Mitophagy in Intestinal Epithelial Cells Triggers Adaptive Immunity during Tumorigenesis.

In colorectal cancer patients, a high density of cytotoxic CD8+ T cells in tumors is associated with better prognosis. Using a Stat3 loss-of-function approach in two wnt/ß-catenin-dependent autochthonous models of sporadic intestinal tumorigenesis, we unravel a complex intracellular process in intestinal epithelial cells (IECs) that controls the induction of a CD8+ T cell based adaptive immune response. Elevated mitophagy in IECs causes iron(II)-accumulation in epithelial lysosomes, in turn, triggering lysosomal membrane permeabilization. Subsequent release of proteases into the cytoplasm augments MHC class I presentation and activation of CD8+ T cells via cross-dressing of dendritic cells. Thus, our findings highlight a so-far-unrecognized link between mitochondrial function, lysosomal integrity, and MHC class I presentation in IECs and suggest that therapies triggering mitophagy or inducing LMP in IECs may prove successful in shifting the balance toward anti-tumor immunity in colorectal cancer.

Ultrasound-based "CEUS-Bosniak"classification for cystic renal lesions: an 8-year clinical experience.

PURPOSE: Renal cysts comprise benign and malignant entities. Risk assessment profits from CT/MRI imaging using the Bosniak classification. While Bosniak-IIF, -III, and -IV cover complex cyst variants, Bosniak-IIF and -III stand out due to notorious overestimation. Contrast-enhanced ultrasound (CEUS) is promising to overcome this deficit but warrants standardization. This study addresses the benefits of a combined CEUS and CT/MRI evaluation of renal cysts. The study provides a realistic account of kidney tumor boards' intricacies in trying to validate renal cysts. METHODS: 247 patients were examined over 8 years. CEUS lesions were graded according to CEUS-Bosniak (IIF, III, IV). 55 lesions were resected, CEUS-Bosniak- and CT/MRI-Bosniak-classification were correlated with histopathological diagnosis. Interobserver agreement between the classifications was evaluated statistically. 105 lesions were followed by ultrasound, and change in CEUS-Bosniak-types and lesion size were documented. RESULTS: 146 patients (156 lesions) were included. CEUS classified 67 lesions as CEUS-Bosniak-IIF, 44 as CEUS-Bosniak-III, and 45 as CEUS-Bosniak-IV. Histopathology of 55 resected lesions revealed benign cysts in all CEUS-Bosniak-IIF lesions (2/2), 40% of CEUS-Bosniak-III and 8% of CEUS-Bosniak-IV, whereas malignancy was uncovered in 60% of CEUS-Bosniak-III and 92% of CEUS-Bosniak-IV. Overall, CEUS-Bosniak-types matched CT/MRI-Bosniak types in 58% (fair agreement, κ = 0.28). CEUS-Bosniak resulted in higher stages than CT/MRI-Bosniak (40%). Ultrasound follow-up of 105 lesions detected no relevant differences between CEUS-Bosniak-types concerning cysts size. 99% of lesions showed the same CEUS-Bosniak-type. CONCLUSION: The CEUS-Bosniak classification is an essential tool in clinical practice to differentiate and monitor renal cystic lesions and empowers diagnostic work-up and patient care.

Bcl3 Couples Cancer Stem Cell Enrichment With Pancreatic Cancer Molecular Subtypes.

BACKGROUND & AIMS: The existence of different subtypes of pancreatic ductal adenocarcinoma (PDAC) and their correlation with patient outcome have shifted the emphasis on patient classification for better decision-making algorithms and personalized therapy. The contribution of mechanisms regulating the cancer stem cell (CSC) population in different subtypes remains unknown. METHODS: Using RNA-seq, we identified B-cell CLL/lymphoma 3 (BCL3), an atypical nf-κb signaling member, as differing in pancreatic CSCs. To determine the biological consequences of BCL3 silencing in vivo and in vitro, we generated bcl3-deficient preclinical mouse models as well as murine cell lines and correlated our findings with human cell lines, PDX models, and 2 independent patient cohorts. We assessed the correlation of bcl3 expression pattern with clinical parameters and subtypes. RESULTS: Bcl3 was significantly down-regulated in human CSCs. Recapitulating this phenotype in preclinical mouse models of PDAC via BCL3 genetic knockout enhanced tumor burden, metastasis, epithelial to mesenchymal transition, and reduced overall survival. Fluorescence-activated cell sorting analyses, together with oxygen consumption, sphere formation, and tumorigenicity assays, all indicated that BCL3 loss resulted in CSC compartment expansion promoting cellular dedifferentiation. Overexpression of BCL3 in human PDXs diminished tumor growth by significantly reducing the CSC population and promoting differentiation. Human PDACs with low BCL3 expression correlated with increased metastasis, and BCL3-negative tumors correlated with lower survival and nonclassical subtypes. CONCLUSIONS: We demonstrate that bcl3 impacts pancreatic carcinogenesis by restraining CSC expansion and by curtailing an aggressive and metastatic tumor burden in PDAC across species. Levels of BCL3 expression are a useful stratification marker for predicting subtype characterization in PDAC, thereby allowing for personalized therapeutic approaches.

