The Five Immune Forces Impacting DNA-Based Cancer Immunotherapeutic Strategy.

DNA-based vaccine strategy is increasingly realized as a viable cancer treatment approach. Strategies to enhance immunogenicity utilizing tumor associated antigens have been investigated in several pre-clinical and clinical studies. The promising outcomes of these studies have suggested that DNA-based vaccines induce potent T-cell effector responses and at the same time cause only minimal side-effects to cancer patients. However, the immune evasive tumor microenvironment is still an important hindrance to a long-term vaccine success. Several options are currently under various stages of study to overcome immune inhibitory effect in tumor microenvironment. Some of these approaches include, but are not limited to, identification of neoantigens, mutanome studies, designing fusion plasmids, vaccine adjuvant modifications, and co-treatment with immune-checkpoint inhibitors. In this review, we follow a Porter's analysis analogy, otherwise commonly used in business models, to analyze various immune-forces that determine the potential success and sustainable positive outcomes following DNA vaccination using non-viral tumor associated antigens in treatment against cancer.

Sodium channel γENaC mediates IL-17 synergized high salt induced inflammatory stress in breast cancer cells.

Chronic inflammation is known to play a critical role in the development of cancer. Recent evidence suggests that high salt in the tissue microenvironment induces chronic inflammatory milieu. In this report, using three breast cancer-related cell lines, we determined the molecular basis of the potential synergistic inflammatory effect of sodium chloride (NaCl) with interleukin-17 (IL-17). Combined treatment of high NaCl (0.15M) with sub-effective IL-17 (0.1 nM) induced enhanced growth in breast cancer cells along with activation of reactive nitrogen and oxygen (RNS/ROS) species known to promote cancer. Similar effect was not observed with equi-molar mannitol. This enhanced of ROS/RNS activity correlates with upregulation of γENaC an inflammatory sodium channel. The similar culture conditions have also induced expression of pro-inflammatory cytokines such as IL-6, TNFα etc. Taken together, these data suggest that high NaCl in the cellular microenvironment induces a γENaC mediated chronic inflammatory response with a potential pro-carcinogenic effect.

Hepatitis C virus-induced changes in microRNA 107 (miRNA-107) and miRNA-449a modulate CCL2 by targeting the interleukin-6 receptor complex in hepatitis.

UNLABELLED: Hepatitis C virus (HCV)-mediated liver diseases are one of the major health issues in the United States and worldwide. HCV infection has been reported to modulate microRNAs (miRNAs) that control various cell surface receptors and gene-regulatory complexes involved in hepatic inflammation and liver diseases. We report here that specific downregulation of miRNA-107 and miRNA-449a following HCV infection in patients with HCV-mediated liver diseases modulates expression of CCL2, an inflammatory chemokine upregulated in patients with chronic liver diseases, by targeting components of the interleukin-6 receptor (IL-6R) complex. Computational analysis for DNA-bound transcription factors in the CCL2 promoter identified adjacent binding sites for CCAAT/CEBPα, spleen focus-forming virus, proviral integration oncogene (SPI1/PU.1), and STAT3. We demonstrate that CEBPα, PU.1, and STAT3 interacted with each other physically to cooperatively bind to the promoter and activate CCL2 expression. Analysis of IL-6R and JAK1 expression in HCV patients by quantitative PCR showed significant upregulation when there was impaired miRNA-107 and miRNA-449a expression, along with upregulation of PU.1 and STAT3, but not CEBPα. miRNA-449a and miRNA-107 target expression of IL-6R and JAK1, respectively, in vitro and also inhibit IL-6 signaling and impair STAT3 activation in human hepatocytes. Taken together, our results demonstrate a novel gene-regulatory mechanism in which HCV-induced changes in miRNAs (miRNA-449a and miRNA-107) regulate CCL2 expression by activation of the IL-6-mediated signaling cascade, which we propose will result in HCV-mediated induction of inflammatory responses and fibrosis. IMPORTANCE: Hepatitis C virus (HCV)-induced hepatitis is a major health concern worldwide. HCV infection results in modulation of noncoding microRNAs affecting major cellular pathways, including inflammatory responses. In this study, we have identified a microRNA-regulated pathway for the chemokine CCL2 in HCV-induced hepatitis. Understanding microRNA-mediated transcriptional-regulatory pathways will result in development of noninvasive biomarkers for better disease prediction and development of effective therapeutics.

