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In this study, attempts were made to utilize date by-product (date fruit pomace; DFP). This study aimed to investigate the health-promoting benefits of the fermented and non-fermented DFP before in vitro digestion and after (bioaccessible fraction). Untargeted metabolomic analyses for bioaccessible fractions were performed by UPLC-QTOF. DPPH percentages were 89.7%-90.3%, 90.1%-91.3%, and 90.8%-91.3% in the control, I. orientalis, and P. kudriazevii samples, respectively, before digestion; α-glucosidase inhibition before digestion was 1.9%-24.4%, 16.3%-30.0%, and 21.3%-31.3%, respectively; antimicrobial activities were 6.1%-13.3%, 13.7%-25.7%, and 20.6%-28.0% against E. coli O157:H7 and 2.2%-11.9%, 7.2%-20.7%, and 11.9%-29.2% against L. monocytogenes, respectively. The DPPH scavenging percentages were ∼63% lower in the bioaccessible fraction. The differentially regulated metabolites classes were benzene and derivatives, amino acids, peptides and analogs, organic acids, and phenols. This study revealed that the fermented DFP exhibited higher health properties than control.

Several studies reported a central role of the endothelin type A receptor (ETAR) in tumor progression leading to the formation of metastasis. Here, we investigated the in vitro and in vivo anti-tumor effects of the FDA-approved ETAR antagonist, Ambrisentan, which is currently used to treat patients with pulmonary arterial hypertension. In vitro, Ambrisentan inhibited both spontaneous and induced migration/invasion capacity of different tumor cells (COLO-357 metastatic pancreatic adenocarcinoma, OvCar3 ovarian carcinoma, MDA-MB-231 breast adenocarcinoma, and HL-60 promyelocytic leukemia). Whole transcriptome analysis using RNAseq indicated Ambrisentan’s inhibitory effects on the whole transcriptome of resting and PAR2-activated COLO-357 cells, which tended to normalize to an unstimulated profile. Finally, in a pre-clinical murine model of metastatic breast cancer, treatment with Ambrisentan was effective in decreasing metastasis into the lungs and liver. Importantly, this was associated with a significant enhancement in animal survival. Taken together, our work suggests a new therapeutic application for Ambrisentan in the treatment of cancer metastasis.

Nanoparticles (NPs) are increasingly being commercialized for use in biomedicine. NP toxicity following acute or chronic exposure has been described, but mechanistic insight into this process remains incomplete. Recent evidence from in vitro studies suggested a role for NLRP3 in NP cytotoxicity. In this study, we investigated the effect of systemic administration of composite inorganic NP, consisting of Ag:Cu:B (dose range 1-20 mg/kg), on the early acute (4-24 h post-exposure) and late phase response (96 h post-exposure) in normal and NLRP3-deficient mice. Our findings indicate that systemic exposure (≥2 mg/kg) was associated with acute liver injury due to preferential accumulation of NP in this organ and resulted in elevated AST, ALT and LDH levels. Moreover, within 24 h of NP administration, there was a dose-dependent increase in intraperitoneal neutrophil recruitment and upregulation in gene expression of several proinflammatory mediators, including TNF-α, IL-1β and S100A9. Histological analysis of liver tissue revealed evidence of dose-dependent hepatocyte necrosis, increase in sinusoidal Kupffer cells, lobular granulomas and foci of abscess formation which were most pronounced at 24 h following NP administration. NP deposition in the liver led to a significant upregulation in gene expression of S100A9, an endogenous danger signal recognition molecule of phagocytes, IL-1β and IL-6. The extent of proinflammatory cytokine activation and hepatotoxicity was significantly attenuated in mice deficient in the NLRP3 inflammasome, demonstrating the critical role of this innate immune system recognition receptor in the response to NP.

