There has been a new interest in using aldehyde dehydrogenase (ALDH) activity as one marker for stem cells since the Aldefluor flow cytometry-based assay has become available. Diethylaminobenzaldehyde (DEAB), used in the Aldeflour assay, has been considered a specific inhibitor for ALDH1A1 isoform. In this study, we explore the effects of human ALDH isoenzymes, ALDH1A2 and ALDH2, on drug resistance and proliferation, and the specificity of DEAB as an inhibitor. We also screened for the expression of 19 ALDH isoenzymes in K562 cells using TaqMan Low Density Array (TLDA). We used lentiviral vectors containing the full cDNA length of either ALDH2 or ALDH1A2 to over express the enzymes in K562 leukemia and H1299 lung cancer cell lines. Successful expression was measured by activity assay, Western blot, RT-PCR, and Aldefluor assay. Both cell lines, with either ALDH1A2 or ALDH2, exhibited higher cell proliferation rates, higher clonal efficiency, and increased drug resistance to 4-hydroperoxycyclophosphamide and doxorubicin. In order to study the specificity of known ALDH activity inhibitors, DEAB and disulfiram, we incubated each cell line with either inhibitor and measured the remaining ALDH enzymatic activity. Both inhibitors reduced ALDH activity of both isoenzymes by 65–90%. Furthermore, our TLDA results revealed that ALDH1, ALDH7, ALDH3 and ALDH8 are expressed in K562 cells. We conclude that DEAB is not a specific inhibitor for ALDH1A1 and that Aldefluor assay is not specific for ALDH1A1 activity. In addition, other ALDH isoenzymes seem to play a major role in the biology and drug resistance of various malignant cells.

BackgroundTherapies directed at augmenting regulatory T cell (Treg) activities in vivo as a systemic treatment for autoimmune disorders and transplantation may be associated with significant off-target effects, including a generalized immunosuppression that may compromise beneficial immune responses to infections and cancer cells. Adoptive cellular therapies using purified expanded Tregs represents an attractive alternative to systemic treatments, with results from animal studies noting increased therapeutic potency of antigen-specific Tregs over polyclonal populations. However, current methodologies are limited in terms of the capacity to isolate and expand a sufficient quantity of endogenous antigen-specific Tregs for therapeutic intervention. Moreover, FOXP3+ Tregs fall largely within the CD4+ T cell subset and are thus routinely MHC class II-specific, whereas class I-specific Tregs may function optimally in vivo by facilitating direct tissue recognition.Methodology/Principal FindingsTo overcome these limitations, we have developed a novel means for generating large numbers of antigen-specific Tregs involving lentiviral T cell receptor (TCR) gene transfer into in vitro expanded polyclonal natural Treg populations. Tregs redirected with a high-avidity class I-specific TCR were capable of recognizing the melanoma antigen tyrosinase in the context of HLA-A*0201 and could be further enriched during the expansion process by antigen-specific reactivation with peptide loaded artificial antigen presenting cells. These in vitro expanded Tregs continued to express FOXP3 and functional TCRs, and maintained the capacity to suppress conventional T cell responses directed against tyrosinase, as well as bystander T cell responses. Using this methodology in a model tumor system, murine Tregs designed to express the tyrosinase TCR effectively blocked antigen-specific effector T cell (Teff) activity as determined by tumor cell growth and luciferase reporter-based imaging.Conclusions/SignificanceThese results support the feasibility of class I-restricted TCR transfer as a promising strategy to redirect the functional properties of Tregs and provide for a more efficacious adoptive cell therapy.