Violacein inhibits matrix metalloproteinase mediated CXCR4 expression: potential anti-tumor effect in cancer invasion and metastasis.

Matrix metalloproteinases (MMP-2 and -9) play an important role in the tumor metastasis through cleavage of proinflammatory cytokines. Violacein a small molecule produced by Chromobacterium violaceum and has been implicated with anti-cancer effects. In this study we investigated the molecular basis of violacein mediated downregulation of CXCL12/CXCR4, chemokine-receptor ligand interaction. Zymography analysis demonstrated that violacein significantly inhibited the cytokine (TNFα and TGFß) mediated MMP-2 activation in MCF-7 breast cancer cell line. MMP-2 plays a critical role in the secretion of inflammatory chemokine, CXCL12, involved in cell migration and cancer metastasis. ELISA analysis demonstrated that violacein inhibited the secretion of CXCL12 from the activated MCF-7 cells. Further, we show that MMP-2/-9 act synergistically at two distinct steps towards the membrane expression of the tumor metastasis chemokine receptor, CXCR4. Violacein efficiently downregulated the CXCR4 membrane expression through MMP-9 inhibition. Taken together, these studies demonstrate a unique anti-tumor mechanism of action of violacein through reduction of CXCL12/CXCR4 interaction. These studies could offer a novel venue for violacein in cancer therapy.

Genomic Features of Homologous Recombination Deficiency in Breast Cancer: Impact on Testing and Immunotherapy.

Genomic instability is one of the well-established hallmarks of cancer. The homologous recombination repair (HRR) pathway plays a critical role in correcting the double-stranded breaks (DSB) due to DNA damage in human cells. Traditionally, the BRCA1/2 genes in the HRR pathway have been tested for their association with breast cancer. However, defects in the HRR pathway (HRD, also termed 'BRCAness'), which has up to 50 genes, have been shown to be involved in tumorigenesis and treatment susceptibility to poly-ADP ribose polymerase inhibitors (PARPis), platinum-based chemotherapy, and immune checkpoint inhibitors (ICIs). A reliable consensus on HRD scores is yet to be established. Emerging evidence suggests that only a subset of breast cancer patients benefit from ICI-based immunotherapy. Currently, albeit with limitations, the expression of programmed death-ligand 1 (PDL1) and tumor mutational burden (TMB) are utilized as biomarkers to predict the favorable outcomes of ICI therapy in breast cancer patients. Preclinical studies demonstrate an interplay between the HRR pathway and PDL1 expression. In this review, we outline the current understanding of the role of HRD in genomic instability leading to breast tumorigenesis and delineate outcomes from various clinical trials. Furthermore, we discuss potential strategies for combining HRD-targeted therapy with immunotherapy to achieve the best healthcare outcomes in breast cancer patients.

Chronic High-Salt Diet Activates Tumor-Initiating Stem Cells Leading to Breast Cancer Proliferation.

