Droplet Tn-Seq identifies the primary secretion mechanism for yersiniabactin in Yersinia pestis.

Nutritional immunity includes sequestration of transition metals from invading pathogens. Yersinia pestis overcomes nutritional immunity by secreting yersiniabactin to acquire iron and zinc during infection. While the mechanisms for yersiniabactin synthesis and import are well-defined, those responsible for yersiniabactin secretion are unknown. Identification of this mechanism has been difficult because conventional mutagenesis approaches are unable to inhibit trans-complementation by secreted factors between mutants. To overcome this obstacle, we utilized a technique called droplet Tn-seq (dTn-seq), which uses microfluidics to isolate individual transposon mutants in oil droplets, eliminating trans-complementation between bacteria. Using this approach, we first demonstrated the applicability of dTn-seq to identify genes with secreted functions. We then applied dTn-seq to identify an AcrAB efflux system as required for growth in metal-limited conditions. Finally, we showed this efflux system is the primary yersiniabactin secretion mechanism and required for virulence during bubonic and pneumonic plague. Together, these studies have revealed the yersiniabactin secretion mechanism that has eluded researchers for over 30 years and identified a potential therapeutic target for bacteria that use yersiniabactin for metal acquisition.

Specific Targeting of STAT3 in B Cells Suppresses Progression of B Cell Lymphoma.

The signal transducer and activator of transcription 3 (STAT3), which regulates multiple oncogenic processes, has been found to be constitutively activated in lymphoma, suggesting its potential as a therapeutic target. Here, we constructed an anti-CD19-N-(4-carboxycyclohexylmethyl) maleimide N-hydroxysuccinimide ester (SMCC)-protamine (CSP)-STAT3 small interfering RNA (siRNA) conjugate and demonstrated that the CSP-STAT3 siRNA conjugate could specifically bind to normal B cells and A20 lymphoma cells in vitro. It decreased the STAT3 expression in B cell lymphoma cell lines (A20, SU-DHL-2 and OCI-Ly3), resulting in reduced proliferation of lymphoma cells featured with lower S-phase and higher apoptosis. Using an A20 transplantable lymphoma model, we found that the CSP-STAT3 siRNA conjugate significantly inhibited tumor growth and weight. Ki-67, p-STAT3, STAT3, and serum IL-6 levels were all significantly reduced in A20-bearing mice treated with CSP-STAT3 siRNA. These findings indicate that specifically targeting STAT3 siRNA to B cell lymphoma cell lines can significantly decrease STAT3 activity and inhibit tumor progression in vitro and in vivo, suggesting its potential utilization for cancer treatment.

Impact of glycoengineering and antidrug antibodies on the anticancer activity of a plant-made lectin-Fc fusion protein.

Plants are an efficient production platform for manufacturing glycoengineered monoclonal antibodies and antibody-like molecules. Avaren-Fc (AvFc) is a lectin-Fc fusion protein or lectibody produced in Nicotiana benthamiana, which selectively recognizes cancer-associated high-mannose glycans. In this study, we report the generation of a glycovariant of AvFc that is devoid of plant glycans, including the core α1,3-fucose and ß1,2-xylose residues. The successful removal of these glycans was confirmed by glycan analysis using HPLC. This variant, AvFcΔXF , has significantly higher affinity for Fc gamma receptors and induces higher levels of luciferase expression in an antibody-dependent cell-mediated cytotoxicity (ADCC) reporter assay against B16F10 murine melanoma cells without inducing apoptosis or inhibiting proliferation. In the B16F10 flank tumour mouse model, we found that systemic administration of AvFcΔXF , but not an aglycosylated AvFc variant lacking affinity for Fc receptors, significantly delayed the growth of tumours, suggesting that Fc-mediated effector functions were integral. AvFcΔXF treatment also significantly reduced lung metastasis of B16F10 upon intravenous challenge whereas a sugar-binding-deficient mutant failed to show efficacy. Lastly, we determined the impact of antidrug antibodies (ADAs) on drug activity in vivo by pretreating animals with AvFcΔXF before implanting tumours. Despite a significant ADA response induced by the pretreatment, we found that the activity of AvFcΔXF was unaffected by the presence of these antibodies. These results demonstrate that glycoengineering is a powerful strategy to enhance AvFc's antitumor activity.

