture better, overgrowth was practically not observed, cells were actively proliferating, оn day 21, the concentration of cells in the culture reached 2.7x105. The PGCs presence was confirmed by expression high level (p≤0.01) specific marker genes PGCs such as CXCR4 (chemokine receptor CXC type 4) and PIWIL1 (Piwi‐like protein 1) in cultured cells. In a control tissue small intestine sample of an adult chicken (330 days old), the above genes expression was completely absent. [ABSTRACT FROM AUTHOR]

R2911 --> 515.7 --> The second messenger cyclic nucleotides cAMP and cGMP are key effectors of thermogenesis in adipocytes in response to a diverse array of cues. Their intracellular levels are normally regulated by the hydrolytic action of phosphodiesterases (PDEs), a conserved superfamily of enzymes composed of 12 families in humans (PDE1‐12) with distinct substrate specificities and tissue distribution profiles. Phosphodiesterase inhibitors are thus promising drugs for the treatment of metabolic disease by promoting cyclic nucleotide‐mediated adipose tissue thermogenesis, but so far mainly those that elevate either cAMP or cGMP signaling alone have been studied. We previously showed that inhibition of the dual‐specificity phosphodiesterase type 10A (PDE10A) with the small molecule drug MP‐10 sufficiently induces a thermogenic gene expression program in differentiated human white pre‐adipocytes (HWP‐ds) to a similar degree as combined cAMP and cGMP treatment. Here, we asked (1) which PDE10A isoforms are expressed in HWP‐ds, (2) the downstream signaling pathways PDE10A controls, and (3) how PDE10A expression is influenced by inflammatory stimuli in this cell type. Immunoblot analysis revealed that both the cytosolic (PDE10A1) and membrane‐bound (PDE10A2) isoforms of PDE10A are highly expressed in HWP‐ds, predominantly in the former subcellular compartment. This was confirmed by fractionation and immunofluorescence analyses. Inhibition of PDE10A with MP‐10 dose‐dependently increased PKA and PKG signaling as revealed by phosphorylation of cyclic response element binding protein (CREB) and vasodilator‐stimulated phosphoprotein (VASP) at S133 and S239, respectively. Consistently, the stimulation of lipolysis by MP‐10 was fully prevented by combined pre‐incubation of HWP‐ds with selective PKA and PKG inhibitors. Finally, mimicking the adipose tissue microenvironment in obesity by applying pro‐inflammatory cytokines onto HWP‐ds caused an early (4‐hour) and sustained (24‐hour) increase in PDE10A mRNA expression. These results provide new insight into the molecular character, function, and regulation of PDE10A in human adipocytes and further justify the use of selective inhibitors for the treatment of metabolic disease through stimulating adipose tissue thermogenesis in a more efficient and targeted manner. [ABSTRACT FROM AUTHOR]

Purpose: The in vitro and in vivo effects of pyrvinium pamoate (PP), a newly identified WNT signaling inhibitor, were evaluated against colon cancer cell lines and primary colon cancer samples. Experimental design: Antiproliferative activity of PP and its effects on protein and RNA levels of WNT targets were evaluated on adenomatous polyposis coli ( APC) and β-catenin cell lines, one WNT colon cancer cell line, as well as six primary colon cancer samples with mutant APC in vitro. In addition, the effect of PP on the growth of liver metastasis was examined. Results: PP blocked colon cancer cell growth in vitro in a dose-dependent manner with great differences in the inhibitory concentration (IC), ranging from 0.6 × 10 to 65 × 10 mol/L for colon cancer cells with mutations in WNT signaling. In addition, PP demonstrated a cytotoxic effect on primary colon cancer samples. A combined cytotoxic effect of PP with 5-fluorouracil (5-FU) was observed for two cell lines. PP decreased messenger RNA (mRNA) and protein levels of known WNT target genes as c-MYC and thereby led to the induction of p21. PP inhibited the migration of HCT116 colon cancer cells in vitro and decreased tumor growth in vivo after intraportal injection of HCT116 cells in nude mice. Conclusions: PP displays promising anticancer activity against a broad panel of human colon cancer cell lines, as well as primary colon cancer samples. However, our findings do not demonstrate a predominant cytotoxic effect of PP on colon cancer cells with mutations in WNT signaling. [ABSTRACT FROM AUTHOR]

OBJECTIVES: Reduced glutathione (GSH) has been shown to improve pulmonary graft preservation. Mitochondrial dysfunction is regarded to be the motor of ischemia–reperfusion injury (IR) in solid organs. We have shown previously that IR induces pulmonary mitochondrial damage. This study elucidates the impact of GSH preconditioning on the integrity and function of pulmonary mitochondria in the setting of warm pulmonary IR. METHODS: Wistar rats were subjected to control, sham, and to two-study-group conditions (IR30/60 and GSH-IR30/60) receiving IR with or without GSH preconditioning. Rats were anesthetized and received mechanical ventilation. Pulmonary in situ clamping followed by reperfusion generated IR. Mitochondria were isolated from pulmonary tissue. Respiratory chain complexes activities (I–IV) were analyzed by polarography. Mitochondrial viability (Ca2+-induced swelling) and membrane integrity (citrate synthase assay) were determined. Subcellular-fractional cytochrome C-content (Cyt C) was quantified by enzyme-linked immunosorbent assay (ELISA). Mitochondrial membrane potential (ΔΨm) was analyzed by fluorescence-activated cell sorting (FACS) after energizing and uncoupling. Inflammatory activation was determined by myeloperoxidase activity (MPO), matrix-metalloproteinase 9 (MMP-9) activity by gel zymography. RESULTS: Pulmonary IR significantly reduced mitochondrial viability in combination with ΔΨm hyper-polarization. GSH preconditioning improved mitochondrial viability and normalized ΔΨm. Cyt C was reduced after IR; GSH protected from Cyt C liberation. Respiratory chain complex activities (I, II, III) declined during IR; GSH protected complex II function. GSH also protected from MMP-9 and neutrophil sequestration (P > .05). CONCLUSIONS: GSH preconditioning is effective to prevent mitochondrial death and improves complex II function during IR, but not mitochondrial membrane stability. GSH-mediated amelioration of ΔΨm hyper-polarization appears to be the key factor of mitochondrial protection. [ABSTRACT FROM AUTHOR]

OBJECTIVE--Low cardiorespiratory fitness (CRF) predisposes one to cardiovascular disease and type 2 diabetes in part independently of body weight. Given the close relationship between intrahepatic lipid content (IHL) and insulin sensitivity, we hypothesized that the direct relationship between fitness and insulin sensitivity may be explained by IHL. RESEARCH DESIGN AND METHODS--We included 138 overweight to obese, otherwise healthy subjects (aged 43.6 ± 8.9 years, BMI 33.8 ± 4 kg/m2). Body composition was estimated by bioimpedance analyses. Abdominal fat distribution, intramyocellular, and IHL were assessed by magnetic resonance spectroscopy and tomography. Incremental exercise testing was performed to estimate an individual's CRF. Insulin sensitivity was determined during an oral glucose tolerance test. RESULTS--For all subjects, CRF was related to insulin sensitivity (r = 0.32, P < 0.05), IHL (r = -0.27, P < 0.05), and visceral (r = -0.25, P < 0.05) and total fat mass (r = -0.32, P < 0.05), but not to intramyocellular lipids (r = -0.08, NS). Insulin sensitivity correlated significantly with all fat depots. In multivariate regression analyses, independent predictors of insulin sensitivity were IHL, visceral fat, and fitness (r2 = -0.43, P < 0.01, 2 = -0.34, and 2 = 0.29, P < 0.05, respectively). However, the positive correlation between fitness and insulin sensitivity was abolished after adjustment for IHL (r = 0.16, NS), whereas it remained significant when adjusted for visceral or total body fat. Further, when subjects were grouped into high versus low IHL, insulin sensitivity was higher in those subjects with low IHL, irrespective of fitness levels. CONCLUSIONS--Our study suggests that the positive effect of increased CRF on insulin sensitivity in overweight to obese subjects may be mediated indirectly through IHL reduction. Diabetes 59:1640-1647, 2010 [ABSTRACT FROM AUTHOR]

