Prognostic significance of myeloid-derived suppressor cells and systemic inflammation in newly diagnosed diffuse large B cell lymphoma treated with chemoimmunotherapy.

The revised International Prognostic Index (R-IPI) is an important prognostic tool in diffuse large B cell lymphoma (DLBCL); however, outcomes can vary markedly within R-IPI groups, and additional prognostic markers are needed. We conducted a prospective observational study to evaluate the circulating immature myeloid (IM) cell subsets and cytokine profiles of 31 patients with newly diagnosed DLBCL before and after chemoimmunotherapy. Among circulating IM cells, myeloid-derived suppressor cells (MDSCs) were the predominant cell type (73.8% ± 26%). At baseline, circulating monocytic MDSCs (M-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs) were predominantly mutually exclusive. Patients with DLBCL clustered into three distinct immunotypes according to MDSC levels and subtype predominance: M-MDSChigh, PMN-MDSChigh, and MDSClow. The M-MDSChigh immunotype was associated with the germinal center B cell-like (GCB) subtype and elevated serum IL-8 and MIP-1α levels. PMN-MDSChigh was associated with the non-GCB subtype and elevated IL-8, MCP-1, IP-10, TNFα, and IL-1Ra levels. Standard chemoimmunotherapy partially reduced M-MDSC distribution across the MDSClow and M-MDSChigh groups. By contrast, among the MDSClow and PMN-MDSChigh groups, PMN-MDSCs persisted after treatment. Two high-risk patients with non-GCB DLBCL and MDSClow immunotype experienced early disease recurrence within 12 months of treatment completion. This study demonstrates that distinct types of MDSCs are associated with subtypes of DLBCL. MDSC levels are dynamic and may be associated with disease status. Persistence of PMN-MDSCs among high-risk patients with DLBCL may be associated with early relapse.

The Immunogenomic Landscape of Peripheral High-Dose IL-2 Pharmacodynamics in Patients with Metastatic Renal Cell Carcinoma: A Benchmark for Next-Generation IL-2-Based Immunotherapies.

High-dose (HD) IL-2 was the first immuno-oncology agent approved for treating advanced renal cell carcinoma and metastatic melanoma, but its use was limited because of substantial toxicities. Multiple next-generation IL-2 agents are being developed to improve tolerability. However, a knowledge gap still exists for the genomic markers that define the target pharmacology for HD IL-2 itself. In this retrospective observational study, we collected PBMC samples from 23 patients with metastatic renal cell carcinoma who were treated with HD IL-2 between 2009 and 2015. We previously reported the results of flow cytometry analyses. In this study, we report the results of our RNA-sequencing immunogenomic survey, which was performed on bulk PBMC samples from immediately before (day 1), during (day 3), and after treatment (day 5) in cycle 1 and/or cycle 2 of the first course of HD IL-2. As part of a detailed analysis of immunogenomic response to HD IL-2 treatment, we analyzed the changes in individual genes and immune gene signatures. By day 3, most lymphoid cell types had transiently decreased, whereas myeloid transcripts increased. Although most genes and/or signatures generally returned to pretreatment expression levels by day 5, certain ones representative of B cell, NK cell, and T cell proliferation and effector functions continued to increase, along with B cell (but not T cell) oligoclonal expansion. Regulatory T cells progressively expanded during and after treatment. They showed strong negative correlation with myeloid effector cells. This detailed RNA-sequencing immunogenomic survey of IL-2 pharmacology complements results of prior flow cytometry analyses. These data provide valuable pharmacological context for assessing PBMC gene expression data from patients dosed with IL-2-related compounds that are currently in development.

DFMO inhibition of neuroblastoma tumorigenesis.