Several chronic inflammatory diseases have been linked to high-salt (HS) diets. Chronic inflammation is an established causative hallmark of cancer. However, a direct role of HS diets in tumorigenesis is yet to be defined. Previous orthotopic murine breast tumor studies have shown that short-term HS diets caused inhibition of tumor growth through the activation of cytotoxic adaptive immune responses. However, there have been experimental challenges in developing a viable chronic HS-diet-based murine tumor model. To address this, we have developed a novel chronic HS diet tumor model through the sequential passaging of tumor cells in mice under HS dietary conditions. Two orthotopic murine triple-negative breast cancer models, 4T1 tumor cells injected into BALB/c mice and Py230 tumor cells injected into C57Bl/6 mice, were utilized in our study. For the HS diet cohort, prior to orthotopic injection with tumor cells, the mice were kept on a 4% NaCl diet for 2 weeks. For the regular salt (RS) diet cohort, the mice were kept on a 1% NaCl diet. Following syngeneic cancer cell injection, tumors were allowed to grow for 28 days, following which they were collected to isolate immune cell-depleted cancer cells (passage 1, P1). The tumor cells from P1 were reinjected into the next set of non-tumor-bearing mice. This procedure was repeated for three cycles (P2-P4). In P1, compared to the RS diet cohort, we observed reduced tumor kinetics in both murine tumor models on the HS diet. In contrast, by P4, there was significantly higher tumor progression in the HS diet cohort over the RS diet cohort. Flow cytometry analysis demonstrated an 8-fold increase in tumor-initiating stem cells (TISCs) from P1 to P4 of the HS diet cohort, while there were no significant change in TISC frequency with sequential passaging in the RS diet cohort. Molecular studies showed enhanced expression of TGFßR2 and CD80 on TISCs isolated from the P4 HS diet cohort. In vitro studies demonstrated that TGFß stimulation of these TISCs increased the cellular expression of CD80 molecules. Further, the chronic HS diet selectively induced the glycolytic metabolic phenotype over the mitochondrial oxidative phosphorylation phenotype in TISCs, which is needed for the production of metabolites during tumor cell differentiation and proliferation. The infiltrating CD8 and CD4 T-lymphocytes in P4 tumors demonstrated increased expression of the immune checkpoint inhibitor (ICI) CTLA4, a known binding partner of CD80, to cause immune exhaustion and pro-tumorigenic effects. Interestingly, anti-TGFß monoclonal antibodies (mAbs) played a synergistic role in further enhancing the anti-tumor effect of anti-CTLA4 mAb. In summary, our findings demonstrated that chronic HS diet increased the frequency of TISCs in tumors leading to blunting of cytotoxic adaptive immune responses causing tumor proliferation. Furthermore, a combination of anti-TGFß with current ICI-based immunotherapies could exert more favorable anti-cancer clinical outcomes.

Food Waste from Campus Dining Hall as a Potential Feedstock for 2,3-Butanediol Production via Non-Sterilized Fermentation.

Food waste is a major issue that is increasingly affecting our environment. More than one-third of food is wasted, resulting in over $400 billion in losses to the U.S. economy. While composting and other small recycling practices are encouraged from person-to-person, it is not enough to balance the net loss of 80 million tons per year. Currently, one of the most promising routes for reducing food waste is through microbial fermentation, which can convert the waste into valuable bioproducts. Among the compounds produced from fermentation, 2,3-butanediol (2,3-BDO) has gained interest recently due to its molecular structure as a building block for many other derivatives used in perfumes, synthetic rubber, fumigants, antifreeze agents, fuel additives, and pharmaceuticals. Waste feedstocks, such as food waste, are a potential source of renewable energy due to their lack of cost and availability. Food waste also possesses microbial requirements for growth such as carbohydrates, proteins, fats, and more. However, food waste is highly inconsistent and the variability in composition may hinder its ability to be a stable source for bioproducts such as 2,3-BDO. This current study focuses specifically on post-consumer food waste and how 2,3-BDO can be produced through a non-model organism, Bacillus licheniformis YNP5-TSU during non-sterile fermentation. From the dining hall at Tennessee State University, 13 food waste samples were collected over a 6-month period and the compositional analysis was performed. On average, these samples consisted of fat (19.7%), protein (18.7%), ash (4.8%), fiber (3.4%), starch (27.1%), and soluble sugars (20.9%) on a dry basis with an average moisture content of 34.7%. Food waste samples were also assessed for their potential production of 2,3-BDO during non-sterile thermophilic fermentation, resulting in a max titer of 12.12 g/L and a 33% g/g yield of 2,3-BDO/carbohydrates. These findings are promising and can lead to the better understanding of food waste as a defined feedstock for 2,3-BDO and other fermentation end-products.