• CD28 Superagonist stimulation leads to a robust expansion of mouse Tregs. • Typical Treg phenotype is not altered by CD28 Superagonist stimulation. • Treg suppressive quality remains unchanged by CD28 Superagonist activation. Regulatory T cells (Tregs) maintain immune homeostasis by regulating the activation of other immune cells. Preclinical studies show that the infusion of Tregs can promote immunological tolerance to allografts and prevent or cure multiple autoimmune diseases. However, Treg therapy is limited by high numbers of cells required to induce tolerance. In this study, we aimed at improving the in vitro expansion of sort purified mouse Tregs using the CD28 Superagonist (CD28-SA) D665 and comparing it to the conventional expansion using anti-CD3/anti-CD28 Dynabeads®. CD28-SA—stimulated Tregs expanded more than Dynabead®-stimulated Tregs while maintaining their phenotype by expressing the same level of CD4, CD25 and Foxp3. CD28-SA—expanded Tregs produced comparable amounts of IL-10 and TGFβ while showing a slightly superior suppressive capacity compared to Dynabead®-stimulated Tregs. Thus, stimulating murine Tregs with the CD28-SA is a promising alternative since it maintains their suppressive capacity without altering their phenotype and yields a higher fold expansion within 14 days. [ABSTRACT FROM AUTHOR]

Allotransplantation of microencapsulated parathyroid tissue is a promising approach to the treatment of permanent hypoparathyroidism. Preoperative assessment of the quality of microencapsulated parathyroid tissue could facilitate selection of the optimal bioartifical graft for human parathyroid allotransplantation. Parathyroid tissue from patients with secondary hyperparathyroidism ( n = 15) was processed mechanically or enzymatically (collagenase type II). Tissue particles and single cells/cell clusters were routinely microencapsulated with amitogenic Ba2+ alginate. Parathyroid secretion dynamics in response to stimulation of nonencapsulated and microencapsulated parathyroid tissue with Ca2+ were evaluated in a perifusion system. The stability of the different types of microcapsule was assessed using an osmotic pressure test. Mechanical cutting of parathyroid tissue led to peripheral necrosis of tissue particles and impaired their vitality. Collagenase digestion, in contrast, resulted in single cells and cell clusters without peripheral necrosis. The quality of microencapsulation of single cells/cell clusters was significantly better than that of tissue particles (deformed and imperfect capsules). Microencapsulation itself did not decrease cell vitality. Nonencapsulated and microencapsulated tissue particles and single cells/cell clusters from different donors maintained their own levels of response to stimulation with low Ca2+. Microcapsules containing tissue particles showed poor stability compared with those containing single cells/cell clusters. Preoperative evaluation of microencapsulated parathyroid tissue can disclose differences in vitality and function and thus facilitate selection of the optimal bioartifical graft for human parathyroid allotransplantation. [ABSTRACT FROM AUTHOR]

The recognition of major histocompatibility complex (MHC) allopeptides by recipient MHC class II-restricted CD4+ T cells via indirect pathway is a prerequisite for the generation of an immune response to the allograft. We tested 13-mer to 24-mer peptides from the MHC class I molecule for their possible immunogenicity in a fully MHC-mismatched rat strain combination. Our results confirm the hierarchical distribution of the immunogenicity of donor MHC class I peptides in the T cell alloactivation via indirect pathway. In addition, we show that allopeptide-induced immune response is critical for acute rejection of heart allografts. Among the seven allopeptides tested, peptide P1 was identified as immunodominant; it induced the greatest T cell proliferation and cytokine production in vitro as well as a significant reduction in allograft survival time. The TCR repertoire of T cells involved in the in vitro and in vivo responses induced by the dominant allopeptide P1 was found to be limited to the Vβ10 and Vβ 19 gene families. The identification of dominant allopeptides should greatly facilitate characterization of the specific T cell population responsible for allograft rejection and may be used to modulate the alloimmune response through antigen-specific therapy. [Copyright &y& Elsevier]

• Roux-en-Y gastric bypass surgery reduces body weight and suppresses food intake in obese zucker fatty rats. • Weight loss from Roux-en-Y gastric bypass surgery, but not from caloric restriction, is associated with increased systemic and portal vein growth differentiation factor 15 levels. • Systemic and portal vein growth differentiation factor 15 levels negatively correlate with body weight and food intake following Roux-en-Y gastric bypass surgery. Weight loss from caloric restriction (i.e. dieting) tends to be modest and short-lived, whereas from bariatric surgeries such as Roux-en-Y gastric bypass (RYGB) is pronounced and generally sustained. The reasons behind these opposing outcomes between interventions remain unclear, but likely involve differential effects on gut-brain communication. Growth differentiation factor 15 (GDF15) is a ubiquitously-induced, centrally-acting, anorexigenic cytokine whose systemic levels are elevated under a variety of conditions associated with a negative energy balance, including in patients following RYGB. We therefore asked whether systemic and portal vein GDF15 levels differ between obese Zucker fatty rats that experienced similar weight loss from RYGB or from forced caloric restriction (CR). Compared with ad libitum fed (ALF) controls, body weight, visceral adiposity and food intake of RYGB and CR rats were markedly lower during the postoperative observation period. Both systemic and portal vein GDF15 levels in RYGB rats at postoperative day 28 were higher compared with ALF rats and particularly compared with CR rats. Further, systemic and portal vein GDF15 levels negatively correlated with body weight and food intake specifically in RYGB rats. These findings provide evidence that, unlike dieting, RYGB might achieve sustained weight loss and appetite suppression partly through increased GDF15 release from epithelial cells of the gastrointestinal tract. [ABSTRACT FROM AUTHOR]

Adipose-derived stromal/stem cells (ASCs) have been shown to exert regenerative functions, which are mainly attributed to the secretion of trophic factors. Upon transplantation, ASCs are facing an ischemic environment characterized by oxygen and nutrient deprivation. However, current knowledge on the secretion capacity of ASCs under such conditions is limited. Thus, the present study focused on the secretory function of ASCs under glucose and oxygen deprivation as major components of ischemia. After exposure to glucose/oxygen deprivation, ASCs maintained distinct viability, but the metabolic activity was greatly reduced by glucose limitation. ASCs were able to secrete a broad panel of factors under glucose/oxygen deprivation as revealed by a cytokine antibody array. Quantification of selected factors by ELISA demonstrated that glucose deprivation in combination with hypoxia led to markedly higher secretion levels of the angiogenic and anti-apoptotic factors IL-6, VEGF, and stanniocalcin-1 as compared to the hypoxic condition alone. A conditioned medium of glucose/oxygen-deprived ASCs promoted the viability and tube formation of endothelial cells, and the proliferation and migration of fibroblasts. These findings indicate that ASCs are stimulated by ischemia-like stress conditions to secrete trophic factors and would be able to exert their beneficial function in an ischemic environment. [ABSTRACT FROM AUTHOR]