Diverse 'just-right' levels of chromosomal instability and their clinical implications in neoadjuvant treated gastric cancer.

BACKGROUND: The Cancer Genome Atlas (TCGA) consortium described EBV positivity(+), high microsatellite instability (MSI-H), genomic stability (GS) and chromosomal instability (CIN) as molecular subtypes in gastric carcinomas (GC). We investigated the predictive and prognostic value of these subtypes with emphasis on CIN in the context of neoadjuvant chemotherapy (CTx) in GC. METHODS: TCGA subgroups were determined for 612 resected adenocarcinomas of the stomach and gastro-oesophageal junction (291 without, 321 with CTx) and 143 biopsies before CTx. EBV and MSI-H were analysed by standard assays. CIN was detected by multiplex PCRs analysing 22 microsatellite markers. Besides the TCGA classification, CIN was divided into four CIN-subgroups: low, moderate, substantial, high. Mutation profiling was performed for 52 tumours by next-generation sequencing. RESULTS: EBV(+) (HR, 0.48; 95% CI, 0.23-1.02), MSI-H (HR, 0.56; 95% CI, 0.35-0.89) and GS (HR, 0.72; 95% CI, 0.45-1.13) were associated with increased survival compared to CIN in the resected tumours. Considering the extended CIN-classification, CIN-substantial was a negative prognostic factor in uni- and multivariable analysis in resected tumours with CTx (each p < 0.05). In biopsies before CTx, CIN-high predicted tumour regression (p = 0.026), but was not prognostically relevant. CONCLUSION: A refined CIN classification reveals tumours with different biological characteristics and potential clinical implications.

[Practical aspects of COVID-19 autopsies]. / Praktische Aspekte von COVID-19-Obduktionen.

BACKGROUND: The COVID-19 pandemic represents a so far unknown challenge for the medical community. Autopsies are important for studying this disease, but their safety was challenged at the beginning of the pandemic. OBJECTIVES: To determine whether COVID-19 autopsies can be performed under existing legal conditions and which safety standards are required. MATERIALS AND METHODS: The autopsy procedure undertaken in five institutions in Germany, Austria, and Switzerland is detailed with respect to legal and safety standards. RESULTS: In all institutions the autopsies were performed in technically feasible rooms. The personal equipment consisted of functional clothing including a disposable gown and apron, a surgical cap, eye protection, FFP­3 masks, and two pairs of gloves. In four institutions, complete autopsies were performed; in one institution the ultrasound-guided biopsy within the postmortal imaging and biopsy program. The latter does not allow the appreciation of gross organ pathology; however, it is able to retrieve standardized biopsies for diagnostic and research purposes. Several scientific articles in highly ranked journals resulted from these autopsies and allowed deep insights into organ damage and conclusions to better understand the pathomechanisms. Viral RNA was frequently detectable in the COVID-19 deceased, but the issue of infectivity remains unresolved and it is questionable if Ct values are greater than 30. CONCLUSIONS: With appropriate safeguards, autopsies of people who have died from COVID-19 can be performed safely and are highly relevant to medical research.

Inhibition of PLK1 by capped-dose volasertib exerts substantial efficacy in MDS and sAML while sparing healthy haematopoiesis.