Inhibition of Sphingosine-1-Phosphate-Induced Th17 Cells Ameliorates Alcohol-Associated Steatohepatitis in Mice.

BACKGROUND AND AIMS: Chronic alcohol consumption is accompanied by intestinal inflammation. However, little is known about how alterations to the intestinal immune system and sphingolipids contribute to the pathogenesis of alcohol-associated liver disease (ALD). APPROACH AND RESULTS: We used wild-type mice, retinoid-related orphan receptor gamma t (RORγt)-deficient mice, sphingosine kinase-deficient mice, and local gut anti-inflammatory, 5-aminosalicyclic acid-treated mice in a chronic-binge ethanol feeding model. Targeted lipidomics assessed the sphingolipids in gut and liver samples. Gut immune cell populations, the amounts of sphingolipids, and the level of liver injury were examined. Alcohol intake induces a pro-inflammatory shift in immune cell populations in the gut, including an increase in Th17 cells. Using RORγt-deficient mice, we found that Th17 cells are required for alcohol-associated gut inflammation and the development of ALD. Treatment with 5-aminosalicyclic acid decreases alcohol-induced liver injury and reverses gut inflammation by the suppression of CD4+ /RORγt+ /interleukin-17A+ cells. Increased Th17 cells were due to up-regulation of sphingosine kinase 1 activity and RORγt activation. We found that S1P/S1PR1 signaling is required for the development of Th17 cell-mediated ALD. Importantly, in vivo intervention blocking of S1P/S1PR1 signaling markedly attenuated alcohol-induced liver inflammation, steatosis, and damage. CONCLUSIONS: Gut inflammation is a functional alteration of immune cells in ALD. Reducing gut Th17 cells leads to reduced liver damage. S1P signaling was crucial in the pathogenesis of ALD in a Th17 cell-dependent manner. Furthermore, our findings suggest that compounds that reduce gut inflammation locally may represent a unique targeted approach in the treatment of ALD.

Lower Respiratory Tract Infection of the Ferret by 2009 H1N1 Pandemic Influenza A Virus Triggers Biphasic, Systemic, and Local Recruitment of Neutrophils.

UNLABELLED: Infection of the lower respiratory tract by influenza A viruses results in increases in inflammation and immune cell infiltration in the lung. The dynamic relationships among the lung microenvironments, the lung, and systemic host responses during infection remain poorly understood. Here we used extensive systematic histological analysis coupled with live imaging to gain access to these relationships in ferrets infected with the 2009 H1N1 pandemic influenza A virus (H1N1pdm virus). Neutrophil levels rose in the lungs of H1N1pdm virus-infected ferrets 6 h postinfection and became concentrated at areas of the H1N1pdm virus-infected bronchiolar epithelium by 1 day postinfection (dpi). In addition, neutrophil levels were increased throughout the alveolar spaces during the first 3 dpi and returned to baseline by 6 dpi. Histochemical staining revealed that neutrophil infiltration in the lungs occurred in two waves, at 1 and 3 dpi, and gene expression within microenvironments suggested two types of neutrophils. Specifically, CCL3 levels, but not CXCL8/interleukin 8 (IL-8) levels, were higher within discrete lung microenvironments and coincided with increased infiltration of neutrophils into the lung. We used live imaging of ferrets to monitor host responses within the lung over time with [(18)F]fluorodeoxyglucose (FDG). Sites in the H1N1pdm virus-infected ferret lung with high FDG uptake had high levels of proliferative epithelium. In summary, neutrophils invaded the H1N1pdm virus-infected ferret lung globally and focally at sites of infection. Increased neutrophil levels in microenvironments did not correlate with increased FDG uptake; hence, FDG uptake may reflect prior infection and inflammation of lungs that have experienced damage, as evidenced by bronchial regeneration of tissues in the lungs at sites with high FDG levels. IMPORTANCE: Severe influenza disease is characterized by an acute infection of the lower airways that may progress rapidly to organ failure and death. Well-developed animal models that mimic human disease are essential to understanding the complex relationships of the microenvironment, organ, and system in controlling virus replication, inflammation, and disease progression. Employing the ferret model of H1N1pdm virus infection, we used live imaging and comprehensive histological analyses to address specific hypotheses regarding spatial and temporal relationships that occur during the progression of infection and inflammation. We show the general invasion of neutrophils at the organ level (lung) but also a distinct pattern of localized accumulation within the microenvironment at the site of infection. Moreover, we show that these responses were biphasic within the lung. Finally, live imaging revealed an early and sustained host metabolic response at sites of infection that may reflect damage and repair of tissues in the lungs.