Growing evidence indicates a link between persistent infections and the development of autoimmune diseases. For instance, the inability to control Salmonella infection due to defective toll-like receptor (TLR) / myeloid differentiation primary response 88 (MyD88) signaling has linked the development of persistent infections to a breakdown in B cell tolerance. However, the extent of immune dysregulation in the absence of TLR-MyD88 signaling remains poorly characterized. Here, we show that MyD88-/- mice areGrowing evidence indicates a link between persistent infections and the development of autoimmune diseases. For instance, the inability to control Salmonella infection due to defective toll-like receptor (TLR) / myeloid differentiation primary response 88 (MyD88) signaling has linked the development of persistent infections to a breakdown in B cell tolerance. However, the extent of immune dysregulation in the absence of TLR-MyD88 signaling remains poorly characterized. Here, we show that MyD88-/- mice are unable to eliminate attenuated Salmonella enterica serovar Typhimurium, even when challenged with a low dose inoculum (200 CFUs/mouse), developing a persistent and progressive infection when compared to wild-type (MyD88+/+) animals. The splenic niche of MyD88-/- mice revealed increased counts of activated, Sca-1-positive, myeloid subpopulations highly expressing BAFF during persistent Salmonella infection. Likewise, the T cell compartment of Salmonella-infected MyD88-/- mice showed increased levels of CD4+ and CD8+ T cells expressing Sca-1 and CD25 and producing elevated amounts of IL-4, IL-10 and IL-21 in response to CD3/CD28 stimulation. This was associated with increased Tfh cell differentiation and the presence of CD4+ T cells co-expressing IFN-/IL-4 and IFN-/IL-10. Noteworthy, infected MyD88-/- mice had enhanced serum titers of both anti-Salmonella antibodies as well as autoantibodies directed against dsDNA, thyroglobulin and IgG rheumatoid factor (RF), positive nuclear staining with HEp2 cells, and immune complex deposition in the kidneys of MyD88-/- mice infected with live but not heat-killed Salmonella. Infection with other microorganisms (Acinetobacter baumanii, Streptococcus agalactiae or Escherichia coli) was unable to trigger the autoimmune phenomenon. Our findings suggest that dysregulation of the immune response in the absence of MyD88 is pathogen-dependent and highlight potentially important genotype-environmental factor correlations.

The use of attenuated bacteria as cancer therapeutic tools has garnered increasing scientific interest over the past 10 years. This is largely due to the development of bacterial strains that maintain good anti-tumor efficacy, but with reduced potential to cause toxicities to the host. Because of its ability to replicate in viable as well as necrotic tissue, cancer therapy using attenuated strains of facultative anaerobic bacteria, such as Salmonella, has several advantages over standard treatment modalities, including chemotherapy and radiotherapy. Despite some findings suggesting that it may operate through a direct cytotoxic effect against cancer cells, there is accumulating evidence demonstrating that bacterial therapy acts by modulating cells of the immune system to counter the growth of the tumor. Herein, we review the experimental evidence underlying the success of bacterial immunotherapy against cancer and highlight the cellular and molecular alterations in the peripheral immune system and within the tumor microenvironment that have been reported following different forms of bacterial therapy. Our improved understanding of these mechanisms should greatly aid in the translational application of bacterial therapy to cancer patients.

Inflammation is a crucial defense mechanism that protects the body from the devastating effects of invading pathogens. However, an unrestrained inflammatory reaction may result in systemic manifestations with dire consequences to the host. The extent of activation of the inflammatory response is tightly regulated through immunological and neural pathways. Previously, we demonstrated that cholinergic stimulation confers enhanced protection in experimental animals orally infected with virulent Salmonella enterica serovar Typhimurium. In this study, we investigated the mechanism by which this enhanced protection takes place. Cholinergic stimulation was induced by a 3-week pretreatment with paraoxon, a highly specific acetylcholinesterase inhibitor. This treatment enhanced host survival following oral-route infection and this correlated with significantly reduced bacterial load in systemic target organs. Enhanced protection was not due to increased gut motility or rapid bacterial clearance from the gastrointestinal tract. Moreover, protection against bacterial infection was not evident when the animals were infected systemically, suggesting that acetylcholine-mediated protective effect was mostly confined to the gut mucosal tissue. In vivo imaging demonstrated a more localized infection and delay in bacterial dissemination into systemic organs in mice pre-treated with paraoxon. Morphological analysis of the small intestine (ileum) showed that acetylcholinesterase inhibition induced the degranulation of goblet cells and Paneth cells, two specialized secretory cells involved in innate immunity. Our findings demonstrate a crucial pathway between neural and immune systems that acts at the mucosal interface to protect the host against oral pathogens.