Small cell lung cancer (SCLC) is the most aggressive lung cancer entity with an extremely limited therapeutic outcome. Most patients are diagnosed at an extensive stage. However, the molecular mechanisms driving SCLC invasion and metastasis remain largely elusive. We used an autochthonous SCLC mouse model and matched samples from patients with primary and metastatic SCLC to investigate the molecular characteristics of tumor metastasis. We demonstrate that tumor cell invasion and liver metastasis in SCLC are triggered by an Angiopoietin-2 (ANG-2)/Integrin β-1-dependent pathway in tumor cells, mediated by focal adhesion kinase/Src kinase signaling. Strikingly, CRISPR-Cas9 KO of Integrin β-1 or blocking Integrin β-1 signaling by an anti-ANG-2 treatment abrogates liver metastasis formation in vivo. Interestingly, analysis of a unique collection of matched samples from patients with primary and metastatic SCLC confirmed a strong increase of Integrin β-1 in liver metastasis in comparison with the primary tumor. We further show that ANG-2 blockade combined with PD-1-targeted by anti-PD-1 treatment displays synergistic treatment effects in SCLC. Together, our data demonstrate a fundamental role of ANG-2/Integrin β-1 signaling in SCLC cells for tumor cell invasion and liver metastasis and provide a potentially new effective treatment strategy for patients with SCLC.

Loss of intestinal epithelial barrier function is a hallmark in digestive tract inflammation. The detailed mechanisms remain unclear due to the lack of suitable cell-based models in barrier research. Here we performed a detailed functional characterization of human intestinal organoid cultures under different conditions with the aim to suggest an optimized ex-vivo model to further analyse inflammation-induced intestinal epithelial barrier dysfunction. Differentiated Caco2 cells as a traditional model for intestinal epithelial barrier research displayed mature barrier functions which were reduced after challenge with cytomix (TNFα, IFN-γ, IL-1ß) to mimic inflammatory conditions. Human intestinal organoids grown in culture medium were highly proliferative, displayed high levels of LGR5 with overall low rates of intercellular adhesion and immature barrier function resembling conditions usually found in intestinal crypts. WNT-depletion resulted in the differentiation of intestinal organoids with reduced LGR5 levels and upregulation of markers representing the presence of all cell types present along the crypt-villus axis. This was paralleled by barrier maturation with junctional proteins regularly distributed at the cell borders. Application of cytomix in immature human intestinal organoid cultures resulted in reduced barrier function that was accompanied with cell fragmentation, cell death and overall loss of junctional proteins, demonstrating a high susceptibility of the organoid culture to inflammatory stimuli. In differentiated organoid cultures, cytomix induced a hierarchical sequence of changes beginning with loss of cell adhesion, redistribution of junctional proteins from the cell border, protein degradation which was accompanied by loss of epithelial barrier function. Cell viability was observed to decrease with time but was preserved when initial barrier changes were evident. In summary, differentiated intestinal organoid cultures represent an optimized human ex-vivo model which allows a comprehensive reflection to the situation observed in patients with intestinal inflammation. Our data suggest a hierarchical sequence of inflammation-induced intestinal barrier dysfunction starting with loss of intercellular adhesion, followed by redistribution and loss of junctional proteins resulting in reduced barrier function with consecutive epithelial death., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kollmann, Buerkert, Meir, Richter, Kretzschmar, Flemming, Kelm, Germer, Otto, Burkard and Schlegel.)

Fibroblast growth factor (FGF)-inducible 14 (Fn14) activates the classical and alternative NFκB (nuclear factor 'kappa-light-chain-enhancer' of activated B-cells) signaling pathway but also enhances tumor necrosis factor (TNF)-induced cell death. Fn14 expression is upregulated in non-hematopoietic cells during tissue injury and is also often highly expressed in solid cancers. In view of the latter, there were and are considerable preclinical efforts to target Fn14 for tumor therapy, either by exploiting Fn14 as a target for antibodies with cytotoxic activity (e.g. antibody-dependent cellular cytotoxicity (ADCC)-inducing IgG variants, antibody drug conjugates) or by blocking antibodies with the aim to interfere with protumoral Fn14 activities. Noteworthy, there are yet no attempts to target Fn14 with agonistic Fc effector function silenced antibodies to unleash the proinflammatory and cell death-enhancing activities of this receptor for tumor therapy. This is certainly not at least due to the fact that anti-Fn14 antibodies only act as effective agonists when they are presented bound to Fcγ receptors (FcγR). Thus, there are so far no antibodies that robustly and selectively engage Fn14 signaling without triggering unwanted FcγR-mediated activities. In this study, we investigated a panel of variants of the anti-Fn14 antibody 18D1 of different valencies and domain architectures with respect to their inherent FcγR-independent ability to trigger Fn14-associated signaling pathways. In contrast to conventional 18D1, the majority of 18D1 antibody variants with four or more Fn14 binding sites displayed a strong ability to trigger the alternative NFκB pathway and to enhance TNF-induced cell death and therefore resemble in their activity soluble (TNF)-like weak inducer of apoptosis (TWEAK), one form of the natural occurring ligand of Fn14. Noteworthy, activation of the classical NFκB pathway, which naturally is predominately triggered by membrane-bound TWEAK but not soluble TWEAK, was preferentially observed with a subset of constructs containing Fn14 binding sites at opposing sites of the IgG scaffold, e.g. IgG1-scFv fusion proteins. A superior ability of IgG1-scFv fusion proteins to trigger classical NFκB signaling was also observed with the anti-Fn14 antibody PDL192 suggesting that we identified generic structures for Fn14 antibody variants mimicking soluble and membrane-bound TWEAK., Competing Interests: The University of Würzburg filed a patent describing novel Fn14 agonists with authors OZ, CO and HW as co-inventors. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zaitseva, Hoffmann, Löst, Anany, Zhang, Kucka, Wiegering, Otto and Wajant.)

Fibroblast growth factor-inducible 14 (Fn14) is a member of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF) and is activated by its ligand TNF-like weak inducer of apoptosis (TWEAK). The latter occurs as a homotrimeric molecule in a soluble and a membrane-bound form. Soluble TWEAK (sTWEAK) activates the weakly inflammatory alternative NF-κB pathway and sensitizes for TNF-induced cell death while membrane TWEAK (memTWEAK) triggers additionally robust activation of the classical NF-κB pathway and various MAP kinase cascades. Fn14 expression is limited in adult organisms but becomes strongly induced in non-hematopoietic cells by a variety of growth factors, cytokines and physical stressors (e.g., hypoxia, irradiation). Since all these Fn14-inducing factors are frequently also present in the tumor microenvironment, Fn14 is regularly found to be expressed by non-hematopoietic cells of the tumor microenvironment and most solid tumor cells. In general, there are three possibilities how the tumor-Fn14 linkage could be taken into consideration for tumor therapy. First, by exploitation of the cancer associated expression of Fn14 to direct cytotoxic activities (antibody-dependent cell-mediated cytotoxicity (ADCC), cytotoxic payloads, CAR T-cells) to the tumor, second by blockade of potential protumoral activities of the TWEAK/Fn14 system, and third, by stimulation of Fn14 which not only triggers proinflammtory activities but also sensitizes cells for apoptotic and necroptotic cell death. Based on a brief description of the biology of the TWEAK/Fn14 system and Fn14 signaling, we discuss the features of the most relevant Fn14-targeting biologicals and review the preclinical data obtained with these reagents. In particular, we address problems and limitations which became evident in the preclinical studies with Fn14-targeting biologicals and debate possibilities how they could be overcome., Competing Interests: The University of Wuerzburg filed a patent describing novel Fn14 agonists with OZ, CO, and HW as co-inventors. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zaitseva, Hoffmann, Otto and Wajant.)