Autonomous transmembrane segment S4 of the voltage sensor domain partitions into the lipid membrane.

The S4 transmembrane segment in voltage-gated ion channels, a highly basic alpha helix, responds to changes in membrane potential and induces channel opening. Earlier work by others indicates that the S4 segment interacts with lipids in plasma membrane, but its mechanism is unclear. Working with synthetic tryptophan-labeled S4 peptides, we characterized binding of autonomous S4 to lipid membranes. The binding free energy (5.2 +/- 0.2 kcal/mol) of the peptide-lipid interaction was estimated from the apparent dissociation constants, determined from the changes in anisotropy of tryptophan fluorescence induced by addition of lipid vesicles with 30 mol% phosphatidylglycerol. The results are in good agreement with the prediction based on the Wimley-White hydrophobicity scale for interfacial (IF) binding of an alpha-helical peptide to the lipid bilayer (6.98 kcal/mol). High salt inhibited the interaction, thus indicating that the peptide/membrane interaction has both electrostatic and non-electrostatic components. Furthermore, the synthetic S4 corresponding to the Shaker potassium channel was found to spontaneously penetrate into the negatively charged lipid membrane to a depth of about 9 A. Our results revealed important biophysical parameters that influence the interaction of S4 with the membrane: they include fluidity, surface charge, and surface pressure of the membrane, and the at helicity and regular spacing of basic amino-acid residues in the S4 sequence.

Mammaglobin-A cDNA vaccination of breast cancer patients induces antigen-specific cytotoxic CD4+ICOShi T cells.

Mammaglobin-A (Mam-A) is a 10 kDa secretory protein that is overexpressed in 80 % of primary and metastatic human breast cancers. Previous studies from our laboratory demonstrated that Mam-A cDNA vaccine can induce Mam-A-specific CD8 T cell responses and mediate regression of human breast cancer xenografts in NOD/SCID mice. In this article, we present our results on a phase I clinical trial of a Mam-A cDNA vaccination in breast cancer patients with stage-IV metastatic disease, including the impact of vaccination on the expression of the inducible co-stimulator molecule (ICOS) on CD4 T cells. Specimens from seven patients with stage-IV metastatic cancer were available for these analyses. Patients were vaccinated with a Mam-A cDNA vaccine on days 0, 28, and 56, and immune responses were assessed at serial time points following vaccination. At 6 months following the first vaccination, flow cytometric analysis demonstrated a significant increase in the frequency of CD4+ICOS(hi) T cells from 5 ± 2 % pre-vaccination to 23 ± 4 % (p < 0.001), with a concomitant decrease in the frequency of CD4+FoxP3+ T cells (regulatory T cells [Treg]) from 19 ± 6 to 10 ± 5 % (p < 0.05). ELISpot analysis of CD4+ICOS(hi) sorted T cells demonstrated that following vaccination the cytokines produced by Mam-A-specific T cells switched from IL-10 (78 ± 21 spm pre-vaccination to 32 ± 14 spm 5 months post-vaccine p < 0.001) to IFN-γ (12 ± 6 spm pre-vaccination to 124 ± 31 spm 5 months post-vaccine p < 0.001). The ratio of CD4+ICOS(hi) T cells to CD4+FoxP3+ T cells increased from 0.37 ± 0.12 before vaccination to 2.3 ± 0.72 (p = 0.021) following vaccination. Further, these activated CD4+ICOS(hi) T cells induced preferential lysis of human breast cancer cells expressing Mam-A protein. We conclude that Mam-A cDNA vaccination is associated with specific expansion and activation of CD4+ICOS(hi) T cells, with a concomitant decrease in Treg frequency. These encouraging results strongly suggest that Mam-A cDNA vaccination can induce antitumor immunity in breast cancer patients.

Kinetic analysis of the interaction between poly(amidoamine) dendrimers and model lipid membranes.