INTRODUCTION: Targeting the cell cycle machinery represents a rational therapeutic approach in myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (sAML). Despite substantial response rates, clinical use of the PLK inhibitor volasertib has been hampered by elevated side effects such as neutropenia and infections. OBJECTIVES: The primary objective was to analyse whether a reduced dose of volasertib was able to limit toxic effects on the healthy haematopoiesis while retaining its therapeutic effect. METHODS: Bone marrow mononuclear cells (BMMNCs) of patients with MDS/sAML (n = 73) and healthy controls (n = 28) were treated with volasertib (1 µM to 1 nM) or vehicle control. Short-term viability analysis was performed by flow cytometry after 72 hours. For long-term viability analysis, colony-forming capacity was assessed after 14 days. Protein expression of RIPK3 and MCL-1 was quantified via flow cytometry. RESULTS: Reduced dose levels of volasertib retained high cell death-inducing efficacy in primary human stem and progenitor cells of MDS/sAML patients without affecting healthy haematopoiesis in vitro. Interestingly, volasertib reduced colony-forming capacity and cell survival independent of clinical stage or mutational status. CONCLUSIONS: Volasertib offers a promising therapeutic approach in patients with adverse prognostic profile. RIPK3 and MCL-1 might be potential biomarkers for sensitivity to volasertib treatment.

IgG4 is Elevated in Eosinophilic Esophagitis but Not in Gastroesophageal Reflux Disease Patients.

BACKGROUND: For eosinophilic esophagitis (EoE) recently an association with immunoglobulin (Ig)G4 rather than IgE has been reported. Gastroesophageal reflux disease (GERD) is the most important differential diagnosis of EoE. We compared esophageal IgG4 plasma cell infiltration and serum IgG4 levels of EoE patients (before and after budesonide therapy) with GERD patients. METHODS: Prospectively collected serum samples of 17 EoE patients before and after 8 weeks of therapy with budesonide (1 mg BID) were analyzed for total and antigen-specific IgG4 and IgE levels. Also, immunohistochemical analysis of total and IgG4-positive plasma cells was performed on esophageal biopsies of these patients. In total, 14 GERD patients without histologic proof of eosinophilic infiltration were taken as a control group. RESULTS: Total IgG4 serum levels in EoE patients were significantly higher than in GERD patients (121.0 vs. 71.2 mg/dL; P=0.038) and decreased under budesonide therapy (121.0 vs. 104.2 mg/dL; P=0.019). IgE levels did not differ significantly between all groups. In EoE patients also a high number of esophageal IgG4-positive plasma cells was detected and significantly reduced under therapy (29.1 vs. 0.1 IgG4-positive cells; P<0.001). In GERD patients no relevant esophageal plasma cell infiltration could be seen. CONCLUSIONS: In EoE patients elevated systemic IgG4 serum levels compared with GERD patients can be seen and decrease under topical steroid therapy. Also, local IgG4 plasma cells expression is high in EoE, but not in GERD patients and normalize under therapy. These findings are further proof for a possible association of EoE with IgG4.

Mass-spectrometry-based draft of the human proteome.

Proteomes are characterized by large protein-abundance differences, cell-type- and time-dependent expression patterns and post-translational modifications, all of which carry biological information that is not accessible by genomics or transcriptomics. Here we present a mass-spectrometry-based draft of the human proteome and a public, high-performance, in-memory database for real-time analysis of terabytes of big data, called ProteomicsDB. The information assembled from human tissues, cell lines and body fluids enabled estimation of the size of the protein-coding genome, and identified organ-specific proteins and a large number of translated lincRNAs (long intergenic non-coding RNAs). Analysis of messenger RNA and protein-expression profiles of human tissues revealed conserved control of protein abundance, and integration of drug-sensitivity data enabled the identification of proteins predicting resistance or sensitivity. The proteome profiles also hold considerable promise for analysing the composition and stoichiometry of protein complexes. ProteomicsDB thus enables navigation of proteomes, provides biological insight and fosters the development of proteomic technology.

STAT3/5 Inhibitors Suppress Proliferation in Bladder Cancer and Enhance Oncolytic Adenovirus Therapy.