BACKGROUND: Most high-risk neuroblastoma patients who relapse succumb to disease despite the existing therapy. We recently reported increased event-free and overall survival in neuroblastoma patients receiving difluoromethylornithine (DFMO) during maintenance therapy. The effect of DFMO on cellular processes associated with neuroblastoma tumorigenesis needs further elucidation. Previous studies have shown cytotoxicity with IC50 values >5-15 mM, these doses are physiologically unattainable in patients, prompting further mechanistic studies at therapeutic doses. METHODS: We characterized the effect of DFMO on cell viability, cell cycle, apoptosis, neurosphere formation, and protein expression in vitro using five established neuroblastoma cell lines (BE2C, CHLA-90, SHSY5Y, SMS-KCNR, and NGP) at clinically relevant doses of 0, 50, 100, 500, 1000, and 2500 µM. Limiting Dilution studies of tumor formation in murine models were performed. Statistical analysis was done using GraphPad and the level of significance set at p = 0.05. RESULTS: There was not a significant loss of cell viability or gain of apoptotic activity in the in vitro assays (p > 0.05). DFMO treatment initiated G1 to S phase cell cycle arrest. There was a dose-dependent decrease in frequency and size of neurospheres and a dose-dependent increase in beta-galactosidase activity in all cell lines. Tumor formation was decreased in xenografts both with DFMO-pretreated cells and in mice treated with DFMO. CONCLUSION: DFMO treatment is cytostatic at physiologically relevant doses and inhibits tumor initiation and progression in mice. This study suggests that DFMO, inhibits neuroblastoma by targeting cellular processes integral to neuroblastoma tumorigenesis at clinically relevant doses.

A Clinical and Correlative Study of Elotuzumab, Carfilzomib, Lenalidomide, and Dexamethasone (Elo-KRd) for Lenalidomide Refractory Multiple Myeloma in First Relapse.

INTRODUCTION: Treatment of patients with multiple myeloma (MM) in first relapse remains a challenge. This phase II study combined elotuzumab (Elo) with carfilzomib, lenalidomide, and dexamethasone (KRd) for treatment of MM in first relapse with the aim of improving efficacy. METHODS: Enrolled patients received Elo-KRd induction for 4 cycles, and Elo-lenalidomide maintenance until progression. The primary endpoint was VGPR or better (≥VGPR) postinduction. Secondary endpoints were MRD by flow cytometry, OS, PFS, and safety. Correlatives included characterization of the impact of Elo-KRd on NK and T cell subsets via flow cytometry. Target accrual of 40 patients was not met due to COVID-19 pandemic. RESULTS: Of 15 patients enrolled, 10 (67%) had high-risk features (del17p, t[4;14], t[14;16], 1q gain/amplification, plasma cell leukemia, extramedullary MM, or functional high risk), 12 (80%) were lenalidomide-refractory, and 5 (33.3%) bortezomib-refractory. Postinduction ≥VGPR was 7/15 (46.7%) and MRD-negative (10-5) rate 20%. Overall response during study was 80%, including ≥VGPR as best response of 53.3%. At median follow-up of 28.2 (range, 3.8 to 44.2) months, the median PFS was 11.5 months (95% CI 1.9, 18), and median OS not reached (95% CI 10.1, NA). No new safety concerns were reported. Elo-KRd treatment did not augment NK cell distribution or activity in blood or bone marrow. Effector CD4+ and CD8+ T cells significantly decreased postinduction, with concomitant acquisition of T central memory phenotype, particularly at a high rate in ≥VGPR group. CONCLUSION: A short course of Elo-KRd induction followed by Elo-lenalidomide maintenance demonstrated activity in predominantly lenalidomide-refractory and / or high-risk MM. The results with this well-tolerated combination are comparable to other contemporary approved triplet combinations.

Standardizing Clinical Workflow for Assessing Minimal Residual Disease by Flow Cytometry in Multiple Myeloma.

INTRODUCTION: Minimal residual disease (MRD) status is an established prognostic biomarker for patients with multiple myeloma. Commonly used MRD testing techniques such as next generation sequencing or next generation flow cytometry can detect as little as one or two multiple myeloma plasma cells in 106 normal bone marrow cells. Early pull of bone marrow aspirates (BMA), necessary to achieve such level of sensitivity, can be difficult to secure in routine clinical practice due to the competing need for early pull samples for clinical response assessment, therefore introducing the risk of analytical interference during MRD testing. METHODS: To overcome this challenge, we standardized our workflow for collecting specimens by using a technical first pull after needle repositioning for MRD testing. To capture a comprehensive picture of MRD assay performance and specimen adequacy, we tested for MRD on 556 technical first pull bone marrow aspirates by next generation flow cytometry. Among the specimens, several key multiple myeloma treatment milestones were represented: end of induction therapy, two to three months post-autologous stem cell transplant, early and late stages of maintenance therapy. RESULTS: By using the technical first pull bone marrow aspirate, we achieved an analytical assay input of 10 million nucleated cells for 97.5% of specimens. Our analytical sensitivity reached 10-6; (i.e., 10 multiple myeloma plasma cells in 10 × 106 bone marrow cells). Twenty-four percent of specimens were significantly hemodiluted. Low assay input or hemodilution quantifiably lowered the assay sensitivity. CONCLUSION: Specimen adequacy is, therefore, an important metric to incorporate into MRD status reporting.

