Immunotherapy in human colorectal cancer: challenges and prospective

Human colorectal cancer(CRC) is the third most commonly diagnosed malignancies and the prognosis for patients with recurrent or metastatic disease is extremely poor. Although new chemotherapeutic regimen improves survival rates, therapy with better efficacy and less adverse effects is drastically needed. Immunotherapy has been investigated in human CRC for decades with limited success. However, recent developments of immunotherapy, particularly immune checkpoint inhibitor therapy, have achieved promising clinical benefits in many types of cancer and revived the hope for utilizing such therapy in human CRC. In this review, we will discuss important immunological landscape within the CRC microenvironment and introduce immunoscore system to better describe immunophenotyping in CRC. We will also discuss different immunotherapeutic approaches currently utilized in different phases of clinical trials. Some of those completed or ongoing trials are summarized. Finally, we provide a brief prospective on the future human CRC immunotherapy.

Fecal microbes, short chain fatty acids, and colorectal cancer across racial/ethnic groups

AIM:To investigate differences in microbes and short chain fatty acid(SCFA) levels in stool samples from Hispanic and non-Hispanic African American,American Indian,and White participants.METHODS:Stool samples from twenty participants were subjected to analysis for relative levels of viable bacteria and for SCFA levels.Additionally,the samples were subjected to 16 S r RNA gene pyrosequencing for identification of bacteria present in the stool.We used a metagenome functional prediction technique to analyze genome copy numbers and estimate the abundance of butyrate kinase in all samples.RESULTS:We found that African Americans had significantly lower levels of acetate,butyrate,and total SCFAs than all other racial/ethnic groups.We also found that participant microbial profiles differed by racial/ethnic group.African Americans had significantly more Firmicutes than Whites,with enriched Ruminococcaceae.The Firmicutes /Bacteroidetes ratio was also significantly higher for African Americans than for Whites(P =0.049).We found Clostridium levels to be significantly and inversely related to total SCFA levels(P =0.019) and we found Bacteroides to be positively associated(P =0.027) and Clostridium to be negatively associated(P =0.012) with levels of butyrate.We also identified a correlation between copy number for a butyrate kinase predicted from 16 S r RNA gene abundance and levels of butyrate in stool.CONCLUSION:The identified differences in gut flora and SCFA levels may relate to colorectal cancer mortality differentials and may be useful as targets for future clinical and behavioral interventions.

Chemoprotective effects of curcumin in esophageal epithelial cells exposed to bile acids

AIM:To investigate the ability of curcumin to counteract the impact of bile acids on gene expression of esophageal epithelial cells.METHODS:An esophageal epithelial cell line(HET1A)was treated with curcumin in the presence of deoxycholic acid.Cell proliferation and viability assays were used to establish an appropriate dose range for curcumin.The combined and individual effects of curcumin and bile acid on cyclooxygenase-2(COX-2)and superoxide dismutase(SOD-1 and SOD-2)gene expression were also assessed.RESULTS:Curcumin in a dose range of 10-100μmol/L displayed minimal inhibition of HET-1A cell viability.Deoxycholic acid at a concentration of 200μmol/L caused a 2.4-fold increase in COX-2 gene expression compared to vehicle control.The increased expression of COX-2 induced by deoxycholic acid was partially reversed by the addition of curcumin,and curcumin reduced COX-2 expression 3.3-to 1.3-fold.HET-1A cells exposed to bile acid yielded reduced expression of SOD-1 and SOD-2 genes with the exception that high dose deoxycholic acid at 200μmol/L led to a 3-fold increase in SOD-2 expression.The addition of curcumin treatment partially reversed the bile acid-induced reduction in SOD-1 expression at all concentrations of curcumin tested.CONCLUSION:Curcumin reverses bile acid suppression of gene expression of SOD-1.Curcumin is also able to inhibit bile acid induction of COX-2 gene expression.

Cell-based therapies for age-related macular degeneration:cell replacement versus paracrine effects

Age-related macular degeneration(AMD)is a leading cause for severe visual loss and legal blindness in seniors worldwide.The molecular basis for the disease remains poorly understood,likely involving genetic and environment-related ocular defects.Its pathogenesis proceeds slowly,started with deposits of fatty proteins(drusen)in the Bruch’s membrane,followed by gradual impairments of the posterior choriocapillaris and the anterior retinal pigment epithelium(RPE),and lead to irreversible degeneration of the light receiving neurons(photoreceptor)and vision decline.Clinically,AMD is divided into two subgroups:dry or atrophy form and wet or exudative form.Twenty percent of AMD patients have the wet form.