Effect of the stop-flow technique on cardiac retention of c-kit positive human cardiac stem cells after intracoronary infusion in a porcine model of chronic ischemic cardiomyopathy.

It is commonly thought that the optimal method for intracoronary administration of cells is to stop coronary flow during cell infusion, in order to prolong cell/vascular wall contact, enhance adhesion, and promote extravasation of cells into the interstitial space. However, occlusion of a coronary artery with a balloon involves serious risks of vascular damage and/or dissection, particularly in non-stented segments such as those commonly found in patients with heart failure. It remains unknown whether the use of the stop-flow technique results in improved donor cell retention. Acute myocardial infarction was produced in 14 pigs. One to two months later, pigs received 10 million indium-111 oxyquinoline (oxine)-labeled c-kit(pos) human cardiac stem cells (hCSCs) via intracoronary infusion with (n = 7) or without (n = 7) balloon inflation. Pigs received cyclosporine to prevent acute graft rejection. Animals were euthanized 24 h later and hearts harvested for radioactivity measurements. With the stop-flow technique, the retention of hCSCs at 24 h was 5.41 ± 0.80 % of the injected dose (n = 7), compared with 4.87 ± 0.62 % without coronary occlusion (n = 7), (P = 0.60). When cells are delivered intracoronarily in a clinically relevant porcine model of chronic ischemic cardiomyopathy, the use of the stop-flow technique does not result in greater myocardial cell retention at 24 h compared with non-occlusive infusion. These results have practical implications for the design of cell therapy trials. Our observations suggest that the increased risk of complications secondary to coronary manipulation and occlusion is not warranted.

Targeted drug delivery to intestinal macrophages by bioactive nanovesicles released from grapefruit.

The gut mucosal immune system is considered to play an important role in counteracting potential adverse effects of food-derived antigens including nanovesicles. Whether nanovesicles naturally released from edible fruit work in a coordinated manner with gut immune cells to maintain the gut in a noninflammatory status is not known. Here, as proof of concept, we demonstrate that grapefruit-derived nanovesicles (GDNs) are selectively taken up by intestinal macrophages and ameliorate dextran sulfate sodium (DSS)-induced mouse colitis. These effects were mediated by upregulating the expression of heme oxygenase-1 (HO-1) and inhibiting the production of IL-1ß and TNF-α in intestinal macrophages. The inherent biocompatibility and biodegradability, stability at wide ranges of pH values, and targeting of intestinal macrophages led us to further develop a novel GDN-based oral delivery system. Incorporating methotrexate (MTX), an anti-inflammatory drug, into GDNs and delivering the MTX-GDNs to mice significantly lowered the MTX toxicity when compared with free MTX, and remarkably increased its therapeutic effects in DSS-induced mouse colitis. These findings demonstrate that GDNs can serve as immune modulators in the intestine, maintain intestinal macrophage homeostasis, and can be developed for oral delivery of small molecule drugs to attenuate inflammatory responses in human disease.

Design and evaluation of new Tc-99m-labeled lactam bridge-cyclized alpha-MSH peptides for melanoma imaging.