Background Patients with X-linked hyper-IgM syndrome caused by CD40 ligand (CD40L) deficiency often present with episodic, cyclic, or chronic neutropenia, suggesting abnormal neutrophil development in the absence of CD40L-CD40 interaction. However, even when not neutropenic and despite immunoglobulin replacement therapy, CD40L-deficient patients are susceptible to life-threatening infections caused by opportunistic pathogens, suggesting impaired phagocyte function and the need for novel therapeutic approaches. Objectives We sought to analyze whether peripheral neutrophils from CD40L-deficient patients display functional defects and to explore the in vitro effects of recombinant human IFN-γ (rhIFN-γ) on neutrophil function. Methods We investigated the microbicidal activity, respiratory burst, and transcriptome profile of neutrophils from CD40L-deficient patients. In addition, we evaluated whether the lack of CD40L in mice also affects neutrophil function. Results Neutrophils from CD40L-deficient patients exhibited defective respiratory burst and microbicidal activity, which were improved in vitro by rhIFN-γ but not soluble CD40L. Moreover, neutrophils from patients showed reduced CD16 protein expression and a dysregulated transcriptome suggestive of impaired differentiation. Similar to CD40L-deficient patients, CD40L knockout mice were found to have impaired neutrophil responses. In parallel, we demonstrated that soluble CD40L induces the promyelocytic cell line HL-60 to proliferate and mature by regulating the expression of genes of the same Gene Ontology categories (eg, cell differentiation) when compared with those dysregulated in peripheral blood neutrophils from CD40L-deficient patients. Conclusion Our data suggest a nonredundant role of CD40L-CD40 interaction in neutrophil development and function that could be improved in vitro by rhIFN-γ, indicating a potential novel therapeutic application for this cytokine.

In addition to its established role in immunoglobulin isotype switching, CD40–CD154 interaction plays a role in the regulation of multiple innate immune system cell types. Of note, we have shown that CD154-deficient (CD154−/−) mice are hyper-susceptible to intraperitoneal infection by attenuated strains of Salmonella enterica serovar Typhimurium. Compared to their normal counterparts, CD154−/− mice exhibited progressive morbidity during the early phases of disease and succumbed to infection 3–4 weeks after inoculation. Since the liver is a key target organ in Salmonella infections, we carried out a comparative analysis of infection-associated hepatic changes at different time-points. Bacterial loads in the liver were significantly higher in CD154−/− compared to WT mice, and this correlated with progressively worsening hepatic damage and rising levels of liver enzymes. Liver histomorphology revealed focal necrosis early in the infection that was self-limiting in WT mice. In contrast, infected CD154−/− mice developed large and generalized necrosis coupled with the appearance of large venous thrombi. Immunohistochemical analysis showed a retarded inflammatory response affecting recruitment of myeloid cells in the liver of CD154−/− animals. Moreover, neutrophil recruitment into the peritoneal cavity, as well as the activation of the respiratory burst, was defective in CD154−/− mice. Taken together, our findings demonstrate that CD154-deficiency results in a failure to mount an appropriate innate immune response to contain bacterial proliferation during the acute phase of disease. This renders the animals incapable of limiting the extent of histopathological damage in the liver resulting, ultimately, in their demise.