Background: Roux-en-Y gastric bypass surgery (RYGB) improves glycemic control in individuals with severe obesity beyond the effects of weight loss alone. To identify potential underlying mechanisms, we asked how equivalent weight loss from RYGB and from chronic caloric restriction impact gut release of the metabolically beneficial cytokine interleukin-22 (Il-22)., Methods: Obese male Zucker fatty rats were randomized into sham-operated (Sham), RYGB, and sham-operated, body weight-matched to RYGB (BWM) groups. Food intake and body weight were measured regularly for 4 weeks. An oral glucose tolerance test (OGTT) was performed on postoperative day 27. Portal vein plasma, systemic plasma, and whole-wall samples from throughout the gut were collected on postoperative day 28. Gut Il- 22 mRNA expression was determined by real-time quantitative PCR. Plasma Il-22 levels were determined by enzyme-linked immunosorbant assay (ELISA)., Results: RYGB and BWM rats had lower food intake and body weight as well as superior blood glucose clearing capability compared with Sham rats. RYGB rats also had superior blood glucose clearing capability compared with BWM rats despite having similar body weights and higher food intake. Il- 22 mRNA expression was approximately 100-fold higher specifically in the upper jejunum in RYGB rats compared with Sham rats. Il-22 protein was only detectable in portal vein (34.1 ± 9.4 pg/mL) and systemic (46.9 ± 10.5 pg/mL) plasma in RYGB rats. Area under the curve of blood glucose during the OGTT, but not food intake or body weight, negatively correlated with portal vein and systemic plasma Il-22 levels in RYGB rats., Conclusions: These results suggest that induction of gut Il-22 release might partly account for the weight loss-independent improvements in glycemic control after RYGB, and further support the use of this cytokine for the treatment of metabolic disease., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors.)

Ribosomal biogenesis and protein synthesis are deregulated in most cancers, suggesting that interfering with translation machinery may hold significant therapeutic potential. Here, we show that loss of the tumor suppressor adenomatous polyposis coli (APC), which constitutes the initiating event in the adenoma carcinoma sequence for colorectal cancer (CRC), induces the expression of RNA polymerase I (RNAPOL1) transcription machinery, and subsequently upregulates ribosomal DNA (rDNA) transcription. Targeting RNAPOL1 with a specific inhibitor, CX5461, disrupts nucleolar integrity, and induces a disbalance of ribosomal proteins. Surprisingly, CX5461-induced growth arrest is irreversible and exhibits features of senescence and terminal differentiation. Mechanistically, CX5461 promotes differentiation in an MYC-interacting zinc-finger protein 1 (MIZ1)- and retinoblastoma protein (Rb)-dependent manner. In addition, the inhibition of RNAPOL1 renders CRC cells vulnerable towards senolytic agents. We validated this therapeutic effect of CX5461 in murine- and patient-derived organoids, and in a xenograft mouse model. These results show that targeting ribosomal biogenesis together with targeting the consecutive, senescent phenotype using approved drugs is a new therapeutic approach, which can rapidly be transferred from bench to bedside., (© 2022 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Unexploded ordnance devices (UXO) pose a potential threat to human life and material during offshore construction activities. Extensive survey activities are conducted to locate, identify, and clear these objects as necessary. For the period thereafter, it is necessary to investigate whether areas that have already been cleared, or even objects that remain in place, may be affected by mobilization under tidal currents or waves, and could thus have an impact on operation and maintenance during the lifetime of the offshore installation. In this study, model simulations based on fluid mechanics are described to derive the loads on the objects caused by currents and waves and combined with knowledge of the known burial condition of the objects. Within the model, the hydrodynamic and hydrostatic loads on the object caused by waves and currents are balanced with inertia and rolling resistance. Thus, the critical current velocity and critical wave conditions for the mobilization of different objects are calculated and compared with the environmental conditions prevailing in the North Sea. As a result, a recurrence interval for the potential mobilization of objects on the seafloor is given, which can now be used to optimize route surveys and thus help accelerate offshore construction work. It is shown that currents are not able to mobilize the objects investigated in the study in almost all regions of the North Sea. Waves can mobilize certain objects in very shallow and extreme conditions.

Background: The hypothalamus is an important brain region for the regulation of energy balance. Roux-en-Y gastric bypass (RYGB) surgery and gut hormone-based treatments are known to reduce body weight, but their effects on hypothalamic gene expression and signaling pathways are poorly studied., Methods: Diet-induced obese male Wistar rats were randomized into the following groups: RYGB, sham operation, sham + body weight-matched (BWM) to the RYGB group, osmotic minipump delivering PYY3-36 (0.1 mg/kg/day), liraglutide s.c. (0.4 mg/kg/day), PYY3-36 + liraglutide, and saline. All groups (except BWM) were kept on a free choice of high- and low-fat diets. Four weeks after interventions, hypothalami were collected for RNA sequencing., Results: While rats in the RYGB, BWM, and PYY3-36 + liraglutide groups had comparable reductions in body weight, only RYGB and BWM treatment had a major impact on hypothalamic gene expression. In these groups, hypothalamic leptin receptor expression as well as the JAK-STAT, PI3K-Akt, and AMPK signaling pathways were upregulated. No significant changes could be detected in PYY3-36 + liraglutide-, liraglutide-, and PYY-treated groups., Conclusions: Despite causing similar body weight changes compared to RYGB and BWM, PYY3-36 + liraglutide treatment does not impact hypothalamic gene expression. Whether this striking difference is favorable or unfavorable to metabolic health in the long term requires further investigation.

Ambitions to improve the connectivity of devices to enable automation and digital representation of processes have been around for some time. Nevertheless, some companies, especially in life science and analytical science, tend to adopt these developments rather slowly. In the field of microbial analysis of drinking and process water, for example, a large part of the work is still carried out manually, although the high number of samples per day and the low fluctuation in work processes would predestine water analysis for a higher degree of automation. Obstacles such as the risk of bottlenecks and possible downtimes after machine failure, the spatial conditions together with the low flexibility of the automated system, a lack of trained personnel, and the high acquisition costs hinder this development, however.To lower these barriers, we have developed a system for the generation of flexibly expandable automated process lines, which handles sample handling and sample transport as a decisive step in the networking of several devices. The system allows the connection of devices that are distributed over the entire laboratory or close to each other, as well as those with a combination of both spatial situations. A functional or throughput expansion of the process can be realized by adding additional devices or storage areas to the network.With this concept, we have established a system for the automatic processing of defined steps of a routine Legionella pneumophila screening in drinking water testing. From this starting point, the process can be extended to cover further steps, such as concentrating or plating, up to the full analytical workflow.