We used fluorescence spectroscopy and surface tensiometry to study the interaction between low-generation (G1 and G4) poly(amidoamine) (PAMAM) dendrimers, potential vehicles for intracellular drug delivery, and model lipid bilayers. Membrane association of fluorescently labeled dendrimers, measured by fluorescence anisotropy, increased with increasing size of the dendrimer and with increasing negative charge density in the membrane, indicating the electrostatic nature of the interaction. When the membrane was doped with pyrene-labeled phosphatidyl glycerol (pyrene-PG), pyrene excimer fluorescence demonstrated a dendrimer-induced selective aggregation of negatively charged lipids when the membrane was in the liquid crystalline state. A nonlinear Stern-Volmer quenching of dendrimer fluorescence with cobalt bromide suggested a dendrimer-induced aggregation of lipid vesicles, which increased with the dendrimer's generation number. Surface tensiometry measurements showed that dendrimers penetrated into the lipid monolayer only at subphysiologic surface pressures (<30mN/m). We conclude that the low-generation PAMAM dendrimers associate with lipid membranes predominantly electrostatically, without significantly compromising the bilayer integrity. They bind stronger to membranes with higher fluidity and lower surface pressure, which are characteristic of rapidly dividing cells.

HIF-1α signaling by airway epithelial cell K-α1-tubulin: role in fibrosis and chronic rejection of human lung allografts.

Long term survival of the human lung allografts are hindered by chronic rejection, manifested clinically as bronchiolitis obliterans syndrome (BOS). We previously demonstrated significant correlation between the development of antibodies (Abs) to K-α1-tubulin (Kα1T) and BOS. In this study, we investigated the molecular basis for fibrinogenesis mediated by ligation of Kα1T expressed on airway epithelial cells by its specific Abs. Using RT-PCR we demonstrate that normal human bronchial epithelial (NHBE) cells upon ligation of Kα1T with specific Abs caused upregulation of pro-fibrotic growth factors. Western blot analysis of NHBE incubated with Kα1T Abs increased hypoxia inducible factor (HIF-1α). Kα1T Ab-mediated growth factor expression is dependent on HIF-1α as inhibition of HIF-1α returned fibrotic growth factor expression to basal levels. In conclusion, we propose that HIF-1α -mediated upregulation of fibrogenic growth factors induced by ligation of Kα1T Abs is critical for development of fibrosis leading to chronic rejection of lung allograft.

The role of molecular chaperonins in warm ischemia and reperfusion injury in the steatotic liver: a proteomic study.

BACKGROUND: The molecular basis of the increased susceptibility of steatotic livers to warm ischemia/reperfusion (I/R) injury during transplantation remains undefined. Animal model for warm I/R injury was induced in obese Zucker rats. Lean Zucker rats provided controls. Two dimensional differential gel electrophoresis was performed with liver protein extracts. Protein features with significant abundance ratios (p < 0.01) between the two cohorts were selected and analyzed with HPLC/MS. Proteins were identified by Uniprot database. Interactive protein networks were generated using Ingenuity Pathway Analysis and GRANITE software. RESULTS: The relative abundance of 105 proteins was observed in warm I/R injury. Functional grouping revealed four categories of importance: molecular chaperones/endoplasmic reticulum (ER) stress, oxidative stress, metabolism, and cell structure. Hypoxia up-regulated 1, calcium binding protein 1, calreticulin, heat shock protein (HSP) 60, HSP-90, and protein disulfide isomerase 3 were chaperonins significantly (p < 0.01) down-regulated and only one chaperonin, HSP-1 was significantly upregulated in steatotic liver following I/R. CONCLUSION: Down-regulation of the chaperones identified in this analysis may contribute to the increased ER stress and, consequently, apoptosis and necrosis. This study provides an initial platform for future investigation of the role of chaperones and therapeutic targets for increasing the viability of steatotic liver allografts.

Modulation of immune responses following solid organ transplantation by microRNA.