Capture Reagent and Strategy for Retrieving Albumin-Bound Ligands from Plasma

A capture strategy is described and demonstrated for retrieving ligand entities in plasma that bind Human Serum Albumin. The method has applications for both exogenous and endogenous ligands. Exogenous ligands include drug candidates, performance enhancing drugs and toxic nerve agents that also interact quite strongly with HSA. Endogenous ligands are natural circulating compounds whose abundance corresponds to normal hemostasis or elevated levels that could be disease-specific molecular biomarkers. Melting curves of plasma solutions measured by differential scanning calorimetry produce “so-called” plasma thermograms that are physical signatures of the plasma solution. Patterns displayed by thermograms can be sensitive indicators of the presence of abnormal levels of exogenous and endogenous ligand components. Effects of ligand interactions on thermodynamic stability of proteins in plasma that they bind, primarily HSA, manifest on the plasma thermogram. The capture strategy is demonstrated for HSA binding in plasma of four “ideal” ligands of different types. The particular ligands were naproxen, bromocresol green, short double stranded and single strand DNA. Thermogram shapes and features were sensitive to the presence of ligands as thermograms of mixtures of plasma and HSA with these ligands were significantly different than thermograms of plasma or HSA alone. These results demonstrated directly that significant perturbations of plasma thermograms corresponded to ligand interactions with HSA in plasma.

AF1q inhibited T cell attachment to breast cancer cell by attenuating Intracellular Adhesion Molecule-1 expression

Aim: To investigate whether AF1q, overexpressed in metastatic cells compared with the primary tumor cells, plays a pivotal role in breast cancer metastasis. Methods: To investigate whether AF1q has a responsibility in the acquisition of a metastatic phenotype, we performed RNA-sequencing (RNA-Seq) to identify the gene signature and applied the Metacore direct interactions network building algorithm with the top 40 amplicons of RNA-Seq. Results: Most genes were directly linked with intercellular adhesion molecule-1 (ICAM-1). Likewise, we identified that ICAM-1 expression is attenuated in metastatic cells compared to primary tumor cells. Moreover, overexpression of AF1q attenuated ICAM-1 expression, whereas suppression of AF1q elicited the opposite effect. AF1q had an effect on ICAM-1 promoter region and regulated its transcription. Decreased ICAM-1 expression ;affected the attachment of T cells to a breast cancer cell monolayer. We confirmed the finding by performing the analysis on Burkitt's lymphoma. Conclusion: Attenuation of ICAM-1 by AF1q on tumor cells disadvantages host anti-tumor defenses through the trafficking of lymphocytes, which affects tumor progression and metastasis.

The NORE1A/RASSF5 Tumor Suppressor Forms a Complex with GSK-3β to Regulate β-Catenin

NORE1A (RASSF5) is a tumor suppressor of the RASSF family that is often down-regulated in human tumors. NORE1A has multiple roles in controlling cellular homeostasis, one of them being regulating levels of β-catenin by binding and modulating the ubiquitin ligase substrate recognition factor β-TrCP. β-catenin is a major executor of the Wnt pathway. The ubiquitin SCF-β-TrCP ligase complex acts on a phospho-degron site in β-catenin that can be phosphorylated by GSK-3β. We now show that in addition to binding β-TrCP, NORE1A also promotes the phosphorylation of the β-catenin phospho-degron by complexing with the kinase GSK-3β. Indeed, NORE1A enhances the formation of a GSK-3β/β-TrCP complex. A structural mutant of NORE1A that retains β-TrCP binding but will no longer interact with GSK-3β inhibits the β-catenin degrading action of NORE1A. The GSK-3β interaction with NORE1A plays an important role in the biology of NORE1A as a GSK-3β inhibitor blocks NORE1A induced senescence. Thus, we identify a new role for the tumor suppressor NORE1A: The regulation of GSK-3β. GSK-3β has many other substrates including multiple transcription factors and co-activators such as p53 and the Hippo component TAZ. The work implies that NORE1A may be able to influence all of them via this new kinase scaffolding interaction.

Stereotactic radiosurgery in the era of novel systemic therapy for lung cancer brain metastases

The emergence of novel systemic therapies has spurred a dramatic paradigm shift in lung cancer treatment.Research has revealed greater intracranial efficacy in targeted agents and immune checkpoint inhibitors(ICI)compared to conventional chemotherapy.Concurrently,advances in stereotactic radiosurgery(SRS)have contributed to the increased use of this highly localized,minimally-invasive treatment modality for local tumor control.In this era of precision medicine,the combination of these novel agents and SRS demands further prospective exploration-particularly as questions regarding their sequence of administration and the risk of neurotoxicity remain unanswered.Presently,although data are limited and largely retrospective,literature supports the concurrent administration of ICI and radiation,with no observed increases in immune-related adverse events or acute neurologic toxicities.In the case of patients with driver mutations,newer generations of tyrosine kinase inhibitors(TKI)display improved intracranial efficacy and are currently preferred alone upfront in patients with asymptomatic brain metastases(BM)due to lack of data.Evidence of combining TKI and SRS is limited with mixed results.In this review,we explore the evidence regarding the use of novel systemic agents and SRS for treatment of lung cancer BM.Clinical practice will continue to be refined as larger,prospective studies yield results.

New insights of T cells in the pathogenesis of psoriasis

Psoriasis is one of the most common immune-mediated chronic, inflammatory skin diseases characterized by hyperproliferative keratinocytes and infiltration of T cells, dendritic cells, macrophages and neutrophils. Although the pathogenesis of psoriasis is not fully understood, there is ample evidence suggesting that the dysregulation of immune cells in the skin, particularly T cells, plays a critical role in psoriasis development. In this review, we mainly focus on the pathogenic T cells and discuss how these T cells are activated and involved in the disease pathogenesis. Newly identified ＇professional＇ IL-17-producing dermal γδ T cells and their potential role in psoriasis will also be included. Finally, we will briefly summarize the recent progress on the T cell and its related cytokine-targeted therapy for psoriasis treatment.