Landscape and clinical impact of metabolic alterations in non-squamous non-small cell lung cancer.

Background: Metabolomics studies to date have described widespread metabolic reprogramming events during the development of non-squamous non-small cell lung cancer (NSCLC). Extending far beyond the Warburg effect, not only is carbohydrate metabolism affected, but also metabolism of amino acids, cofactors, lipids, and nucleotides. Methods: We evaluated the clinical impact of metabolic reprogramming. We performed comparative analysis of publicly available data on non-squamous NSCLC, to identify concensus altered metabolic pathways. We investigated whether alterations of metabolic genes controlling those consensus metabolic pathways impacted clinical outcome. Using the clinically annotated lung adenocarcinoma (LUAD) cohort from The Cancer Genome Atlas, we surveyed the distribution and frequency of function-altering mutations in metabolic genes and their impact on overall survival (OS). Results: We identified 42 metabolic genes of clinical significance, the majority of which (37 of 42) clustered across three metabolic superpathways (carbohydrates, amino acids, and nucleotides) and most functions (40 of 42) were associated with shorter OS. Multivariate analyses showed that dysfunction of carbohydrate metabolism had the most profound impact on OS [hazard ratio (HR) =5.208; 95% confidence interval (CI): 3.272 to 8.291], false discovery rate (FDR)-P≤0.0001, followed by amino acid metabolism (HR =3.346; 95% CI: 2.129 to 5.258), FDR-P≤0.0001 and nucleotide metabolism (HR =2.578; 95% CI: 1.598 to 4.159), FDR-P=0.0001. The deleterious effect of metabolic reprogramming on non-squamous NSCLC was observed independently of disease stage and across treatments groups. Conclusions: By providing a detailed landscape of metabolic alterations in non-squamous NSCLC, our findings offer new insights in the biology of the disease and metabolic adaptation mechanisms of clinical significance.

Phase II Study of Pembrolizumab in Combination with Doxorubicin in Metastatic and Unresectable Soft-Tissue Sarcoma.

PURPOSE: Doxorubicin is standard therapy for advanced soft-tissue sarcoma (STS) with minimal improvement in efficacy and increased toxicity with addition of other cytotoxic agents. Pembrolizumab monotherapy has demonstrated modest activity and tolerability in previous advanced STS studies. This study combined pembrolizumab with doxorubicin to assess safety and efficacy in frontline and relapsed settings of advanced STS. PATIENTS AND METHODS: This single-center, single-arm, phase II trial enrolled patients with unresectable or metastatic STS with no prior anthracycline therapy. Patients received pembrolizumab 200 mg i.v. and doxorubicin (60 mg/m2 cycle 1 with subsequent escalation to 75 mg/m2 as tolerated). The primary endpoint was safety. Secondary endpoints included overall survival (OS), objective response rate (ORR), and progression-free survival (PFS) based on RECIST v1.1 guidelines. RESULTS: Thirty patients were enrolled (53.3% female; median age 61.5 years; 87% previously untreated) with 4 (13.3%) patients continuing treatment. The study met its primary safety endpoint by prespecified Bayesian stopping rules. The majority of grade 3+ treatment-emergent adverse events were hematologic (36.7% 3+ neutropenia). ORR was 36.7% [95% confidence interval (CI), 19.9-56.1%], with documented disease control in 80.0% (95% CI, 61.4-92.3%) of patients. Ten (33.3%) patients achieved partial response, 1 (3.3%) patient achieved complete response, and 13 (43.3%) patients had stable disease. Median PFS and OS were 5.7 months (6-month PFS rate: 44%) and 17 months (12-month OS rate: 62%), respectively. Programmed cell death ligand-1 (PD-L1) expression was associated with improved ORR, but not OS or PFS. CONCLUSIONS: Combination pembrolizumab and doxorubicin has manageable toxicity and preliminary promising activity in treatment of patients with anthracycline-naive advanced STS.