Organ transplantation, an accepted treatment for end stage organ failure, is often complicated by allograft rejection and disease recurrence. In this review we will discuss the potential role of microRNAs in allograft immunity especially leading to rejection of the transplanted organ. microRNAs (miRNAs), originally identified in C. elegans, are short non-coding 21-24 nucleotide sequences that bind to its complementary sequences in functional messenger RNAs and inhibits post-translational processes through RNA duplex formation resulting in gene silencing (Lau et al., 2001). Gene specific translational silencing by miRNAs regulates pathways for immune responses such as development of innate immunity, inflammation, T-cell and B-cell differentiation and signaling that are implicated in various stages of allograft rejection. miRNAs also play a role in development of post-transplant complicacies like fibrosis, cirrhosis, carcinogenesis often leading to graft loss and poor patient outcome. Recent advancements in the methods for detecting and quantifying miRNA in tissue biopsies, as well as in serum and urine samples, has led to identification of specific miRNA signatures in patients with allograft rejection and have been utilized to predict allograft status and survival. Therefore, miRNAs play a significant role in post-transplant events including allograft rejection, disease recurrence and tumor development impacting patient outcome.

Complex Role of Microbiome in Pancreatic Tumorigenesis: Potential Therapeutic Implications.

Pancreatic cancer (PC) is the fourth leading cause of cancer-related mortality with limited diagnostic and therapeutic options. Although immunotherapy has shown promise in the treatment of several cancers, its role in pancreatic cancer is rather limited. Several studies have focused on determining the role of the tumor microenvironment with cancer-cell-intrinsic events and tumor-infiltrating immune cellular properties. However, in the past decade, there has been emerging research aimed at delineating the role of the host microbiome, including the metabolites from microbes and host responses, on pancreatic tumorigenesis. Importantly, there is emerging evidence suggesting the beneficial role of a gut microbiome transplant to improve immunotherapeutic outcomes in cancer patients. In this review, we summarize the recent understanding of the role of the microbiome in pancreatic cancer progression, along with its clinical diagnostic and therapeutic implications.

Low-Salt Diet Reduces Anti-CTLA4 Mediated Systemic Immune-Related Adverse Events while Retaining Therapeutic Efficacy against Breast Cancer.

Immune checkpoint inhibitor (ICI) therapy has revolutionized the breast cancer treatment landscape. However, ICI-induced systemic inflammatory immune-related adverse events (irAE) remain a major clinical challenge. Previous studies in our laboratory and others have demonstrated that a high-salt (HS) diet induces inflammatory activation of CD4+T cells leading to anti-tumor responses. In our current communication, we analyzed the impact of dietary salt modification on therapeutic and systemic outcomes in breast-tumor-bearing mice following anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) monoclonal antibody (mAb) based ICI therapy. As HS diet and anti-CTLA4 mAb both exert pro-inflammatory activation of CD4+T cells, we hypothesized that a combination of these would lead to enhanced irAE response, while low-salt (LS) diet through blunting peripheral inflammatory action of CD4+T cells would reduce irAE response. We utilized an orthotopic murine breast tumor model by injecting Py230 murine breast cancer cells into syngeneic C57Bl/6 mice. In an LS diet cohort, anti-CTLA4 mAb treatment significantly reduced tumor progression (day 35, 339 ± 121 mm3), as compared to isotype mAb (639 ± 163 mm3, p < 0.05). In an HS diet cohort, treatment with anti-CTLA4 reduced the survival rate (day 80, 2/15) compared to respective normal/regular salt (NS) diet cohort (8/15, p < 0.05). Further, HS plus anti-CTLA4 mAb caused an increased expression of inflammatory cytokines (IFNγ and IL-1ß) in lung infiltrating and peripheral circulating CD4+T cells. This inflammatory activation of CD4+T cells in the HS plus anti-CTLA4 cohort was associated with the upregulation of inflammasome complex activity. However, an LS diet did not induce any significant irAE response in breast-tumor-bearing mice upon treatment with anti-CTLA4 mAb, thus suggesting the role of high-salt diet in irAE response. Importantly, CD4-specific knock out of osmosensitive transcription factor NFAT5 using CD4cre/creNFAT5flox/flox transgenic mice caused a downregulation of high-salt-mediated inflammatory activation of CD4+T cells and irAE response. Taken together, our data suggest that LS diet inhibits the anti-CTLA4 mAb-induced irAE response while retaining its anti-tumor efficacy.