The JAK-STAT signalling pathway regulates cellular processes like cell division, cell death and immune regulation. Dysregulation has been identified in solid tumours and STAT3 activation is a marker for poor outcome. The aim of this study was to explore potential therapeutic strategies by targeting this pathway in bladder cancer (BC). High STAT3 expression was detected in 51.3% from 149 patient specimens with invasive bladder cancer by immunohistochemistry. Protein expression of JAK, STAT and downstream targets were confirmed in 10 cell lines. Effects of the JAK inhibitors Ruxolitinib and BSK-805, and STAT3/5 inhibitors Stattic, Nifuroxazide and SH-4-54 were analysed by cell viability assays, immunoblotting, apoptosis and cell cycle progression. Treatment with STAT3/5 but not JAK1/2 inhibitors reduced survival, levels of phosphorylated STAT3 and Cyclin-D1 and increased apoptosis. Tumour xenografts, using the chicken chorioallantoic membrane (CAM) model responded to Stattic monotherapy. Combination of Stattic with Cisplatin, Docetaxel, Gemcitabine, Paclitaxel and CDK4/6 inhibitors showed additive effects. The combination of Stattic with the oncolytic adenovirus XVir-N-31 increased viral replication and cell lysis. Our results provide evidence that inhibitors against STAT3/5 are promising as novel mono- and combination therapy in bladder cancer.

BarrettNET-a prospective registry for risk estimation of patients with Barrett's esophagus to progress to adenocarcinoma.

Risk stratification in patients with Barrett's esophagus (BE) to prevent the development of esophageal adenocarcinoma (EAC) is an unsolved task. The incidence of EAC and BE is increasing and patients are still at unknown risk. BarrettNET is an ongoing multicenter prospective cohort study initiated to identify and validate molecular and clinical biomarkers that allow a more personalized surveillance strategy for patients with BE. For BarrettNET participants are recruited in 20 study centers throughout Germany, to be followed for progression to dysplasia (low-grade dysplasia or high-grade dysplasia) or EAC for >10 years. The study instruments comprise self-administered epidemiological information (containing data on demographics, lifestyle factors, and health), as well as biological specimens, i.e., blood-based samples, esophageal tissue biopsies, and feces and saliva samples. In follow-up visits according to the individual surveillance plan of the participants, sample collection is repeated. The standardized collection and processing of the specimen guarantee the highest sample quality. Via a mobile accessible database, the documentation of inclusion, epidemiological data, and pathological disease status are recorded subsequently. Currently the BarrettNET registry includes 560 participants (23.1% women and 76.9% men, aged 22-92 years) with a median follow-up of 951 days. Both the design and the size of BarrettNET offer the advantage of answering research questions regarding potential causes of disease progression from BE to EAC. Here all the integrated methods and materials of BarrettNET are presented and reviewed to introduce this valuable German registry.

[Disseminated tuberculosis mimicking metastatic gastric cancer - a case report]. / Disseminierte Tuberkulose imitiert metastasiertes Magenkarzinom ­ ein Fallbericht.

Increasing numbers of active tuberculosis in Germany were recorded in the last years. Thus, also extrapulmonary manifestations of tuberculosis gain clinical significance as differential diagnoses, especially when a metastatic tumor disease is suspected. We report the case of a 77-year-old male patient who presented with unilateral leg pain and B symptoms. Further investigations revealed an osteolytic mass in the sacrum as well as CT-morphological findings consistent with metastatic gastric cancer. However, transgastric biopsies showed necrotising granuloma with giant cells leading to molecular and cultural detection of Mycobacterium tuberculosis instead of suspected neoplastic tissue. A nine-month treatment regimen for suspected disseminated tuberculosis with bone involvement was initiated according to national guidelines. Clinical and radiological follow up examinations after treatment completion showed complete remission.

Gut microbiota modulate T cell trafficking into human colorectal cancer.

OBJECTIVE: Tumour-infiltrating lymphocytes (TILs) favour survival in human colorectal cancer (CRC). Chemotactic factors underlying their recruitment remain undefined. We investigated chemokines attracting T cells into human CRCs, their cellular sources and microenvironmental triggers. DESIGN: Expression of genes encoding immune cell markers, chemokines and bacterial 16S ribosomal RNA (16SrRNA) was assessed by quantitative reverse transcription-PCR in fresh CRC samples and corresponding tumour-free tissues. Chemokine receptor expression on TILs was evaluated by flow cytometry on cell suspensions from digested tissues. Chemokine production by CRC cells was evaluated in vitro and in vivo, on generation of intraperitoneal or intracecal tumour xenografts in immune-deficient mice. T cell trafficking was assessed on adoptive transfer of human TILs into tumour-bearing mice. Gut flora composition was analysed by 16SrRNA sequencing. RESULTS: CRC infiltration by distinct T cell subsets was associated with defined chemokine gene signatures, including CCL5, CXCL9 and CXCL10 for cytotoxic T lymphocytes and T-helper (Th)1 cells; CCL17, CCL22 and CXCL12 for Th1 and regulatory T cells; CXCL13 for follicular Th cells; and CCL20 and CCL17 for interleukin (IL)-17-producing Th cells. These chemokines were expressed by tumour cells on exposure to gut bacteria in vitro and in vivo. Their expression was significantly higher in intracecal than in intraperitoneal xenografts and was dramatically reduced by antibiotic treatment of tumour-bearing mice. In clinical samples, abundance of defined bacteria correlated with high chemokine expression, enhanced T cell infiltration and improved survival. CONCLUSIONS: Gut microbiota stimulate chemokine production by CRC cells, thus favouring recruitment of beneficial T cells into tumour tissues.