Genomic analysis of response to bacillus Calmette-Guérin (BCG) treatment in high-grade stage 1 bladder cancer patients.

BACKGROUND: Intravesical bacillus Calmette-Guérin (BCG) therapy is standard treatment for high-risk non-muscle invasive bladder cancer (NMIBC) but overall efficacy is low, and no reliable predictive biomarkers currently exist to refine patient selection. We performed genomic analysis on high-grade (HG) T1 NMIBCs to determine if response to therapy is predicted by certain mutational and/or expressional changes. METHODS: Patients with HG T1 NMIBC treated with induction BCG were stratified by response into durable and non-durable responders. Baseline tumor samples were subjected to targeted DNA sequencing and whole-exome RNAseq. Genomic variants differing significantly between response groups were analyzed using Ingenuity Pathway Analysis (IPA) software. Variant selection was refined to target potential biomarker candidates for responsiveness to BCG. RESULTS: Among 42 patients, the median follow-up was 51.7 months and 40.5% (n=17) were durable BCG responders. Deleterious mutations in the RNA sequence of JCHAIN, S100A7, CLEC2B, and ANXA10 were more common in non-durable responders. Mutations in MCL1 and MSH6 detected on targeted sequencing were more commonly found in durable responders. Of all deleterious DNA and RNA mutations identified, only MCL1 was significantly associated with longer recurrence free survival (RFS) (P=0.031). CONCLUSIONS: Differences in the genomic profiles of HG T1 NMIBC tumors exist between those who show durable response to BCG and those who do not. Using pathway analysis, those differences imply upregulation of several interconnected inflammatory pathways among responders. Specific variants identified here, namely MCL1, are candidates for further study and, if clinically validated, may serve as useful biomarkers in the future.

Ex vivo efficacy of BCMA-bispecific antibody TNB-383B in relapsed/refractory multiple myeloma.

TNB-383B is a fully human BCMA-targeting T-cell engaging bispecific monoclonal antibody (T-BsAb). We assessed ex vivo efficacy of this drug to mediate killing of bone marrow mononuclear cells (BMMCs) freshly isolated from 10 patients with relapsed multiple myeloma (MM). BMMC were treated ex vivo with TNB-383B at doses ranging from 0.001-1 µg. Plasma cell (PC) lysis, viability, BCMA expression, CTL distribution, and degranulation were assessed by flow cytometry. Cytokine response to TNB-383B was quantified by multiplex protein assay. Dose-dependent PC lysis was triggered in all cases by TNB-383B at doses as low as 0.001 µg (P = .0102). Primary MM cells varied in BCMA expression. High BCMA+ PC count correlated with increased PC lysis (P = .005) and significant CTL degranulation specific to TNB-383B treatment (P = .0153 at 1 µg). High E:T ratio in bone marrow specimens led to lower viable and higher apoptotic PC compared with low E:T ratio (P < .001). Three cytokines were significantly modulated by TNB-383B: IL-2/TNFα increased by ∼4 ± 3.5-fold average (P < .005 at 1 µg) and IP10 increased by ∼50 ± 15-fold (P < .001 at 1 µg). We conclude that TNB-383B triggers primary PC lysis and CTL degranulation in a dose-dependent fashion ex vivo with no T cell expansion and mild increase of CRS-associated cytokines.

Extramedullary multiple myeloma.

Extramedullary multiple myeloma (EMM) is an aggressive subentity of multiple myeloma, characterized by the ability of a subclone to thrive and grow independent of the bone marrow microenvironment, resulting in a high-risk state associated with increased proliferation, evasion of apoptosis and treatment resistance. Despite improvement in survival for most patients with multiple myeloma over recent decades, outcomes are generally poor when EMM develops. Understanding the molecular underpinnings leading to homing of plasma cells in ecosystems outside the bone marrow will be crucial for therapeutically manipulating the microenvironment and targeting key signaling pathways. Herein, we discuss the evolutionary biology of EMM, underscore the importance of a uniform definition, discuss prognostic significance, and provide current and emerging treatment strategies for managing this rare subentity of multiple myeloma.

A Repeated Measures Pilot Comparison of Trajectories of Fluctuating Endogenous Hormones in Young Women with Traumatic Brain Injury, Healthy Controls.

