Biological and clinical significance of tryptophan-catabolizing enzymes in cutaneous T-cell lymphomas

Indoleamine 2,3-deoxygenase 1 (IDO1) induces immune tolerance in the tumor microenvironment (TME) and is recognized as a potential therapeutic target. We studied the expression of both IDO1 and the related tryptophan 2,3-dioxygenase (TDO) in several different subtypes of cutaneous T-cell lymphoma (CTCL), and evaluated the kynurenine (KYN) pathway in the local TME and in patient sera. Specimens from the total of 90 CTCL patients, including mycosis fungoides (MF, n = 37), lymphomatoid papulosis (LyP, n = 36), primary cutaneous anaplastic large cell lymphoma (pcALCL, n = 4), subcutaneous panniculitis-like T-cell lymphoma (SPTCL n = 13), and 10 patients with inflammatory lichen ruber planus (LRP), were analyzed by immunohistochemistry (IHC), immunofluorescence (IF), quantitative PCR, and/or liquid chromatography-tandem mass spectrometry (LC-MS/MS). Three CTCL cell lines also were studied. Expression of both IDO1 and TDO was upregulated in CTCL. In MF specimens and in the MF cell line MyLa2000, IDO1 expression exceeded that of TDO, whereas the opposite was true for LyP, ALCL, and corresponding Mac1/2A cell lines. The spectrum of IDO1-expressing cell types differed among CTCL subtypes and was reflected in the clinical behavior. In MF, SPTCL, and LyP, IDO1 was expressed by malignant cells and by CD33(+) myeloid-derived suppressor cells, whereas in SPTCL CD163(+) tumor-associated macrophages also expressed IDO1. Significantly elevated serum KYN/Trp ratios were found in patients with advanced stages of MF. Epacadostat, an IDO1 inhibitor, induced a clear decrease in KYN concentration in cell culture. These results show the importance of IDO1/TDO-induced immunosuppression in CTCL and emphasize its role as a new therapeutic target.Peer reviewe

Metabolomic Analysis of Campylobacter jejuni by Direct-Injection Electrospray Ionization Mass Spectrometry

Direct-injection mass spectrometry (DIMS) is a means of rapidly obtaining metabolomic phenotype data in both prokaryotes and eukaryotes. Given our generally poor understanding of Campylobacter metabolism, the high-throughput and relatively simple sample preparation of DIMS has made this an attractive technique for metabolism-related studies and hypothesis generation, especially when attempting to analyze metabolic mutants with no clear phenotype. Here we describe a metabolomic fingerprinting approach with sampling and extraction methodologies optimized for direct-injection electrospray ionization mass spectrometry (ESI-MS), which we have used as a means of comparing wild-type and isogenic mutant strains of C. jejuni with various metabolic blocks

Metabolomic Analysis of Campylobacter jejuni by Direct-Injection Electrospray Ionization Mass Spectrometry

Direct-injection mass spectrometry (DIMS) is a means of rapidly obtaining metabolomic phenotype data in both prokaryotes and eukaryotes. Given our generally poor understanding of Campylobacter metabolism, the high-throughput and relatively simple sample preparation of DIMS has made this an attractive technique for metabolism-related studies and hypothesis generation, especially when attempting to analyze metabolic mutants with no clear phenotype. Here we describe a metabolomic fingerprinting approach with sampling and extraction methodologies optimized for direct-injection electrospray ionization mass spectrometry (ESI-MS), which we have used as a means of comparing wild-type and isogenic mutant strains of C. jejuni with various metabolic blocks

Two parallel pathways connect glutamine metabolism and mTORC1 activity to regulate glutamoptosis.

Glutamoptosis is the induction of apoptotic cell death as a consequence of the aberrant activation of glutaminolysis and mTORC1 signaling during nutritional imbalance in proliferating cells. The role of the bioenergetic sensor AMPK during glutamoptosis is not defined yet. Here, we show that AMPK reactivation blocks both the glutamine-dependent activation of mTORC1 and glutamoptosis in vitro and in vivo. We also show that glutamine is used for asparagine synthesis and the GABA shunt to produce ATP and to inhibit AMPK, independently of glutaminolysis. Overall, our results indicate that glutamine metabolism is connected with mTORC1 activation through two parallel pathways: an acute alpha-ketoglutarate-dependent pathway; and a secondary ATP/AMPK-dependent pathway. This dual metabolic connection between glutamine and mTORC1 must be considered for the future design of therapeutic strategies to prevent cell growth in diseases such as cancer.This work was supported by funds from the following institutions: Agencia Estatal de Investigación/European Regional Development Fund, European Union (PGC2018-096244- B-I00, SAF2016-75442-R), Ministry of Science, Innovation and Universities of Spain, Spanish National Research Council—CSIC, Institut National de la Santé et de la Recherche Médicale —INSERM, Université de Bordeaux, Fondation pour la Recherche Médicale, the Conseil Régional d’Aquitaine, SIRIC-BRIO, Fondation ARC, and Institut Européen de Chimie et Biologie. C.B. was recipient of fellowships from the Minister of Higher Education, Research and Innovation (France) and the Fondation ARC (France). We thank Prof. Patricia Boya (Centro de Investigaciones Biologicas, Madrid, Spain) for kindly providing with the ATG5+/+ and ATG5−/− MEFs. We thank Prof. Benoit Viollet (Institute Cochin, Paris, France) for kindly providing with the AMPK+/+ and AMPK−/− MEFs, and the CA-AMPK plasmid