The Meharry-Vanderbilt-Tennessee State University Cancer Partnership (MVTCP): History and Highlights of 20 Years of Accomplishments.

Cancer health disparities among populations are the result of a combination of socioeconomic, environmental, behavioral, and biological factors, which affect cancer incidence, prevalence, mortality, survivorship, financial burden, and screening rates. The long-standing Meharry Medical College (MMC), Vanderbilt-Ingram Cancer Center (VICC), Tennessee State University (TSU) Cancer Partnership has built an exceptional cancer research and training environment to support the efforts of diverse investigators in addressing disparities. Over the past 20 years, collaborative partnership efforts across multiple disciplines have supported research into the determinants of cancer health disparities at a National Cancer Institute-designated comprehensive cancer center (VICC) along with enhancing research infrastructure and training at MMC and TSU, two institutions that serve predominantly underserved populations and underrepresented students. Moreover, the geographical placement of this partnership in Tennessee, a region with some of the highest cancer incidence and mortality in the United States, has provided an especially important opportunity to positively affect outcomes for cancer patients.

Identification of immunodominant HLA-B7-restricted CD8+ cytotoxic T cell epitopes derived from mammaglobin-A expressed on human breast cancers.

Mammaglobin-A (MGBA), a 10-kD protein, is over expressed in 80% of primary and metastatic human breast cancers. Breast cancer patients demonstrate high frequencies of CD8(+) cytotoxic T lymphocytes (CTL) specific to MGBA. Defining CD8(+) CTL responses to HLA class I-restricted MGBA-derived epitopes assumes significance in the context of our ongoing efforts to clinically translate vaccine strategies targeting MGBA for prevention and/or treatment of human breast cancers. In this study, we define the CD8(+) CTL response to MGBA-derived candidate epitopes presented in the context of HLA-B7, which has a frequency of 17.7% in Caucasian and 15.5% in African American populations. We identified seven MGBA-derived candidate epitopes with high predicted binding scores for HLA-B7 using a computer algorithm. Membrane stabilization studies with TAP-deficient T2 cells transfected with HLA-B7 indicated that MGBA B7.3 (VSKTEYKEL), B7.6 (KLLMVLMLA), B7.7 (NPQVSKTEY), and B7.1 (YAGSGCPLL) have the highest HLA-B7 binding affinities. Further, two CD8(+) CTL cell lines generated in vitro against T2.B7 cells individually loaded with MGBA-derived candidate epitopes showed significant cytotoxic activity against MGBA B7.1, B7.3, B7.6, and B7.7. In addition, the same CD8(+) CTL lines lysed the HLA-B7(+)/MGBA(+) human breast cancer cell line DU-4475 but had no significant cytotoxicity against HLA-B7(-) or MGBA(-) breast cancer cell lines. Cold-target inhibition studies strongly suggest that MGBA B7.3 is an immunodominant epitope. In summary, our results define HLA-B7-restriced, MGBA-derived, CD8(+) CTL epitopes with all of the necessary features for developing novel vaccine strategies against HLA-B7 expressing breast cancer patients.

Endoplasmic reticulum stress is a mediator of posttransplant injury in severely steatotic liver allografts.