Inflammation-induced reduction of intestinal desmosomal cadherin Desmoglein 2 (Dsg2) is linked to changes of tight junctions (TJ) leading to impaired intestinal epithelial barrier (IEB) function by undefined mechanisms. We characterized the interplay between loss of Dsg2 and upregulation of pore-forming TJ protein Claudin2. Intraperitoneal application of Dsg2-stablising Tandem peptide (TP) attenuated impaired IEB function, reduction of Dsg2 and increased Claudin2 in DSS-induced colitis in C57Bl/6 mice. TP blocked loss of Dsg2-mediated adhesion and upregulation of Claudin2 in Caco2 cells challenged with TNFα. In Dsg2-deficient Caco2 cells basal expression of Claudin2 was increased which was paralleled by reduced transepithelial electrical resistance and by augmented phosphorylation of AKT Ser473 under basal conditions. Inhibition of phosphoinositid-3-kinase proved that PI-3-kinase/AKT-signaling is critical to upregulate Claudin2. In immunostaining PI-3-kinase dissociated from Dsg2 under inflammatory conditions. Immunoprecipitations and proximity ligation assays confirmed a direct interaction of Dsg2 and PI-3-kinase which was abrogated following TNFα application. In summary, Dsg2 regulates Claudin2 expression by sequestering PI-3-kinase to the cell borders in intestinal epithelium., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Burkard, Meir, Kannapin, Otto, Petzke, Germer, Waschke and Schlegel.)

Abdominal aortic aneurysm (AAA) is a disease with high morbidity and mortality, especially when ruptured. The rationale of this study was to evaluate the repurposing of lenvatinib, a multi-tyrosine kinase inhibitor, in limiting experimental AAA growth targeting vascular smooth muscle cells (VSMCs) and angiogenesis. We applied systemic and local lenvatinib treatment to elastase-induced murine aortic aneurysms, and RNA profiling identified myosin heavy chain 11 (Myh11) as the most deregulated transcript. Daily oral treatment substantially reduced aneurysm formation in 2 independent mouse models. In addition, a large animal aneurysm model in hypercholesterolemic low-density lipoprotein receptor-knockout (LDLR-/-) Yucatan minipigs was applied to endovascularly deliver lenvatinib via drug-eluting balloons (DEBs). Here, a single local endovascular delivery blocked AAA progression successfully compared with a DEB-delivered control treatment. Reduced VSMC proliferation and a restored contractile phenotype were observed in animal tissues (murine and porcine), as well as AAA patient-derived cells. Apart from increasing MYH11 levels, lenvatinib reduced downstream ERK signaling. Hence, lenvatinib is a promising therapy to limit aortic aneurysm expansion upon local endovascular delivery. The tyrosine kinase inhibitor was able to positively affect pathways of key relevance to human AAA disease, even in a potentially new local delivery using DEBs.

Sensitization to the adipokine leptin is a promising therapeutic strategy against obesity and its comorbidities and has been proposed to contribute to the lasting metabolic benefits of Roux-en-Y gastric bypass (RYGB) surgery. We formally tested this idea using Zucker fatty fa/fa rats as an established genetic model of obesity, glucose intolerance, and fatty liver due to leptin receptor deficiency. We show that the changes in body weight in these rats following RYGB largely overlaps with that of diet-induced obese Wistar rats with intact leptin receptors. Further, food intake and oral glucose tolerance were normalized in RYGB-treated Zucker fatty fa/fa rats to the levels of lean Zucker fatty fa/+ controls, in association with increased glucagon-like peptide 1 (GLP-1) and insulin release. In contrast, while fatty liver was also normalized in RYGB-treated Zucker fatty fa/fa rats, their circulating levels of the liver enzyme alanine aminotransferase (ALT) remained elevated at the level of obese Zucker fatty fa/fa controls. These findings suggest that the leptin system is not required for the normalization of energy and glucose homeostasis associated with RYGB, but that its potential contribution to the improvements in liver health postoperatively merits further investigation.

Background: Combination therapies of anorectic gut hormones partially mimic the beneficial effects of bariatric surgery. Thus far, the effects of a combined chronic systemic administration of Glucagon-like peptide-1 (GLP-1) and peptide tyrosine tyrosine 3-36 (PYY 3-36 ) have not been directly compared to Roux-en-Y gastric bypass (RYGB) in a standardized experimental setting., Methods: High-fat diet (HFD)-induced obese male Wistar rats were randomized into six treatment groups: (1) RYGB, (2) sham-operation (shams), (3) liraglutide, (4) PYY 3-36 , (5) PYY 3-36 +liraglutide (6), saline. Animals were kept on a free choice high- and low-fat diet. Food intake, preference, and body weight were measured daily for 4 weeks. Open field (OP) and elevated plus maze (EPM) tests were performed., Results: RYGB reduced food intake and achieved sustained weight loss. Combined PYY 3-36 +liraglutide treatment led to similar and plateaued weight loss compared to RYGB. Combined PYY 3-36 +liraglutide treatment was superior to PYY 3-36 (p ≤ 0.0001) and liraglutide (p ≤ 0.05 or p ≤ 0.01) mono-therapy. PYY 3-36 +liraglutide treatment and RYGB also reduced overall food intake and (less pronounced) high-fat preference compared to controls. The animals showed no signs of abnormal behavior in OF or EPM., Conclusions: Liraglutide and PYY 3-36 combination therapy vastly mimics reduced food intake, food choice and weight reducing benefits of RYGB., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Dischinger, Hasinger, Königsrainer, Corteville, Otto, Fassnacht, Hankir and Seyfried.)

Regional changes to the intestinal microenvironment brought about by Roux-en-Y gastric bypass (RYGB) surgery may contribute to some of its potent systemic metabolic benefits through favorably regulating various local cellular processes. Here, we show that the intestinal contents of RYGB-operated compared with sham-operated rats region-dependently confer superior glycemic control to recipient germ-free mice in association with suppression of endotoxemia. Correspondingly, they had direct barrier-stabilizing effects on an intestinal epithelial cell line which, bile-exposed intestinal contents, were partly farnesoid X receptor (FXR)-dependent. Further, circulating fibroblast growth factor 19 levels, a readout of intestinal FXR activation, negatively correlated with endotoxemia severity in longitudinal cohort of RYGB patients. These findings suggest that various host- and/or microbiota-derived luminal factors region-specifically and synergistically stabilize the intestinal epithelial barrier following RYGB through FXR signaling, which could potentially be leveraged to better treat endotoxemia-induced insulin resistance in obesity in a non-invasive and more targeted manner., Competing Interests: The authors declare no competing interests., (© 2020 The Authors.)

In enterocytes, protein RS1 ( RSC1A1 ) mediates an increase of glucose absorption after ingestion of glucose-rich food via upregulation of Na + -d-glucose cotransporter SGLT1 in the brush-border membrane (BBM). Whereas RS1 decelerates the exocytotic pathway of vesicles containing SGLT1 at low glucose levels between meals, RS1-mediated deceleration is relieved after ingestion of glucose-rich food. Regulation of SGLT1 is mediated by RS1 domain RS1-Reg, in which Gln-Ser-Pro (QSP) is effective. In contrast to QSP and RS1-Reg, Gln-Glu-Pro (QEP) and RS1-Reg with a serine to glutamate exchange in the QSP motif downregulate the abundance of SGLT1 in the BBM at high intracellular glucose concentrations by about 50%. We investigated whether oral application of QEP improves diabetes in db/db mice and affects the induction of diabetes in New Zealand obese (NZO) mice under glucolipotoxic conditions. After 6-day administration of drinking water containing 5 mM QEP to db/db mice, fasting glucose was decreased, increase of blood glucose in the oral glucose tolerance test was blunted, and insulin sensitivity was increased. When QEP was added for several days to a high fat/high carbohydrate diet that induced diabetes in NZO mice, the increase of random plasma glucose was prevented, accompanied by lower plasma insulin levels. QEP is considered a lead compound for development of new antidiabetic drugs with more rapid cellular uptake. In contrast to SGLT1 inhibitors, QEP-based drugs may be applied in combination with insulin for the treatment of type 1 and type 2 diabetes, decreasing the required insulin amount, and thereby may reduce the risk of hypoglycemia., Competing Interests: The authors declare no competing financial interest., (© 2020 American Chemical Society.)