The proteasome inhibitor bortezomib controls indoleamine 2,3-dioxygenase 1 breakdown and restores immune regulation in autoimmune diabetes

Bortezomib (BTZ) is a first-in-class proteasome inhibitor approved for the therapy of multiple myeloma that also displays unique regulatory activities on immune cells. The enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is a tryptophan metabolizing enzyme exerting potent immunoregulatory effects when expressed in dendritic cells (DCs), the most potent antigen-presenting cells capable of promoting either immunity or tolerance. We previously demonstrated that, in inflammatory conditions, IDO1 is subjected to proteasomal degradation in DCs, turning these cells from immunoregulatory to immunostimulatory. In non-obese diabetic (NOD) mice, an experimental model of autoimmune diabetes, we also identified an IDO1 defect such that the DCs do not develop tolerance toward pancreatic islet autoantigens. We found that BTZ rescues IDO1 protein expression in vitro in a particular subset of DCs, i.e., plasmacytoid DCs (pDCs) from NOD mice. When administered in vivo to prediabetic mice, the drug prevented diabetes onset through IDO1- and pDC-dependent mechanisms. Although the drug showed no therapeutic activity when administered alone to overtly diabetic mice, its combination with otherwise suboptimal dosages of autoimmune-preventive anti-CD3 antibody resulted in disease reversal in 70% diabetic mice, a therapeutic effect similar to that afforded by full-dosage anti-CD3. Thus, our data indicate a potential for BTZ in the immunotherapy of autoimmune diabetes and further underline the importance of IDO1-mediated immune regulation in such disease

Randomized phase 2 trial of intravenous oncolytic virus JX-594 combined with low-dose cyclophosphamide in patients with advanced soft-tissue sarcoma

JX-594 is an oncolytic vaccinia virus genetically modified to replicate selectively in tumor cells. Metronomic chemotherapy has shown preclinical synergy with oncolytic viruses. We report here the results of the METROMAJX which is a randomized phase II clinical trial investigating the combination of JX-594 combined with metronomic cyclophosphamide (arm 1) or metronomic cyclophosphamide (arm 2) in patients with advanced STS. A two-stage Simon design was used. JX-594 was administered intra-venously at the dose 1.109 every 2 weeks for the first 3 injections and then every 3 weeks. Cyclophosphamide was given orally at the dose of 50 mg BID 1 week on 1 week off. The primary endpoint was the 6-month non progression rate. 20 patients were included (arm 1:15, arm 2:5). The two most frequent toxicities were grade 1 fatigue and fever and grade 2 fatigue and grade 2 lymphopenia in arms 1 and 2, respectively. In arm 1, 12 patients were assessable for the efficacy analysis. None of them were progression-free at 6 months indicating that the first stage of the Simon's design was not satisfied. One patient out 4 assessable for efficacy was progression-free at 6 months in arm 2. High throughput analysis of sequential plasma samples revealed an upregulation of protein biomarkers reflecting immune induction such as CXCL10 and soluble CD8 antigen in arm 1. Systemic treatment with JX-594 is safe in patients with advanced STS. Further investigations are needed to improve immune response to oncolytic viruses and define their therapeutic potential in patients with STS

Vascular Reconstructions in Kidney Transplantation

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Nanolitre real-time PCR detection of bacterial, parasitic, and viral agents from patients with diarrhoea in Nunavut, Canada

Background. Little is known about the microbiology of diarrhoeal disease in Canada's Arctic regions. There are a number of limitations of conventional microbiology testing techniques for diarrhoeal pathogens, and these may be further compromised in the Arctic, given the often long distances for specimen transport. Objective. To develop a novel multiple-target nanolitre real-time reverse transcriptase (RT)-PCR platform to simultaneously test diarrhoeal specimens collected from residents of the Qikiqtani (Baffin Island) Region of Nunavut, Canada, for a wide range of bacterial, parasitic and viral agents. Study design/methods. Diarrhoeal stool samples submitted for bacterial culture to Qikiqtani General Hospital in Nunavut over an 18-month period were tested with a multiple-target nanolitre real-time PCR panel for major diarrhoeal pathogens including 8 bacterial, 6 viral and 2 parasitic targets. Results. Among 86 stool specimens tested by PCR, a total of 50 pathogens were detected with 1 or more pathogens found in 40 (46.5%) stool specimens. The organisms detected comprised 17 Cryptosporidium spp., 5 Clostridium difficile with toxin B, 6 Campylobacter spp., 6 Salmonella spp., 4 astroviruses, 3 noroviruses, 1 rotavirus, 1 Shigella spp. and 1 Giardia spp. The frequency of detection by PCR and bacterial culture was similar for Salmonella spp., but discrepant for Campylobacter spp., as Campylobacter was detected by culture from only 1/86 specimens. Similarly, Cryptosporidium spp. was detected in multiple samples by PCR but was not detected by microscopy or enzyme immunoassay. Conclusions. Cryptosporidium spp., Campylobacter spp. and Clostridium difficile may be relatively common but possibly under-recognised pathogens in this region. Further study is needed to determine the regional epidemiology and clinical significance of these organisms. This method appears to be a useful tool for gastrointestinal pathogen research and may also be helpful for clinical diagnostics and outbreak investigation in remote regions where the yield of routine testing may be compromised