A20 deletion in T cells modulates acute graft-versus-host disease in mice.

The NF-κB regulator A20 limits inflammation by providing negative feedback in myeloid cells and B cells. Functional lack of A20 has been linked to several inflammatory and autoimmune diseases. To define how A20 affects the functionality of T effector cells in a highly inflammatory environment, we performed conventional allogeneic hematopoietic stem cell transplantation (allo-HSCT) with A20-deficient CD4+ and CD8+ donor T cells in mice. Severity and mortality of graft-versus-host disease (GVHD) after allo-HSCT was drastically reduced in recipients transplanted with conventional doses of A20-deficient T cells. Consistently, we found that the A20-deficient donor T-cell compartment was strongly diminished at various timepoints after allo-HSCT. However, proportionally more A20-deficient donor T cells produced IFN-γ and systemic inflammation was elevated early after allo-HSCT. Consequently, increasing the dose of transplanted A20-deficient T cells reversed the original phenotype and resulted in enhanced GVHD mortality compared to recipients that received A20+/+ T cells. Still, A20-deficient T cells, activated either through T cell receptor-dependent or -independent mechanisms, were less viable than control A20+/+ T cells, highlighting that A20 balances both, T-cell activation and survival. Thus, our findings suggest that targeting A20 in T cells may allow to modulate T-cell-mediated inflammatory diseases like GVHD.

Correction to: MicroRNA expression profiling for the prediction of resistance to neoadjuvant radiochemotherapy in squamous cell carcinoma of the esophagus.

Following publication of the original article [1], the authors reported that for one of the authors, Stephanie E. Combs, the middle name was accidentally omitted. They also reported that for two of the authors, Daniel Habermehl and Stephanie E. Combs, two affiliations were accidentally omitted. In this Correction the incorrect and correct author name are shown and the two omitted affiliations are listed.

MicroRNA expression profiling for the prediction of resistance to neoadjuvant radiochemotherapy in squamous cell carcinoma of the esophagus.

BACKGROUND: MicroRNAs (miRNAs) play an important role in cancer biology. Neoadjuvant radiochemotherapy followed by surgery is a standard treatment for locally advanced esophageal squamous cell carcinoma (ESCC). However, a subset of patients do not respond. We evaluated whether miRNA profiles can predict resistance to radiochemotherapy. METHODS: Formalin-fixed, paraffin-embedded pretherapeutic biopsies of patients treated by radiochemotherapy followed by esophagectomy were analyzed. The response was determined by histopathological tumor regression grading. miRNA profiling was performed by microarray analysis (Agilent platform) in 16 non-responders and 15 responders. Differentially expressed miRNAs were confirmed by real-time quantitative PCR (qRT-PCR) in an expanded cohort of 53 cases. RESULTS: The miRNA profiles within and between non-responders and responders were highly similar (r = 0.96, 0.94 and 0.95). However, 12 miRNAs were differentially expressed (> twofold; p ≤ 0.025): non-responders showed upregulation of hsa-miR-1323, hsa-miR-3678-3p, hsv2-miR-H7-3p, hsa-miR-194*, hsa-miR-3152, kshv-miR-K12-4-3p, hsa-miR-665 and hsa-miR-3659 and downregulation of hsa-miR-126*, hsa-miR-484, hsa-miR-330-3p and hsa-miR-3653. qRT-PCR analysis confirmed the microarray findings for hsa-miR-194* and hsa-miR-665 (p < 0.001 each) with AUC values of 0.811 (95% CI 0.694-0.927) and 0.817 (95% CI 0.704-0.930), respectively, in ROC analysis. CONCLUSIONS: Our results indicate that miRNAs are involved in the therapeutic response in ESCC and suggest that miRNA profiles could facilitate pretherapeutic patient selection.