The purpose of this study was to examine the melanoma targeting and imaging properties of new (99m)Tc-labeled lactam bridge-cyclized alpha-melanocyte stimulating hormone (α-MSH) peptides using bifunctional chelating agents. MAG3-GGNle-CycMSH(hex), AcCG3-GGNle-CycMSH(hex), and HYNIC-GGNle-CycMSH(hex) peptides were synthesized, and their melanocortin-1 (MC1) receptor binding affinities were determined in B16/F1 melanoma cells. The biodistribution of (99m)Tc-MAG3-GGNle-CycMSH(hex), (99m)Tc-AcCG3-GGNle-CycMSH(hex), (99m)Tc(CO)3-HYNIC-GGNle-CycMSH(hex), and (99m)Tc(EDDA)-HYNIC-GGNle-CycMSH(hex) were determined in B16/F1 melanoma-bearing C57 mice at 2 h postinjection to select a lead peptide for further evaluation. The melanoma targeting and imaging properties of (99m)Tc(EDDA)-HYNIC-GGNle-CycMSH(hex) were further examined because of its high melanoma uptake and fast urinary clearance. The IC50 values of MAG3-GGNle-CycMSH(hex), AcCG3-GGNle-CycMSH(hex), and HYNIC-GGNle-CycMSH(hex) were 1.0 ± 0.05, 1.2 ± 0.19, and 0.6 ± 0.04 nM in B16/F1 melanoma cells, respectively. Among these four (99m)Tc-peptides, (99m)Tc(EDDA)-HYNIC-GGNle-CycMSH(hex) exhibited the highest melanoma uptake (14.14 ± 4.90% ID/g) and fastest urinary clearance (91.26 ± 1.96% ID) at 2 h postinjection. (99m)Tc(EDDA)-HYNIC-GGNle-CycMSH(hex) showed high tumor to normal organ uptake ratios except for the kidneys. The tumor/kidney uptake ratios of (99m)Tc(EDDA)-HYNIC-GGNle-CycMSH(hex) were 2.50 and 3.55 at 4 and 24 h postinjection. The melanoma lesions were clearly visualized by SPECT/CT using (99m)Tc(EDDA)-HYNIC-GGNle-CycMSH(hex) as an imaging probe at 2 h postinjection. Overall, high melanoma uptake coupled with fast urinary clearance of (99m)Tc(EDDA)-HYNIC-GGNle-CycMSH(hex) highlighted its potential for metastatic melanoma detection in the future.

Melanoma targeting property of a Lu-177-labeled lactam bridge-cyclized alpha-MSH peptide.

The purpose of this study was to determine the melanoma targeting property of (177)Lu-DOTA-GGNle-CycMSHhex in B16/F1 melanoma-bearing C57 mice. (177)Lu-DOTA-GGNle-CycMSHhex exhibited high receptor-mediated melanoma uptake and fast urinary clearance. The tumor uptake of (177)Lu-DOTA-GGNle-CycMSHhex was 20.25 ± 4.59 and 21.63 ± 6.27% ID/g at 0.5 and 2h post-injection, respectively. Approximately 83% of injected dose cleared out the body via urinary system at 2h post-injection. (177)Lu-DOTA-GGNle-CycMSHhex showed high tumor to normal organ uptake ratios except for the kidneys. The tumor/kidney uptake ratios of (177)Lu-DOTA-GGNle-CycMSHhex were 2.76 and 1.74 at 2 and 24h post-injection. The melanoma lesions were clearly visualized by SPECT/CT using (177)Lu-DOTA-GGNle-CycMSHhex as an imaging probe at 2h post-injection. Overall, high melanoma uptake coupled with fast urinary clearance of (177)Lu-DOTA-GGNle-CycMSHhex underscored its potential for melanoma treatment in the future.

Influences of hydrocarbon linkers on the receptor binding affinities of gonadotropin-releasing hormone peptides.

Three new DOTA-conjugated GnRH peptides with various hydrocarbon linkers were synthesized to evaluate the influences of the linkers on their receptor binding affinities. The hydrocarbon linker displayed a profound impact on the receptor binding affinities of DOTA-conjugated GnRH peptides. The Aun linker was better than Gaba, Ahx and Aoc linkers in retaining strong receptor binding affinity of the GnRH peptide. DOTA-Aun-(D-Lys(6)-GnRH) displayed 22.8 nM GnRH receptor binding affinity. (111)In-DOTA-Aun-(D-Lys(6)-GnRH) exhibited fast tumor uptake and urinary clearance in MDA-MB-231 human breast cancer-xenografted nude mice. The cellular and biological results provided an insight into the design of new GnRH peptides in the future.

Gallium-67-labeled lactam bridge-cyclized alpha-MSH peptides with enhanced melanoma uptake and reduced renal uptake.