Epidemiological evidence suggests that obesity is linked to increased risk of developing several types of cancer. However, the mechanisms underlying this phenomenon remain unknown. Successful growth of cancer cells depends on developing mechanisms to evade host immunosurveillance, particularly within the tumor microenvironment. In the current study, we utilized a high-fat-diet (HFD)-induced obese mouse model to gain mechanistic insight into the effect of obesity on progression of implantable syngeneic tumors. When lean or obese mice were implanted with B16.F1 melanoma or MC38 colon carcinoma, tumor growth was more robust in obese group than lean controls, suggesting that obese conditions promote tumor growth. Multi-color flow cytometric analysis and gene expression profiling were carried out on distinct subpopulations of intratumoral myeloid cells. Tumors of lean mice had higher percentages of myeloid cells compared to obese counterparts with characteristics typical of activated inflammatory macrophages. In contrast, tumors of obese mice had disproportionately higher frequency of myeloid derived suppressor cells (MDSC) with a gene signature suggestive of immunosuppressive potential. These findings highlight the role of metaflammation in tumor growth promotion via the regulation of frequency and activation status of distinct subpopulations of intratumoral myeloid cells.

Obesity is a metabolic disorder characterized by a state of chronic inflammation. Based on epidemiological evidence, obesity has been linked to increased risk of developing several types of cancer. However, the mechanisms underlying this phenomenon remain unknown. Successful growth of cancer cells hinges on developing mechanisms to evade host immunosurveillance, particularly within the tumor microenvironment. In the current study, we utilized a high-fat-diet (HFD)-induced obesity mouse model to gain mechanistic insight into the effect of obesity on progression of implantable syngeneic tumors. When lean or obese mice were implanted with B16.F1 melanoma or MC38 colon carcinoma, tumor growth was more robust in obese group than lean controls, suggesting that obese conditions promote tumor growth. Multi-color flow cytometric analysis and gene expression profiling were carried out on distinct subpopulations of intratumoral myeloid cells. Tumors of lean mice had higher percentages of myeloid cells compared to obese counterparts with characteristics typical of activated inflammatory macrophages. In contrast, tumors of obese mice had disproportionately higher frequency of myeloid derived suppressor cells (MDSC) with a gene signature suggestive of immunosuppressive potential. These findings highlight the role of metaflammation in tumor growth promotion via the regulation of frequency and activation status of distinct subpopulations of intratumoral myeloid cells. Citation Format: Basel K. al-Ramadi, Suneesh Kaimala, Yassir A. Mohamed, Ashraf Al-Sbiei, Ghada Bashir, Maria J. Fernandez-Cabezudo. Tumor growth enhancement under obese conditions: Role of intratumoral myeloid cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A70.

Susceptibility to infection by the intracellular bacterial pathogen, Salmonella enterica serovar Typhimurium (S. typhimurium), is controlled by many genes of innate and adaptive immunity. One of the most critical genes is IFN-γ and mice deficient in IFN-γ synthesis are highly susceptible to Salmonella. Previously, we also demonstrated that mice deficient in MyD88, an adaptor that regulates TLR signaling, are susceptible to Salmonella infection. In the current study, we compared immune responses in mice deficient in IFN-γ or MyD88 with wild-type controls following infection with an attenuated strain of S. typhimurium (designated BRD509) or a recombinant derivative engineered to express murine IFN-γ (GIDIFN). Infection studies with BRD509 or GIDIFN revealed that the latter strain was significantly less virulent in immunodeficient mice than BRD509 and correlated with decreased bacterial loads in systemic organs. Enhanced responsiveness was due to GIDIFN strain’s ability to activate effector macrophages, as shown by increased synthesis of inflammatory cytokines and anti-microbial effector molecules, including NO. Gene expression profiling by qPCR demonstrated stronger induction of key inflammatory modulators by GIDIFN in macrophages of immunodeficient animals. These findings suggest that immunotherapeutic approaches using attenuated bacterial strains expressing immunomodulatory genes is more efficacious and offers a superior safety profile even in severely immunodeficient hosts.