OBJECTIVE: To compare baseline and 72-hour hormone levels in women with traumatic brain injury (TBI) and controls. SETTING: Hospital emergency department. PARTICIPANTS: 21 women ages 18-35 with TBI and 21 controls. DESIGN: Repeated measures. MAIN MEASURES: Serum samples at baseline and 72 hours; immunoassays for estradiol (E2), progesterone (PRO), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and cortisol (CORT); and health history. RESULTS: Women with TBI had lower E2 (p = 0.042) and higher CORT (p = 0.028) levels over time. Lower Glasgow Coma Scale (GSC) and OCs were associated with lower FSH (GCS p = 0.021; OCs p = 0.016) and higher CORT (GCS p = 0.001; OCs p = 0.008). CONCLUSION: Acute TBI may suppress E2 and increase CORT in young women. OCs appeared to independently affect CORT and FSH responses. Future work is needed with a larger sample to characterize TBI effects on women's endogenous hormone response to injury and OC use's effects on post-TBI stress response and gonadal function, as well as secondary injury.

Correction: Cytokine profiles in acute liver injury-Results from the US Drug-Induced Liver Injury Network (DILIN) and the Acute Liver Failure Study Group.

[This corrects the article DOI: 10.1371/journal.pone.0206389.].

Cytokine profiles in acute liver injury-Results from the US Drug-Induced Liver Injury Network (DILIN) and the Acute Liver Failure Study Group.

Changes in levels of cytokines and chemokines have been proposed as possible biomarkers of tissue injury, including liver injury due to drugs. Recently, in acute drug-induced liver injury (DILI), we showed that 19 of 27 immune analytes were differentially expressed and that disparate patterns of immune responses were evident. Lower values of serum albumin (< 2.8 g/dL) and lower levels of only four analytes, namely, IL-9, IL-17, PDGF-bb, and RANTES, were highly predictive of early death [accuracy = 96%]. The goals of this study were to assess levels of the same 27 immune analytes in larger numbers of subjects to learn whether the earlier findings would be confirmed in new and larger cohorts of subjects, compared with a new cohort of healthy controls. We studied 127 subjects with acute DILI enrolled into the US DILIN. We also studied 118 subjects with severe acute liver injury of diverse etiologies, enrolled into the ALF SG registry of subjects. Controls comprised 63 de-identified subjects with no history of liver disease and normal liver tests. Analytes associated with poor outcomes [death before 6 months, n = 32 of the total of 232 non-acetaminophen (Apap) subjects], were lower serum albumin [2.6 vs 3.0 g/dL] and RANTES [6,458 vs 8,999 pg/mL] but higher levels of IL-6 [41 vs 18], IL-8 [78 vs 48], and MELD scores [30 vs 24]. Similar patterns were observed for outcome of death/liver transplant within 6 months. A model that included only serum albumin < 2.8 g/dL and RANTES below its median value of 11,349 had 83% (or 81%) accuracy for predicting early death (or early death/liver transplant) in 127 subjects from DILIN. No patterns of serum immune analytes were reflective of the etiologies of acute liver failure, but there were cytokine patterns that predicted prognosis in both acute DILI and ALF.

MiR-122 decreases HCV entry into hepatocytes through binding to the 3' UTR of OCLN mRNA.

BACKGROUND & AIMS: Analysis in silico suggests that occludin (OCLN), a key receptor for HCV, is a candidate target of miR-122; the most abundant hepatic micro RNA. We aimed to determine if miR-122 can decrease HCV entry through binding to the 3' UTR of OCLN mRNA. DESIGN: Huh7.5 cells were cotransfected with luciferase construct containing 3' UTR of OCLN (pLuc-OCLN) and with selected miRNAs (0-50 nM) and luciferase activity was measured. Huh7.5 cells were also infected by viral particles containing lenti-miR122 genome or control virus. After 48 h, the cells were infected with HCV pseudo-particles (HCVpp) and VSV pseudo-particles (VSVpp). After 72 h of infection, luciferase activity was measured and HCVpp activity was normalized to VSVpp activity. RESULTS: miR-122 binds to the 3'-UTR of OCLN and down-regulates its expression; cotransfection of miR-122 mimic with pLuc-OCLN resulted in a significant decrease in luciferase activity [by 55% (P < 0.01)], while a non-specific miRNA and a mutant miR-122 did not have any effect. miR-122 mimic significantly down-regulated [by 80% (P < 0.01)] OCLN protein in Huh7.5 cells. Accordingly, patients with chronic hepatitis C and higher levels of hepatic miR-122 have lower hepatic expression of OCLN. Immuno-fluorescence imaging showed a decrease in colocalization of OCLN and CLDN following miR-122 over-expression in HCV infected cells. Huh7.5 cells transiently expressing Lenti-miR122 system showed 42% (P < 0.01) decrease in HCV entry. CONCLUSION: This study uncovers a novel antiviral effect of miR-122 on human liver cells and shows that over-expression of miR-122 can decrease HCV entry into hepatocytes through down-regulation of OCLN.