The purpose of this study was to examine the melanoma targeting and pharmacokinetic properties of (67)Ga-DOTA-GGNle-CycMSHhex {(67)Ga-1,4,7,10-tetraazacyclononane-1,4,7,10-tetraacetic acid-Gly-Gly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and (67)Ga-NOTA-GGNle-CycMSHhex {(67)Ga-1,4,7-triazacyclononane-1,4,7-triacetic acid-Gly-Gly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and compare with (67)Ga-DOTA-GlyGlu-CycMSH {(67)Ga-DOTA-Gly-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]} we previously reported. DOTA-GGNle-CycMSHhex and NOTA-GGNle-CycMSHhex were synthesized using fluorenylmethyloxy carbonyl (Fmoc) chemistry. The melanocortin-1 (MC1) receptor binding affinity of NOTA-GGNle-CycMSHhex was determined in B16/F1 melanoma cells and compared with DOTA-GGNle-CycMSHhex. The melanoma targeting and pharmacokinetic properties of (67)Ga-NOTA-GGNle-CycMSHhex and (67)Ga-DOTA-GGNle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice. NOTA-GGNle-CycMSHhex and DOTA-GGNle-CycMSHhex displayed comparable MC1 receptor binding affinities (1.6 vs 2.1 nM) in B16/F1 melanoma cells. Both (67)Ga-NOTA-GGNle-CycMSHhex and (67)Ga-DOTA-GGNle-CycMSHhex exhibited dramatically enhanced melanoma uptake and reduced renal uptake than (67)Ga-DOTA-GlyGlu-CycMSH in B16/F1 melanoma-bearing C57 mice. Furthermore, (67)Ga-NOTA-GGNle-CycMSHhex exhibited more favorable radiolabeling conditions (>85% radiolabeling yields started at 37 °C), as well as higher tumor/kidney uptake ratios than (67)Ga-DOTA-GGNle-CycMSHhex at 0.5, 2, and 24 h postinjection. High melanoma uptake coupled with low renal uptake highlighted the potential of (67)Ga-NOTA-GGNle-CycMSHhex for melanoma imaging and therapy.

Cu-64-labeled lactam bridge-cyclized α-MSH peptides for PET imaging of melanoma.

The purpose of this study was to examine and compare the melanoma targeting and imaging properties of (64)Cu-NOTA-GGNle-CycMSH(hex) {(64)Cu-1,4,7-triazacyclononane-1,4,7-triacetic acid-Gly-Gly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and (64)Cu-DOTA-GGNle-CycMSH(hex) {(64)Cu-1,4,7,10-tetraazacyclononane-1,4,7,10-tetraacetic acid-GGNle-CycMSH(hex)}. Two lactam bridge-cyclized peptides, NOTA-GGNle-CycMSH(hex) and DOTA-GGNle-CycMSH(hex), were synthesized using fluorenylmethyloxy carbonyl (Fmoc) chemistry. The melanocortin-1 (MC1) receptor binding affinity of NOTA-GGNle-CycMSH(hex) was determined in B16/F1 melanoma cells and compared with DOTA-GGNle-CycMSH(hex). The melanoma targeting and imaging properties of (64)Cu-NOTA-GGNle-CycMSH(hex) and (64)Cu-DOTA-GGNle-CycMSH(hex) were determined in B16/F1 melanoma-bearing C57 mice. NOTA-GGNle-CycMSH(hex) and DOTA-GGNle-CycMSH(hex) displayed comparable MC1 receptor binding affinities (1.6 vs 2.1 nM). The substitution of DOTA with NOTA dramatically increased the melanoma uptake and decreased the renal and liver uptake of (64)Cu-NOTA-GGNle-CycMSH(hex). The tumor uptake of (64)Cu-NOTA-GGNle-CycMSH(hex) was between 12.39 ± 1.61 and 12.71 ± 2.68% ID/g at 0.5, 2, and 4 h postinjection. The accumulation of (64)Cu-NOTA-GGNle-CycMSH(hex) activity in normal organs was lower than 1.02% ID/g except for the kidneys 2, 4, and 24 h postinjection. The tumor/liver uptake ratios of (64)Cu-NOTA-GGNle-CycMSHhex were 17.96, 16.95, and 8.02, whereas the tumor/kidney uptake ratios of (64)Cu-NOTA-GGNle-CycMSH(hex) were 2.52, 3.60, and 5.74 at 2, 4, and 24 h postinjection, respectively. Greater than 91% of the injected radioactivity cleared through the urinary system by 2 h postinjection. The substitution of DOTA with NOTA resulted in a dramatic increase in melanoma uptake and decrease in renal and liver uptake of (64)Cu-NOTA-GGNle-CycMSH(hex) as compared to (64)Cu-DOTA-GGNle-CycMSH(hex). High melanoma uptake coupled with low accumulation in nontarget organs suggested (64)Cu-NOTA-GGNle-CycMSH(hex) as a lead radiolabeled peptide for melanoma imaging and therapy.