Background: The aim of this study is to evaluate perioperative as well as long-term outcomes in patients operated with carotid endarterectomy (CEA) or stenting (CAS) due to symptomatic or asymptomatic high-grade restenosis of the internal carotid artery (ICA). Patients and methods: In a retrospective analysis of our electronic database including 2980 patients who underwent carotid endarterectomy or stenting due to a symptomatic or asymptomatic high-grade stenosis of the ICA, between 2000 and 2016, we enrolled 111 patients with recurrent ICA stenosis. Results: An ipsilateral 2nd time restenosis (> 80 % in the asymptomatic and > 50 % in the symptomatic patients according to NASCET criteria) of ICA was detected in 13 patients (12 %); 3 of them were symptomatic. These patients were managed with either CEA (n = 5/38 %) or CAS (n = 8/62 %) with no perioperative stroke or death. The stroke-free survival rates at 2 and 8 years for CEA were 98 % and 98 % versus 100 % and 100 % for CAS respectively (P = .271). The type of the initial procedure (patch, CAS or interposition) did not play any significant role for the development of a 2nd time restenosis (P = .841). Conclusions : Redo-CEA/CAS seem to have similar results as primary procedures (as reported in the literature) with favorable periprocedural and long-term outcomes.

In vitro models represent a critical tool in cancer research to study tumor biology and to evaluate new treatment options. Unfortunately, there are no effective preclinical models available that represent Wilms tumor (WT) - the most common pediatric renal tumor. Especially the high-risk blastemal WT subtype is not represented by the few primary cell lines established until now. Here, we describe a new 3D approach for in vitro cultivation of blastemal WT cells, where primary cultures grown in suspension as spheroids could be propagated long-term. Besides blastemal cultures, we could generate spheroids representing epithelial and stromal WT. Spheroid cultures were analyzed by immunohistochemistry in comparison to corresponding tumor sections and were further characterized by RNA sequencing. Histological appearance of spheroids resembled the original tumor and they expressed marker genes characteristic of early renal development and blastemal WT elements. The cultures were amenable to genetic manipulation and they formed xenograft tumors, which resemble the primary human tumor. This collection of WT spheroids that carry different genetic drivers forms a long-sought tool for drug testing and in vitro modeling.

Tumours depend on altered rates of protein synthesis for growth and survival, which suggests that mechanisms controlling mRNA translation may be exploitable for therapy. Here, we show that loss of APC, which occurs almost universally in colorectal tumours, strongly enhances the dependence on the translation initiation factor eIF2B5. Depletion of eIF2B5 induces an integrated stress response and enhances translation of MYC via an internal ribosomal entry site. This perturbs cellular amino acid and nucleotide pools, strains energy resources and causes MYC-dependent apoptosis. eIF2B5 limits MYC expression and prevents apoptosis in APC-deficient murine and patient-derived organoids and in APC-deficient murine intestinal epithelia in vivo. Conversely, the high MYC levels present in APC-deficient cells induce phosphorylation of eIF2α via the kinases GCN2 and PKR. Pharmacological inhibition of GCN2 phenocopies eIF2B5 depletion and has therapeutic efficacy in tumour organoids, which demonstrates that a negative MYC-eIF2α feedback loop constitutes a targetable vulnerability of colorectal tumours.

Background: Roux-en-Y gastric bypass (RYGB) modifies various aspects of eating behavior in morbidly obese individuals to cause marked and lasting weight loss and improvements in metabolic health, but the underlying mechanisms remain poorly understood., Objectives: To assess the relative contributions of the gut hormones glucagon-like peptide 1 (GLP-1) and peptide tyrosine tyrosine 3-36 (PYY 3-36 ), whose circulating levels are enhanced by RYGB, in the reduced high-fat (HF) food preference that develops postoperatively., Setting: University hospital, Würzburg, Germany., Methods: HF diet-induced obese male Wistar rats underwent RYGB (n = 11) or sham (n = 7) surgeries and were subsequently maintained on a choice of low-fat (10% calories from fat) and HF (60% calories from fat) diets. From postoperative weeks 4 to 6, acute feeding studies were performed in which the selective GLP-1 receptor antagonist exendin-9 (30 μg/kg), the second-generation selective Y2 receptor antagonist JNJ-31020028 (10 mg/kg), or a combination of both drugs was administered intraperitoneally., Results: During the observational period weight, adiposity and total food intake were lower while postprandial plasma GLP-1 and peptide tyrosine tyrosine levels were higher for RYGB-operated compared with sham-operated rats. There was a gradual shift in preference from HF to low-fat food in RYGB-operated rats by postoperative week 3. Single antagonist treatments had a relatively modest impact on HF food preference in rats from both surgical groups. However, dual antagonist treatment caused a striking increase in HF food preference specifically in RYGB-operated rats., Conclusions: GLP-1 and peptide tyrosine tyrosine 3-36 reduce HF food preference additively after RYGB supporting the use of gut hormone combination strategies for healthier feeding behavior., (Copyright © 2019 American Society for Bariatric Surgery. Published by Elsevier Inc. All rights reserved.)

TNF-like weak inducer of apoptosis (TWEAK) and inhibition of protein synthesis with cycloheximide (CHX) sensitize for poly(I:C)-induced cell death. Notably, although CHX preferentially enhanced poly(I:C)-induced apoptosis, TWEAK enhanced primarily poly(I:C)-induced necroptosis. Both sensitizers of poly(I:C)-induced cell death, however, showed no major effect on proinflammatory poly(I:C) signaling. Analysis of a panel of HeLa-RIPK3 variants lacking TRADD, RIPK1, FADD, or caspase-8 expression revealed furthermore similarities and differences in the way how poly(I:C)/TWEAK, TNF, and TRAIL utilize these molecules for signaling. RIPK1 turned out to be essential for poly(I:C)/TWEAK-induced caspase-8-mediated apoptosis but was dispensable for this response in TNF and TRAIL signaling. TRADD-RIPK1-double deficiency differentially affected poly(I:C)-triggered gene induction but abrogated gene induction by TNF completely. FADD deficiency abrogated TRAIL- but not TNF- and poly(I:C)-induced necroptosis, whereas TRADD elicited protective activity against all three death inducers. A general protective activity against poly(I:C)-, TRAIL-, and TNF-induced cell death was also observed in FLIP L and FLIP S transfectrants.