Arylamine N-acetyltransferase 2 genotype-dependent N-acetylation of isoniazid in cryopreserved human hepatocytes

Cryopreserved human hepatocytes were used to investigate the role of arylamine Nacetyltransferase 2(NAT2; EC 2.3.1.5) polymorphism on the N-acetylation of isoniazid(INH). NAT2 genotype was determined by Taqman allelic discrimination assay and INH N-acetylation was measured by high performance liquid chromatography. INH N-acetylation rates in vitro exhibited a robust and highly significant(P o0.005) NAT2 phenotype-dependent metabolism. N-acetylation rates in situ were INH concentration-and time-dependent. Following incubation for 24 h with 12.5 or 100 mmol/L INH, acetylINH concentrations varied significantly(P = 0.0023 and P = 0.0002) across cryopreserved human hepatocytes samples from rapid, intermediate, and slow acetylators, respectively. The clear association between NAT2 genotype and phenotype supports use of NAT2 genotype to guide INH dosing strategies in the treatment and prevention of tuberculosis.

Transcriptional Analysis of Normal Human Fibroblast Responses to Microgravity Stress

To understand the molecular mechanism（s） of how spaceflight affects cellular signaling pathways, quiescent normal human WI-38 fibroblasts were flown on the STS-93 space shuttle mission. Subsequently, RNA samples from the spaceflown and ground-control cells were used to construct two cDNA libraries, which were then processed for suppression subtractive hybridization （SSH） to identify spaceflight-specific gene expression. The SSH data show that key genes related to oxidative stress, DNA repair, and fatty acid oxidation are activated by spaceflight, suggesting the induction of cellular oxidative stress. This is further substantiated by the up-regulation of neuregulin 1 and the calcium-binding protein calmodulin 2. Another obvious stress sign is that spaceflight evokes the Ras/mitogen-activated protein kinase and phosphatidylinositol-3 kinase signaling pathways, along with up-regulating several Gl-phase cell cycle traverse genes. Other genes showing upregulation of expression are involved in protein synthesis and pro-apoptosis, as well as pro-survival. Interactome analysis of functionally related genes shows that c-Myc is the ＂hub＂ for those genes showing significant changes. Hence, our results suggest that microgravity travel may impact changes in gene expression mostly associated with cellular stress signaling, directing cells to either apoptotic death or premature senescence.

Microbiome data analysis with applications to pre-clinical studies using QIIME2: Statistical considerations

Diversity analysis and taxonomic profiles can be generated from marker-gene sequence data with the help of many available computational tools.The Quantitative Insights into Microbial Ecology Version 2(QIIME2)has been widely used for 16S rRNA data analysis.While many articles have demonstrated the use of QIIME2 with suitable datasets,the application to preclinical data has rarely been talked about.The issues involved in the pre-clinical data include the low-quality score and small sample size that should be addressed properly during analysis.In addition,there are few articles that discuss the detailed statistical methods behind those alpha and beta diversity significance tests that researchers are eager to find.Running the program without knowing the logic behind it is extremely risky.In this article,we first provide a guideline for analyzing 16S rRNA data using QIIME2.Then we will talk about issues in pre-clinical data,and how they could impact the outcome.Finally,we provide brief explanations of statistical methods such as group significance tests and sample size calculation.

Proteomics and metabolic phenotyping define principal roles for the aryl hydrocarbon receptor in mouse liver

Dioxin-like molecules have been associated with endocrine disruption and liver disease.To better understand aryl hydrocarbon receptor(AHR)biology,metabolic phenotyping and liver proteomics were performed in mice following ligand-activation or whole-body genetic ablation of this receptor.Male wild type(WT)and Ahr^(-/-) mice(Taconic)were fed a control diet and exposed to 3,3',4,4',5-pentachlorobiphenyl(PCB126)(61 nmol/kg by gavage)or vehicle for two weeks.PCB126 increased expression of canonical AHR targets(Cyp1 a1 and Cyp1 a2)in WT but not Ahr^(-/-).Knockouts had increased adiposity with decreased glucose tolerance;smaller livers with increased steatosis and perilipin-2;and paradoxically decreased blood lipids.PCB126 was associated with increased hepatic triglycerides in Ahr^(-/-).The liver proteome was impacted more so by Ahr^(-/-) genotype than ligandactivation,but top gene ontology(GO)processes were similar.The PCB126-associated liver proteome was Ahr-dependent.Ahr principally regulated liver metabolism(e.g.,lipids,xenobiotics,organic acids)and bioenergetics,but it also impacted liver endocrine response(e.g.,the insulin receptor)and function,including the production of steroids,hepatokines,and pheromone binding proteins.These effects could have been indirectly mediated by interacting transcription factors or microRNAs.The biologic roles of the AHR and its ligands warrant more research in liver metabolic health and disease.