Pharmacoproteomic characterisation of human colon and rectal cancer.

Most molecular cancer therapies act on protein targets but data on the proteome status of patients and cellular models for proteome-guided pre-clinical drug sensitivity studies are only beginning to emerge. Here, we profiled the proteomes of 65 colorectal cancer (CRC) cell lines to a depth of > 10,000 proteins using mass spectrometry. Integration with proteomes of 90 CRC patients and matched transcriptomics data defined integrated CRC subtypes, highlighting cell lines representative of each tumour subtype. Modelling the responses of 52 CRC cell lines to 577 drugs as a function of proteome profiles enabled predicting drug sensitivity for cell lines and patients. Among many novel associations, MERTK was identified as a predictive marker for resistance towards MEK1/2 inhibitors and immunohistochemistry of 1,074 CRC tumours confirmed MERTK as a prognostic survival marker. We provide the proteomic and pharmacological data as a resource to the community to, for example, facilitate the design of innovative prospective clinical trials.

Expression patterns of programmed death-ligand 1 in esophageal adenocarcinomas: comparison between primary tumors and metastases.

Expression analysis of programmed death-ligand 1 (PD-L1) may be helpful in guiding clinical decisions for immune checkpoint inhibition therapy, but testing by immunohistochemistry may be hampered by heterogeneous staining patterns within tumors and expression changes during metastatic course. PD-L1 expression (clone SP142) was investigated in esophageal adenocarcinomas using tissue microarrays (TMA) from 112 primary resected tumors, preoperative biopsies and full slide sections from a subset of these cases (n = 24), corresponding lymph node (n = 55) and distant metastases (n = 17). PD-L1 expression was scored as 0.1-1, >1, >5, >50% positive membranous staining of tumor cells and any positive staining of tumor-associated inflammatory infiltrates and/or stroma cells. There was a significant correlation with overall PD-L1 expression between the full slide sections and the TMA (p = 0.001), but not with the corresponding biopsies. PD-L1 expression in tumor cells >1% was detected in 8.0% of cases (9/112) and 51.8% of cases (58/112) in tumor-associated inflammatory infiltrates and/or stroma cells of primary tumors. Epithelial expression in metastases was found in 5.6% of cases (4/72) and immune cell expression in 18.1% of cases (13/72), but did not correlate with the expression pattern in the primary tumor. Overall PD-L1 expression in the primary tumor did not influence survival. However, PD-L1 expression was correlated with the number of CD3+ tumor-infiltrating lymphocytes in the tumor center, and a combinational score of PD-L1 status/CD3+ tumor-infiltrating lymphocytes was correlated with patients' overall survival.

Neural influences on human intestinal epithelium in vitro.

KEY POINTS: We present the first systematic and, up to now, most comprehensive evaluation of the basic features of epithelial functions, such as basal and nerve-evoked secretion, as well as tissue resistance, in over 2200 surgical specimens of human small and large intestine. We found no evidence for impaired nerve-evoked epithelial secretion or tissue resistance with age or disease pathologies (stomach, pancreas or colon cancer, polyps, diverticulitis, stoma reversal). This indicates the validity of future studies on epithelial secretion or resistance that are based on data from a variety of surgical specimens. ACh mainly mediated nerve-evoked and basal secretion in the small intestine, whereas vasoactive intestinal peptide and nitric oxide were the primary pro-secretory transmitters in the large intestine. The results of the present study revealed novel insights into regional differences in nerve-mediated secretion in the human intestine and comprise the basis by which to more specifically target impaired epithelial functions in the diseased gut. ABSTRACT: Knowledge on basic features of epithelial functions in the human intestine is scarce. We used Ussing chamber techniques to record basal tissue resistance (R-basal) and short circuit currents (ISC; secretion) under basal conditions (ISC-basal) and after electrical field stimulation (ISC-EFS) of nerves in 2221 resectates from 435 patients. ISC-EFS was TTX-sensitive and of comparable magnitude in the small and large intestine. ISC-EFS or R-basal were not influenced by the patients' age, sex or disease pathologies (cancer, polyps, diverticulitis). Ion substitution, bumetanide or adenylate cyclase inhibition studies suggested that ISC-EFS depended on epithelial cAMP-driven chloride and bicarbonate secretion but not on amiloride-sensitive sodium absorption. Although atropine-sensitive cholinergic components prevailed for ISC-EFS of the duodenum, jejunum and ileum, PG97-269-sensitive [vasoactive intestinal peptide (VIP) receptor 1 antagonist] VIPergic together with L-NAME-sensitive nitrergic components dominated the ISC-EFS in colonic preparations. Differences in numbers of cholinergic or VIPergic neurons, sensitivity of epithelial muscarinic or VIP receptors, or stimulus frequency-dependent transmitter release were not responsible for the region-specific transmitter contribution to ISC-EFS. Instead, the low atropine-sensitivity of ISC-EFS in the colon was the result of high cholinesterase activity because neostigmine revealed cholinergic components. Colonic ISC-EFS remained unchanged after tachykinin, P2X, P2Y or A1 and A2 receptor blockade. R-basal was smaller and ISC-basal was higher in the small intestine. TTX and bumetanide decreased ISC-basal in all regions, suggesting nerve-dependent secretory tone. ISC-basal was atropine-sensitive in the small intestine and PG97-269-sensitive in the large intestine. This comprehensive study reveals novel insights into region-specific nerve-mediated secretion in the human small and large intestine.