Alcohol-driven metabolic reprogramming promotes development of RORγt-deficient thymic lymphoma.

RORγt is a master regulator of Th17 cells. Despite evidence linking RORγt deficiency/inhibition with metastatic thymic T cell lymphomas, the role of RORγt in lymphoma metabolism is unknown. Chronic alcohol consumption plays a causal role in many human cancers. The risk of T cell lymphoma remains unclear in humans with alcohol use disorders (AUD) after chronic RORγt inhibition. Here we demonstrated that alcohol consumption accelerates RORγt deficiency-induced lymphomagenesis. Loss of RORγt signaling in the thymus promotes aerobic glycolysis and glutaminolysis and increases allocation of glutamine carbon into lipids. Importantly, alcohol consumption results in a shift from aerobic glycolysis to glutaminolysis. Both RORγt deficiency- and alcohol-induced metabolic alterations are mediated by c-Myc, as silencing of c-Myc decreases the effects of alcohol consumption and RORγt deficiency on glutaminolysis, biosynthesis, and tumor growth in vivo. The ethanol-mediated c-Myc activation coupled with increased glutaminolysis underscore the critical role of RORγt-Myc signaling and translation in lymphoma.

Microbial metabolite restricts 5-fluorouracil-resistant colonic tumor progression by sensitizing drug transporters via regulation of FOXO3-FOXM1 axis.

The survival rate of colorectal cancer patients is adversely affected by the selection of tumors resistant to conventional anti-cancer drugs such as 5-fluorouracil (5FU). Although there is mounting evidence that commensal gut microbiota is essential for effective colon cancer treatment, the detailed molecular mechanisms and the role of gut microbial metabolites remain elusive. The goal of this study is to decipher the impact and mechanisms of gut microbial metabolite, urolithin A (UroA) and its structural analogue, UAS03 on reversal of 5FU-resistant (5FUR) colon cancers. Methods: We have utilized the SW480 and HCT-116 parental (5FU-sensitive) and 5FUR colon cancer cells to examine the chemosensitization effects of UroA or UAS03 by using both in vitro and in vivo models. The effects of mono (UroA/UAS03/5FU) and combinatorial therapy (UroA/UAS03 + 5FU) on cell proliferation, apoptosis, cell migration and invasion, regulation of epithelial mesenchymal transition (EMT) mediators, expression and activities of drug transporters, and their regulatory transcription factors were examined using molecular, cellular, immunological and flowcytometric methods. Further, the anti-tumor effects of mono/combination therapy (UroA or UAS03 or 5FU or UroA/UAS03 + 5FU) were examined using pre-clinical models of 5FUR-tumor xenografts in NRGS mice and azoxymethane (AOM)-dextran sodium sulfate (DSS)-induced colon tumors. Results: Our data showed that UroA or UAS03 in combination with 5FU significantly inhibited cell viability, proliferation, invasiveness as well as induced apoptosis of the 5FUR colon cancer cells compared to mono treatments. Mechanistically, UroA or UAS03 chemosensitized the 5FUR cancer cells by downregulating the expression and activities of drug transporters (MDR1, BCRP, MRP2 and MRP7) leading to a decrease in the efflux of 5FU. Further, our data suggested the UroA or UAS03 chemosensitized 5FUR cancer cells to 5FU treatment through regulating FOXO3-FOXM1 axis. Oral treatment with UroA or UAS03 in combination with low dose i.p. 5FU significantly reduced the growth of 5FUR-tumor xenografts in NRGS mice. Further, combination therapy significantly abrogated colonic tumors in AOM-DSS-induced colon tumors in mice. Conclusions: In summary, gut microbial metabolite UroA and its structural analogue UAS03 chemosensitized the 5FUR colon cancers for effective 5FU chemotherapy. This study provided the novel characteristics of gut microbial metabolites to have significant translational implications in drug-resistant cancer therapeutics.

The induction of peripheral trained immunity in the pancreas incites anti-tumor activity to control pancreatic cancer progression.