Sequential immune monitoring in patients with melanoma and renal cell carcinoma treated with high-dose interleukin-2: immune patterns and correlation with outcome.

Interleukin-2 (IL-2) therapy leads to clinically relevant responses in 10-16 % of patients with metastatic melanoma (MMEL) or 10-30 % of patients with metastatic renal cell carcinoma (MRCC). To date, no biomarkers have been validated to identify patients who are likely to respond. We hypothesized that changes in T cell subset distribution in patients undergoing IL-2 therapy may correlate with treatment outcomes. Immune profiles of 64 patients (27-MMEL, 37-MRCC) were evaluated using flow cytometry at baseline, during (≥three doses) and at the end of treatment cycle (30 ± 6 h after last dose), through two courses of IL-2 therapy. Changes in distribution and phenotype of circulating CD4 and CD8 lymphocyte subsets were compared (1) based on cancer types and (2) intra-patient during the course of the IL-2 therapy. Exploratory analysis of immunologic profiles was also performed based on treatment outcome. Independent of cancer type, IL-2 led to a transient decrease of circulating effector lymphocytes, while regulatory T cells gradually increased. Interleukin-2 differentially affected a subset of CD8 T cell expressing Foxp3, depending on malignancy type. In MMEL patients, IL-2 gradually expanded circulating CD8 Foxp3+ cells; in MRCC patients, IL-2 transiently increased expression of CD103 and CCR4 homing markers. Monitoring of adaptive immune variables early on and during the course of IL-2 therapy revealed transient alterations in immune profiles, specific to MMEL and MRCC patients, related to immune balance (and ultimately response to IL-2 therapy) or T cell egress from the circulation.

Drug-induced liver injury with autoimmune features.

Drug-induced liver injury (DILI) with features of autoimmunity (AI) represents an important category of hepatotoxicity due to medication exposure. Drugs repeatedly associated with AI-DILI include diclofenac, α-methyl DOPA, hydralazine, nitrofurantoin, minocycline, and more recently statins and anti-TNF-α agents. Usually, symptoms of acute liver injury occur within a few months after initiation of a culprit medication, but a longer latency period is possible. Like idiopathic autoimmune hepatitis, circulating autoantibodies and a hypergammaglobulinemia are frequently present in sera from patients with AI-DILI. If performed, a liver biopsy should demonstrate interface hepatitis with a prominent plasma cell infiltrate. The severity of AI-DILI is variable, but a complete resolution after withdrawal of the offending medication is the expectation. A response to corticosteroid therapy supports the diagnosis, whereas a lack of recurrence of symptoms or signs following corticosteroid cessation distinguishes AI-DILI from idiopathic autoimmune hepatitis.

Profiles of serum cytokines in acute drug-induced liver injury and their prognostic significance.