Targeted positron emission tomography imaging of CXCR4 expression in patients with acute myeloid leukemia.

Acute myeloid leukemia originates from leukemia-initiating cells that reside in the protective bone marrow niche. CXCR4/CXCL12 interaction is crucially involved in recruitment and retention of leukemia-initiating cells within this niche. Various drugs targeting this pathway have entered clinical trials. To evaluate CXCR4 imaging in acute myeloid leukemia, we first tested CXCR4 expression in patient-derived primary blasts. Flow cytometry revealed that high blast counts in patients with acute myeloid leukemia correlate with high CXCR4 expression. The wide range of CXCR4 surface expression in patients was reflected in cell lines of acute myeloid leukemia. Next, we evaluated the CXCR4-specific peptide Pentixafor by positron emission tomography imaging in mice harboring CXCR4 positive and CXCR4 negative leukemia xenografts, and in 10 patients with active disease. [(68)Ga]Pentixafor-positron emission tomography showed specific measurable disease in murine CXCR4 positive xenografts, but not when CXCR4 was knocked out with CRISPR/Cas9 gene editing. Five of 10 patients showed tracer uptake correlating well with leukemia infiltration assessed by magnetic resonance imaging. The mean maximal standard uptake value was significantly higher in visually CXCR4 positive patients compared to CXCR4 negative patients. In summary, in vivo molecular CXCR4 imaging by means of positron emission tomography is feasible in acute myeloid leukemia. These data provide a framework for future diagnostic and theranostic approaches targeting the CXCR4/CXCL12-defined leukemia-initiating cell niche.

A new model of implant-related osteomyelitis in the metaphysis of rat tibiae.

BACKGROUND: Animal models serve as an important tool to understand peri-implant infection. Most of the models use high bacterial loads (>10(4) colony forming units, CFU) to provide high infection rates. Therefore these animals evolve rather similarly, making comparison between groups and statistical analysis possible. On the other hand, to mimic clinical constellation of surgery-related infections the use of low amounts of bacteria would be more advantageous. METHODS: We developed a metaphyseal rat model of peri-implant bone infection with low amount of bacterial loads (10(2) and 10(3) CFU of Staphylococcus aureus) and investigated osseointegration of the implants coated with hydroxyapatite (HA) and low-dosed HA-silver (HA-Ag). Non-infected implants served as controls. After 6 weeks rats were sacrificed and implants evaluated for osseointegration and infection. RESULTS: Infection of implanted devices was reliably induced, independently whether 10(2) or 10(3) CFU of S. aureus were inoculated and HA or HA-Ag coated implants were used. No systemic infection was present in any of the animals at the time of sacrifice, and no animal developed acute infection requiring premature sacrifice. All CFU counts of the implant and the bone at sacrifice were significantly higher than the inoculated load (p < .05). All sterilely inserted implants showed excellent osseointegration and no infection. CONCLUSIONS: Our present study of a rat tibia model reliably induced osteomyelitis in the metaphysis with low-doses of bacteria. The addition of low-dosed Ag to the implant coating was not able to reduce the infection rates. The results demonstrate that it is possible to develop a model of implant-related osteomyelitis in rats with low amounts of bacteria to better mimic clinical constellations. No other promoters of infection besides insertion of the screw implant were used in this model.