Objectives: Glycemic control by medical treatment represents one therapeutic strategy for diabetic patients. The Na+-d-glucose cotransporter 1 (SGLT1) is currently of high interest in this context. SGLT1 is known to mediate glucose absorption and incretin secretion in the small intestine. Recently, inhibition of SGLT1 function was shown to improve postprandial hyperglycemia. In view of the lately demonstrated SGLT1 expression in pancreatic islets, we investigated if loss of SGLT1 affects islet morphology and function., Methods: Effects associated with the loss of SGLT1 on pancreatic islet (cyto) morphology and function were investigated by analyzing islets of a SGLT1 knockout mouse model, that were fed a glucose-deficient, fat-enriched diet (SGLT1 -/- -GDFE) to circumvent the glucose-galactose malabsorption syndrome. To distinguish diet- and Sglt1 -/- -dependent effects, wildtype mice on either standard chow (WT-SC) or the glucose-free, fat-enriched diet (WT-GDFE) were used as controls. Feeding a glucose-deficient, fat-enriched diet further required the analysis of intestinal SGLT1 expression and function under diet-conditions., Results: Consistent with literature, our data provide evidence that small intestinal SGLT1 mRNA expression and function is regulated by nutrition. In contrast, pancreatic SGLT1 mRNA levels were not affected by the applied diet, suggesting different regulatory mechanisms for SGLT1 in diverse tissues. Morphological changes such as increased islet sizes and cell numbers associated with changes in proliferation and apoptosis and alterations of the β- and α-cell population are specifically observed for pancreatic islets of SGLT1 -/- -GDFE mice. Glucose stimulation revealed no insulin response in SGLT1 -/- -GDFE mice while WT-GDFE mice displayed only a minor increase of blood insulin. Irregular glucagon responses were observed for both, SGLT1 -/- -GDFE and WT-GDFE mice. Further, both animal groups showed a sustained release of GLP-1 compared to WT-SC controls., Conclusion: Loss or impairment of SGLT1 results in abnormal pancreatic islet (cyto)morphology and disturbed islet function regarding the insulin or glucagon release capacity from β- or α-cells, respectively. Consequently, our findings propose a new, additional role for SGLT1 maintaining proper islet structure and function., (Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Obesity is associated with several detrimental health consequences, among them an increased risk for development of cancer, and an overall elevated mortality. Multiple factors like hyperinsulinemia, chronic microinflammation and oxidative stress may be involved. The comet assay has been proven to be very sensitive for detection of DNA damage and has been used to explore the relationship between overweight/obesity and DNA damage, but results are controversial. Very few investigations have been performed to correlate weight loss of obese individuals and possible reduction of DNA damage and these studies have not provided clear results. As currently, only surgical interventions (metabolic/bariatric surgery) enable substantial and sustained weight loss in the vast majority of morbidly obese patients, we analyzed whole blood samples of 56 subsequent patients prior, 6 and 12 months after bariatric surgery. No reduction of DNA damage was observed in comet assay analysis after 6 months despite efficient weight loss, but a significant reduction was observed 12 months after surgery. Concurrently, the ferric-reducing antioxidant power assay showed a significant reduction after 6 and 12 months. The level of oxidised glutathione and lipid peroxidation products were increased at 6 months but normalised at 12 months after surgery. As conclusion, a significant weight reduction in obese patients may help to diminish existing DNA damage besides improving many other health aspects in these patients.

Background: Roux-en-Y gastric bypass (RYGB) may improve beta cell function by mechanisms other than caloric restriction and body weight loss. We aimed to assess the impact of anatomical and hormonal alterations specific to RYGB on glucose homeostasis, β cell function and morphology., Methods: Male Zucker(fa/fa) rats underwent either RYGB (n = 11) or sham surgeries (n = 10). Five of the shams were then food restricted and body weight matched (BWM) to the RYGB rats. Six male Zucker(fa/+) rats underwent sham surgery and served as additional lean controls. Twenty-seven days after surgery, an oral glucose tolerance test (OGTT) was performed and plasma levels of glucose, insulin and glucagon-like peptide-1 (GLP-1) were measured. Immunohistological analysis of pancreatic islets was performed, and GLP-1 receptor and PDX-1 mRNA content were quantified., Results: Shams consumed more food and gained more weight compared to both RYGB and BWM (p < 0.001). Hyperglycaemia was evident in ad libitum-fed shams, whilst postprandial glucose levels were lower in RYGB compared to the BWM sham group (p < 0.05). During the OGTT, RYGB rats responded with >2.5-fold increase of GLP-1. Histology revealed signs of islet degeneration in ad libitum-fed shams, but not in RYGB and sham BWM controls (p < 0.001). GLP-1 receptor and PDX-1 mRNA content was similar between the RYGB and BWM shams but higher compared to ad libitum shams (p < 0.05)., Conclusions: Combined molecular, cellular and histological analyses of pancreatic function suggest that weight loss alone, and not the enhancement of GLP-1 responses, is predominant for the short-term β cell protective effects of RYGB.

Deregulated expression of MYC is a driver of colorectal carcinogenesis, necessitating novel strategies to inhibit MYC function. The ubiquitin ligase HUWE1 (HECTH9, ARF-BP1, MULE) associates with both MYC and the MYC-associated protein MIZ1. We show here that HUWE1 is required for growth of colorectal cancer cells in culture and in orthotopic xenograft models. Using high-throughput screening, we identify small molecule inhibitors of HUWE1, which inhibit MYC-dependent transactivation in colorectal cancer cells, but not in stem and normal colon epithelial cells. Inhibition of HUWE1 stabilizes MIZ1. MIZ1 globally accumulates on MYC target genes and contributes to repression of MYC-activated target genes upon HUWE1 inhibition. Our data show that transcriptional activation by MYC in colon cancer cells requires the continuous degradation of MIZ1 and identify a novel principle that allows for inhibition of MYC function in tumor cells., (© 2014 The Authors. Published under the terms of the CC BY 4.0 license.)

Expression of fibroblast growth factor (FGF)-inducible 14 (Fn14), a member of the tumor necrosis factor receptor superfamily, is typically low in healthy adult organisms, but strong Fn14 expression is induced in tissue injury and tissue remodeling. High Fn14 expression is also observed in solid tumors, which is why this receptor is under consideration as a therapeutic target in oncology. Here, we describe various novel mouse-human cross-reactive llama-derived recombinant Fn14-specific antibodies (5B6, 18D1, 4G5) harboring the human IgG1 Fc domain. In contrast to recombinant variants of the established Fn14-specific antibodies PDL192 and P4A8, all three llama-derived antibodies efficiently bound to the W42A and R56P mutants of human Fn14. 18D1 and 4G5, but not 5B6, efficiently blocked TNF-like weak inducer of apoptosis(TWEA K) binding at low concentrations (0.2–2 μg/ml). Oligomerization and Fcγ receptor (FcγR) binding converted all antibodies into strong Fn14 agonists. Variants of 18D1 with enhanced and reduced antibody-dependent cell-mediated cytotoxicity (ADCC) activity were further analyzed in vivo with respect to their effect on metastasis. In a xenogeneic model using human colon carcinoma cancer cells, both antibody variants were effective in reducing metastasis to the liver. In contrast, only the 18D1 variant with enhanced ADCC activity, but not its ADCC-defective counterpart, suppressed lung metastasis in the RE NCA model. In sum, this suggests that Fn14 targeting might primarily act by triggering of antibody effector functions, but also by blockade of TWEA K-Fn14 interaction in some cases