Despite the remarkable success of immunotherapy in many types of cancer, pancreatic ductal adenocarcinoma has yet to benefit. Innate immune cells are critical to anti-tumor immunosurveillance and recent studies have revealed that these populations possess a form of memory, termed trained innate immunity, which occurs through transcriptomic, epigenetic, and metabolic reprograming. Here we demonstrate that yeast-derived particulate ß-glucan, an inducer of trained immunity, traffics to the pancreas, which causes a CCR2-dependent influx of monocytes/macrophages to the pancreas that display features of trained immunity. These cells can be activated upon exposure to tumor cells and tumor-derived factors, and show enhanced cytotoxicity against pancreatic tumor cells. In orthotopic models of pancreatic ductal adenocarcinoma, ß-glucan treated mice show significantly reduced tumor burden and prolonged survival, which is further enhanced when combined with immunotherapy. These findings characterize the dynamic mechanisms and localization of peripheral trained immunity and identify an application of trained immunity to cancer.

Validated Preclinical Mouse Model for Therapeutic Testing against Multidrug-Resistant Pseudomonas aeruginosa Strains.

The rise in infections caused by antibiotic-resistant bacteria is outpacing the development of new antibiotics. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) are a group of clinically important bacteria that have developed resistance to multiple antibiotics and are commonly referred to as multidrug resistant (MDR). The medical and research communities have recognized that, without new antimicrobials, infections by MDR bacteria will soon become a leading cause of morbidity and death. Therefore, there is an ever-growing need to expedite the development of novel antimicrobials to combat these infections. Toward this end, we set out to refine an existing mouse model of pulmonary Pseudomonas aeruginosa infection to generate a robust preclinical tool that can be used to rapidly and accurately predict novel antimicrobial efficacy. This refinement was achieved by characterizing the virulence of a panel of genetically diverse MDR P. aeruginosa strains in this model, by both 50% lethal dose (LD50) analysis and natural history studies. Further, we defined two antibiotic regimens (aztreonam and amikacin) that can be used as comparators during the future evaluation of novel antimicrobials, and we confirmed that the model can effectively differentiate between successful and unsuccessful treatments, as predicted by in vitro inhibitory data. This validated model represents an important tool in our arsenal to develop new therapies to combat MDR P. aeruginosa strains, with the ability to provide rapid preclinical evaluation of novel antimicrobials and support data from clinical studies during the investigational drug development process. IMPORTANCE The prevalence of antibiotic resistance among bacterial pathogens is a growing problem that necessitates the development of new antibiotics. Preclinical animal models are important tools to facilitate and speed the development of novel antimicrobials. Successful outcomes in animal models not only justify progression of new drugs into human clinical trials but also can support FDA decisions if clinical trial sizes are small due to a small population of infections with specific drug-resistant strains. However, in both cases the preclinical animal model needs to be well characterized and provide robust and reproducible data. Toward this goal, we have refined an existing mouse model to better predict the efficacy of novel antibiotics. This improved model provides an important tool to better predict the clinical success of new antibiotics.

Synthesis and evaluation of novel gonadotropin-releasing hormone receptor-targeting peptides.

The purpose of this study was to develop novel radiolabeled gonadotropin-releasing hormone (GnRH) receptor-targeting peptides for breast cancer imaging. Three novel 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated GnRH peptides were designed and synthesized. The radiometal chelator DOTA was conjugated to the epsilon or alpha amino group of D-lysine, or the epsilon amino group of L-lysine via an Ahx {aminohexanoic acid} linker to generate DOTA-Ahx-(D-Lys(6)-GnRH1), DOTA-Ahx-(D-Lys(6)-GnRH2) and DOTA-Ahx-(L-Lys(6)-GnRH3), respectively. The conjugation of the DOTA to the epsilon amino group of D-lysine (rather than alpha amino group of D-lysine nor epsilon amino group of L-lysine) maintained the nanomolar GnRH receptor binding affinity. The IC(50) values of DOTA-Ahx-(D-Lys(6)-GnRH1), DOTA-Ahx-(D-Lys(6)-GnRH2) and DOTA-Ahx-(L-Lys(6)-GnRH3) were 36.1 nM, 10.6 mM and 4.3 mM, respectively. Since only DOTA-Ahx-(D-Lys(6)-GnRH1) displayed nanomolar receptor binding affinity, the specific GnRH receptor binding of (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1) was determined in human GnRH receptor membrane preparations. Furthermore, the biodistribution and tumor imaging properties of (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1) were examined in MDA-MB-231 human breast cancer-xenografted nude mice. (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1) exhibited specific GnRH receptor binding and rapid tumor uptake (1.76 ± 0.58% ID/g at 0.5 h postinjection) coupled with fast whole-body clearance through the urinary system. The MDA-MB-231 human breast cancer-xenografted tumor lesions were clearly visualized by single photon emission computed tomography (SPECT)/CT at 1 h postinjection of (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1). The profound impact of DOTA position on the binding affinity of the GnRH peptide provided a new insight into the design of novel radiolabeled GnRH peptides. The successful imaging of MDA-MB-231 human breast cancer-xenografted tumor lesions using (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1) suggested its potential as a novel imaging probe for human breast cancer imaging.