Hepatic steatosis continues to present a major challenge in liver transplantation. These organs have been shown to have increased susceptibility to cold ischemia/reperfusion (CIR) injury in comparison with otherwise comparable lean livers; the mechanisms governing this increased susceptibility to CIR injury are not fully understood. Endoplasmic reticulum (ER) stress is an important link between hepatic steatosis, insulin resistance, and metabolic syndrome. In this study, we investigated ER stress signaling and blockade in the mediation of CIR injury in severely steatotic rodent allografts. Steatotic allografts from genetically leptin-resistant rodents had increased ER stress responses and increased markers of hepatocellular injury after liver transplantation into strain-matched lean recipients. ER stress response components were reduced by the chemical chaperone taurine-conjugated ursodeoxycholic acid (TUDCA), and this resulted in an improvement in the allograft injury. TUDCA treatment decreased nuclear factor kappa B activation and the proinflammatory cytokines interleukin-6 and interleukin-1ß. However, the predominant response was decreased expression of the ER stress cell death mediator [CCAAT/enhancer-binding protein homologous protein (CHOP)]. Furthermore, activation of inflammation-associated caspase-11 was decreased, and this linked ER stress/CHOP to proinflammatory cytokine production after steatotic liver transplantation. These data confirm ER stress in steatotic allografts and implicate this as a mediating mechanism of inflammation and hepatocyte death in the steatotic liver allograft.

Cancer Salt Nostalgia.

High-salt (sodium chloride) diets have been strongly associated with disease states and poor health outcomes. Traditionally, the impact of salt intake is primarily studied in cardiovascular diseases, hypertension and renal diseases; however, recently there has been increasing evidence demonstrating the role of salt in autoimmune diseases. Salt has been shown to modulate the inflammatory activation of immune cells leading to chronic inflammation-related ailments. To date, there is minimal evidence showing a direct correlation of salt with cancer incidence and/or cancer-related adverse clinical outcomes. In this review article, we will discuss the recent understanding of the molecular role of salt, and elucidate the apparent double-edged sword nature of the relationship between salt and cancer progression.

Ex Vivo High Salt Activated Tumor-Primed CD4+T Lymphocytes Exert a Potent Anti-Cancer Response.

Cell based immunotherapy is rapidly emerging as a promising cancer treatment. A modest increase in salt (sodium chloride) concentration in immune cell cultures is known to induce inflammatory phenotypic differentiation. In our current study, we analyzed the ability of salt treatment to induce ex vivo expansion of tumor-primed CD4 (cluster of differentiation 4)+T cells to an effector phenotype. CD4+T cells were isolated using immunomagnetic beads from draining lymph nodes and spleens from tumor bearing C57Bl/6 mice, 28 days post-injection of Py230 syngeneic breast cancer cells. CD4+T cells from non-tumor bearing mice were isolated from splenocytes of 12-week-old C57Bl/6 mice. These CD4+T cells were expanded ex vivo with five stimulation cycles, and each cycle comprised of treatment with high salt (Δ0.035 M NaCl) or equimolar mannitol controls along with anti-CD3/CD28 monoclonal antibodies for the first 3 days, followed by the addition of interleukin (IL)-2/IL-7 cytokines and heat killed Py230 for 4 days. Ex vivo high salt treatment induced a two-fold higher Th1 (T helper type 1) expansion and four-fold higher Th17 expansion compared to equimolar mannitol treatment. Importantly, the high salt expanded CD4+T cells retained tumor-specificity, as demonstrated by higher in vitro cytotoxicity against Py230 breast cancer cells and reduced in vivo syngeneic tumor growth. Metabolic studies revealed that high salt treatment enhanced the glycolytic reserve and basal mitochondrial oxidation of CD4+T cells, suggesting a role of high salt in enhanced pro-growth anabolic metabolism needed for inflammatory differentiation. Mechanistic studies demonstrated that the high salt induced switch to the effector phenotype was mediated by tonicity-dependent transcription factor, TonEBP/NFAT5. Using a transgenic murine model, we demonstrated that CD4 specific TonEBP/NFAT5 knock out (CD4cre/creNFAT5flox/flox) abrogated the induction of the effector phenotype and anti-tumor efficiency of CD4+T cells following high salt treatment. Taken together, our data suggest that high salt-mediated ex vivo expansion of tumor-primed CD4+T cells could induce effective tumor specific anti-cancer responses, which may have a novel cell-based cancer immunotherapeutic application.