Background: Ascorbic acid demonstrates a cytotoxic effect by generating hydrogen peroxide, a reactive oxygen species (ROS) involved in oxidative cell stress. A panel of eleven human cancer cell lines, glioblastoma and carcinoma, were exposed to serial dilutions of ascorbic acid (5-100 mmol/L). The purpose of this study was to analyse the impact of catalase, an important hydrogen peroxide-detoxifying enzyme, on the resistance of cancer cells to ascorbic acid mediated oxidative stress., Methods: Effective concentration (EC(50)) values, which indicate the concentration of ascorbic acid that reduced the number of viable cells by 50%, were detected with the crystal violet assay. The level of intracellular catalase protein and enzyme activity was determined. Expression of catalase was silenced by catalase-specific short hairpin RNA (sh-RNA) in BT-20 breast carcinoma cells. Oxidative cell stress induced apoptosis was measured by a caspase luminescent assay., Results: The tested human cancer cell lines demonstrated obvious differences in their resistance to ascorbic acid mediated oxidative cell stress. Forty-five percent of the cell lines had an EC(50) > 20 mmol/L and fifty-five percent had an EC(50) < 20 mmol/L. With an EC(50) of 2.6-5.5 mmol/L, glioblastoma cells were the most susceptible cancer cell lines analysed in this study. A correlation between catalase activity and the susceptibility to ascorbic acid was observed. To study the possible protective role of catalase on the resistance of cancer cells to oxidative cell stress, the expression of catalase in the breast carcinoma cell line BT-20, which cells were highly resistant to the exposure to ascorbic acid (EC(50): 94,9 mmol/L), was silenced with specific sh-RNA. The effect was that catalase-silenced BT-20 cells (BT-20 KD-CAT) became more susceptible to high concentrations of ascorbic acid (50 and 100 mmol/L)., Conclusions: Fifty-five percent of the human cancer cell lines tested were unable to protect themselves against oxidative stress mediated by ascorbic acid induced hydrogen peroxide production. The antioxidative enzyme catalase is important to protect cancer cells against cytotoxic hydrogen peroxide. Silenced catalase expression increased the susceptibility of the formerly resistant cancer cell line BT-20 to oxidative stress.

Unlabelled: Obesity-related hepatic steatosis is a major risk factor for metabolic and cardiovascular disease. Fat reduced hypocaloric diets are able to relieve the liver from ectopically stored lipids. We hypothesized that the widely used low carbohydrate hypocaloric diets are similarly effective in this regard. A total of 170 overweight and obese, otherwise healthy subjects were randomized to either reduced carbohydrate (n = 84) or reduced fat (n = 86), total energy restricted diet (-30% of energy intake before diet) for 6 months. Body composition was estimated by bioimpedance analyses and abdominal fat distribution by magnetic resonance tomography. Subjects were also submitted to fat spectroscopy of liver and oral glucose tolerance testing. In all, 102 subjects completed the diet intervention with measurements of intrahepatic lipid content. Both hypocaloric diets decreased body weight, total body fat, visceral fat, and intrahepatic lipid content. Subjects with high baseline intrahepatic lipids (>5.56%) lost ≈7-fold more intrahepatic lipids compared with those with low baseline values (<5.56%) irrespective of diet composition. In contrast, changes in visceral fat mass and insulin sensitivity were similar between subgroups, with low and high baseline intrahepatic lipids., Conclusion: A prolonged hypocaloric diet low in carbohydrates and high in fat has the same beneficial effects on intrahepatic lipid accumulation as the traditional low-fat hypocaloric diet. The decrease in intrahepatic lipids appears to be independent of visceral fat loss and is not tightly coupled with changes in whole body insulin sensitivity during 6 months of an energy restricted diet., (Copyright © 2011 American Association for the Study of Liver Diseases.)

Disseminated tumor cells (DTC) are a potential contributor to relapse of cancer. In the present study we developed a model for induction of disseminated tumor cells in nude mice, which can aid in the search for therapeutic approaches as well as improve our understanding of metastasizing gastric cancer. To detect DTC in blood and bone marrow we established a modified animal model of orthotopic transplantation. Two groups of nude mice were used for xenotransplantation of gastric cancer specimens. In group I tumor specimens originating from a gastric adenocarcinoma cell line were transplanted onto the stomach; in group II they were transplanted subcutaneously into both axillaries. Tumor growth, metastases and presence of DTC were compared in both groups. For detection of DTC a nested reverse transcriptase polymerase chain reaction (RT-PCR) for human cytokeratin (CK)-20 was performed on blood and bone marrow of all mice. Tumor growth occurred in both groups (9/10 animals in group I, 10/10 in group II) within 14 weeks. Only animals in group I developed local invasive tumor growth, stenosis of the stomach and distant metastases. Tumors in animals of group II grew with local displacement only and developed no metastases. There were no signals of CK-20 detected in the blood in both groups. In group I, 5 of 9 animals had positive signals of human CK-20 in their bone marrow as a sign of DTC. In group II no DTC were detected in bone marrow. We conclude that orthotopic transplantation is a prerequisite for the development of DTC and metastasizing tumor growth in this modified gastric cancer model.

It has been extensively documented the role of the indirect pathway of allorecognition in allograft rejection. However, recent data demonstrate that the manipulation of this pathway could be also sufficient to promote prolongation of allograft survival. In the present study we evaluated the effect of preoperative immunization with the WF-specific MHC class II peptides RT1.D2 and RT1.B2 in combination with low-dose CsA from days 0 to 7 (5 mg/kg/day) and from days 8 to 30 (1 mg/kg/day) after WF small bowel transplantation. Seven days before and on the day of transplantation, LEW recipients were immunized with the two WF MHC class II peptides RT1.B2 and RT1.D2. The CsA monotherapy induced an allograft survival of 49.3 +/- 6.1 days. MHC class II peptide immunization had a limited effect on allograft survival for RT1.D2 (47.1 +/- 3.8 days) and induced prolongation of allograft survival for RT1.B2 (73.6 +/- 34.6 days). This effect seems to be based on the absence or silence of RT1.B2-reactive T cells and rejection seems to be correlated with the presence of RT1.B2-specific T cells in the late phase. Therefore, the combination of RT1.B2 with low-dose CsA shifts the immunological response and protects small bowel allograft rejection.

Increasing evidence supports the existence of regulatory T cells that may inhibit the allogeneic immune response after transplantation by secreting regulatory cytokines. To determine whether rat liver tolerance is associated with intrahepatic regulatory T cells secreting a characteristic cytokine profile, we analyzed the cytokine production of freshly isolated intragraft CD4(+) T cells at different times postoperatively by semiquantitative reverse transcription-polymerase chain reaction and by enzyme-linked immunosorbent assay before and after in vitro stimulation. Orthotopic arterialized liver transplantation was performed in two allogeneic rat strain combinations, one with fatal acute rejection (DA-to-LEW) and one with long-lasting tolerance (LEW-to-DA) without immunosuppression despite a complete major histocompatibility complex mismatch (spontaneous liver tolerance). Liver allografts of both groups showed continuously increasing cellular infiltration between day 3 and day 7 after transplantation. In this inflammatory situation, very low levels of interleukin-13 were detectable directly after cell isolation, as well as after in vitro stimulation. However, after 30 days, intrahepatic CD4(+)T cells in the tolerance group were then able to express elevated messenger RNA levels of the anti-inflammatory cytokine interleukin-13 in response to stimulation. This result indicates the presence of an intragraft Th2-like CD4(+) T cell population, which may have a regulatory function in the induction of liver tolerance.