A novel indium-111-labeled gonadotropin-releasing hormone peptide for human prostate cancer imaging.

The purpose of this study was to evaluate the tumor targeting and imaging properties of a novel (111)In-labeled gonadotropin-releasing hormone (GnRH) peptide {1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-Ahx-(D-Lys(6)-GnRH1)} for human prostate cancer. The biodistribution and tumor imaging properties of (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1) were determined in DU145 human prostate cancer-xenografted nude mice. (111)In-DOTA-Ahx-(d-Lys(6)-GnRH1) exhibited rapid tumor uptake (1.27 ± 0.40% ID/g at 0.5h post-injection) coupled with fast whole-body clearance through the urinary system. The DU145 human prostate cancer-xenografted tumor lesions were clearly visualized by single photon emission computed tomography (SPECT)/CT at 0.5h post-injection of (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1). The successful imaging of DU145 human prostate cancer-xenografted tumor lesions using (111)In-DOTA-Ahx-(d-Lys(6)-GnRH1) highlighted its potential as a novel imaging probe for human prostate cancer imaging.

Tumor-derived exosomes drive immunosuppressive macrophages in a pre-metastatic niche through glycolytic dominant metabolic reprogramming.

One of the defining characteristics of a pre-metastatic niche, a fundamental requirement for primary tumor metastasis, is infiltration of immunosuppressive macrophages. How these macrophages acquire their phenotype remains largely unexplored. Here, we demonstrate that tumor-derived exosomes (TDEs) polarize macrophages toward an immunosuppressive phenotype characterized by increased PD-L1 expression through NF-kB-dependent, glycolytic-dominant metabolic reprogramming. TDE signaling through TLR2 and NF-κB leads to increased glucose uptake. TDEs also stimulate elevated NOS2, which inhibits mitochondrial oxidative phosphorylation resulting in increased conversion of pyruvate to lactate. Lactate feeds back on NF-κB, further increasing PD-L1. Analysis of metastasis-negative lymph nodes of non-small-cell lung cancer patients revealed that macrophage PD-L1 positively correlates with levels of GLUT-1 and vesicle release gene YKT6 from primary tumors. Collectively, our study provides a novel mechanism by which macrophages within a pre-metastatic niche acquire their immunosuppressive phenotype and identifies an important link among exosomes, metabolism, and metastasis.

Synthesis and biological evaluation of a novel 99mTc(CO)3 complex of ciprofloxacin dithiocarbamate as a potential agent to target infection.

The ciprofloxacin dithiocarbamate (CPFXDTC) was radiolabeled with [(99m)Tc(CO)(3)(H(2)O)(3)](+) intermediate to form the (99m)Tc(CO)(3)-CPFXDTC complex in high yield. The (99m)Tc(CO)(3)-CPFXDTC complex was characterized by HPLC and its stability in serum was studied. Its partition coefficient indicated that it was a lipophilic complex. The bacterial binding efficiency of (99m)Tc(CO)(3)-CPFXDTC was almost the same as that of (99m)TcN-CPFXDTC, and was higher than that of (99m)Tc-ciprofloxacin. Biodistribution results in induced infection mice showed (99m)Tc(CO)(3)-CPFXDTC had higher uptake at the sites of infection and better abscess/blood and abscess/muscle ratios than those of (99m)Tc-ciprofloxacin and (99m)TcN-CPFXDTC. Single photon emission computed tomography (SPECT) static imaging study in infected rabbits demonstrated the uptake in the left thigh infection lesion was observable, while no accumulation in the right thigh muscle was found. These results suggested (99m)Tc(CO)(3)-CPFXDTC would be a promising candidate for further evaluation as infection imaging agent.