UNLABELLED: Drug-induced liver injury (DILI) is the most common cause of acute liver failure in the United-States. The aim of the study was to describe serum immune profiles associated with acute DILI, to investigate whether there are profiles associated with clinical features or types of DILI and/or with prognosis, and to assess temporal changes in levels. Twenty-seven immune analytes were measured in the sera of 78 DILI subjects in the Drug-Induced Liver Injury Network (DILIN) and compared with 40 healthy controls. Immune analytes (14 cytokines, 7 chemokines and 6 growth factors) were measured by BioPlex multiplex ELISA at DILI onset and after 6 months. A modeling process utilizing immune principles was used to select a final set of variables among 27 immune analytes and several additional clinical lab values for prediction of early death (within 6 months of DILI onset). Nineteen of the 27 immune analytes were differentially expressed among healthy control, DILI onset and 6-month cohorts. Disparate patterns of immune responses, especially innate and adaptive cellular (mostly TH17) immunity were evident. Low values of four immune analytes (IL-9, IL-17, PDGF-bb and RANTES) and serum albumin are predictive of early death [PPV = 88% (95% CI, 65%-100%), NPV = 97% (95% CI, 93%-100%), accuracy = 96% (95% CI, 92%-100%)]. CONCLUSIONS: Acute DILI is associated with robust and varying immune responses. High levels of expression of cytokines associated with innate immunity are associated with a poor prognosis, whereas high levels of expression of adaptive cytokines are associated with good long-term prognosis and eventual recovery. Serum immune analyte profiles at DILI onset appear to be of prognostic, and perhaps, diagnostic significance.

Silibinin (Milk Thistle) potentiates ethanol-dependent hepatocellular carcinoma progression in male mice.

Hepatocellular carcinoma (HCC) is a global health burden with limited treatment options and poor prognosis. Silibinin, an antioxidant derived from the Milk Thistle plant (Silybum marianum), is reported to exert hepatoprotective and antitumorigenic effects in vitro and in vivo by suppressing oxidative stress and proliferation. Using a DEN-initiated mouse model of HCC, this study examined the effects of dietary silibinin supplementation alone, or in combination with chronic ethanol consumption on HCC progression. Our data demonstrate silibinin exerted marginal hepatoprotective effects in early stages of hepatocarcinogenesis but, when co-administered with ethanol, exacerbated the promotional effects of ethanol in HCC bearing mice, but only in males.

Structure and function of proteins in hydrated choline dihydrogen phosphate ionic liquid.

Ionic liquids are being intensely studied as promising media for the stabilization of proteins and other biomolecules. Choline dihydrogen phosphate (CDHP) has been identified as one of the most promising candidates for this application. In this work we have probed in more detail the effects that CDHP may have on the thermodynamics, structure, and stability of proteins, including one of therapeutic interest. Microcalorimetry and circular dichroism spectropolarimetry (CD) were used to assess the thermal stability of protein solutions in CDHP/water mixtures at various concentrations. Increasing thermal stability of lysozyme and interleukin-2 in proportion to CDHP concentration was observed. Isothermal titration calorimetry (ITC) was used to quantify binding interactions, and indicate that the mechanism for stability does not appear to be dependent upon CDHP binding to protein. CD and small angle X-ray scattering (SAXS) analyses were used to probe for structural changes due to the presence of CDHP. SAXS indicates charge effects on the surface of the protein play a role in protein stability in ionic liquids, and no significant alteration of the overall tertiary conformation of lysozyme was observed at 25 °C. However, after incubation at 37 °C or at higher concentrations of CDHP, small changes in protein structure were seen. Effects on protein activity were monitored using turbidity assays, and CDHP decreases protein activity but does not eliminate it. Protein solubility was also monitored using a turbidity assay and was found to be inversely proportional to the concentration of CDHP in solution.

In vitro assessment of choline dihydrogen phosphate (CDHP) as a vehicle for recombinant human interleukin-2 (rhIL-2).

Choline dihydrogen phosphate (CDHP) is an ionic liquid reported to increase thermal stability of model proteins. The current work investigated CDHP effect on structural integrity and biological activity of recombinant human interleukin-2 (rhIL-2), a therapeutic protein used for treating advanced melanoma. In vitro CDHP biocompatibility was also evaluated using primary cell cultures, or B16-F10 cell line, chronically exposed to the ionic liquid. Formulation of rhIL-2 in an aqueous 680mM CDHP pH 7.4 solution resulted in a 12.5°C increase in the Tm of rhIL-2 compared to a basic buffer formulation, and provided conformational rhIL-2 stabilization when the solution was heated to 23.3°C above the Tm. CDHP solutions (≤80mM), exhibited no cytotoxic activity toward primary splenocytes or B16-F10 cells in culture. However, a 10-fold loss in biological activity was observed when rhIL-2 was used in a 30mM CDHP aqueous solution with NaHCO3 (pH≥7.2) compared to controls without CDHP. While increased Tm is associated with a diminished rhIL-2 biological activity, the therapeutic protein remains structurally intact and functional.