IMPORTANCE Ovarian cancer has the highest mortality rate among gynecologic malignant tumors. Data are lacking on the survival benefit of hyperthermic intraperitoneal chemotherapy (HIPEC) in women with ovarian cancer who underwent primary or interval cytoreductive surgery.OBJECTIVE To assess the clinical benefit of HIPEC after primary or interval maximal cytoreductive surgery in women with stage III or IV primary advanced ovarian cancer. DESIGN, SETTING, AND PARTICIPANTSIn this single-blind randomized clinical trial performed at 2 institutions in South Korea from March 2, 2010, to January 22, 2016, a total of 184 patients with stage III or IV ovarian cancer with residual tumor size less than 1 cm were randomized (1:1) to a HIPEC (41.5 °C, 75 mg/m 2 of cisplatin, 90 minutes) or control group. The primary end point was progression-free survival. Overall survival and adverse events were key secondary end points. The date of the last follow-up was January 10, 2020, and the data were locked on February 17, 2020. EXPOSURES Hyperthermic intraperitoneal chemotherapy after cytoreductive surgery.MAIN OUTCOMES AND MEASURES Progression-free and overall survival. RESULTSOf the 184 Korean women who underwent randomization, 92 were randomized to the HIPEC group (median age, 52.0 years; IQR, 46.0-59.5 years) and 92 to the control group (median age, 53.5 years; IQR, 47.5-61.0 years). After a median follow-up of 69.4 months (IQR, 54.4-86.3 months), median progression-free survival was 18.8 months (IQR, 13.0-43.2 months) in the control group and 19.8 months (IQR, 13.7-55.4 months) in the HIPEC group (P = .43), and median overall survival was 61.3 months (IQR, 34.3 months to not reported) in the control group and 69.5 months (IQR, 45.6 months to not reported) in the HIPEC group (P = .52). In the subgroup of interval cytoreductive surgery after neoadjuvant chemotherapy, the median progression-free survival was 15.4 months (IQR, 10.6-21.1 months) in the control group and 17.4 months (IQR, in the HIPEC group (hazard ratio for disease progression or death, 0.60; 95% CI, 0.37-0.99; P = .04), and the median overall survival was 48.2 months (IQR, 33.8-61.3 months) in the control group and 61.8 months (IQR, 46.7 months to not reported) in the HIPEC group (hazard ratio, 0.53; 95% CI, 0.29-0.96; P = .04). In the subgroup of primary cytoreductive surgery, median progression-free survival was 29.7 (IQR, 17.2-90.1 months) in the control group and 23.9 months (IQR, 12.3-71.5 months) in the HIPEC group, and the median overall survival was not reached in the control group and 71.3 months (IQR, 45.6 months to not reported) in the HIPEC group. CONCLUSIONS AND RELEVANCEThe addition of HIPEC to cytoreductive surgery did not improve progression-free and overall survival in patients with advanced epithelial ovarian cancer. Although the results are from a subgroup analysis, the addition of HIPEC to interval cytoreductive surgery provided an improvement of progression-free and overall survival.

Objective Diffuse alveolar hemorrhage (DAH) in lupus patients is >50% fatal. The cause is unknown. The pathogenesis of DAH in C57BL/6 mice with pristane-induced lupus, a model of human lupus-associated DAH, was examined. Methods Clinical/pathological and immunological manifestations DAH in pristane-lupus were compared with human DAH. Tissue distribution of pristane was examined by mass spectrometry. Cell types responsible for disease were determined by in vivo depletion using clodronate liposomes (CloLip) and anti-neutrophil monoclonal antibodies (GR1). The effect of complement depletion with cobra venom factor (CVF) was examined. Results After i.p. injection, pristane migrated to the lung, causing cell death, small vessel vasculitis, and alveolar hemorrhage similar to human DAH. B-cell-deficient mice were resistant to induction of DAH, but susceptibility was restored by infusing IgM. C3-deficient and CD18-deficient mice also were resistant and DAH was prevented in wild-type mice by CVF. Induction of DAH was independent of TLRs, inflammasomes, and inducible nitric oxide (iNOS). Mortality was increased in IL-10-deficient mice and pristane treatment decreased IL-10 receptor expression in monocytes and Stat3 phosphorylation in lung macrophages. In vivo neutrophil depletion was not protective, whereas treatment with CloLip prevented DAH, suggesting that macrophage activation is central to DAH pathogenesis. Conclusion The pathogenesis of DAH involves opsonization of dead cells by natural IgM and complement followed by complement receptor-mediated lung inflammation. The disease is macrophage-dependent and IL-10 is protective. Complement inhibition and/or macrophage-targeted therapies may reduce mortality in lupus-associated DAH.

Objective To define the pathogenesis of bone marrow (BM) involvement in systemic lupus erythematosus (SLE). Methods Tumor necrosis factor-α (TNFα), cell death, and cellular damage in BM from SLE patients, controls, and mice with pristane-induced lupus were analyzed morphologically and using immunohistochemistry. The pathogenesis of BM abnormalities was studied in wild-type, and TNFα-, TLR7-, and interferon-α receptor-deficient, along with B cell-deficient (µmt) mice treated with pristane. Flow cytometry was used to examine TNFα production (intracellular staining) and plasma cell/plasmablast development. CXCL12 expression was determined by quantitative PCR. Results SLE patients’ BM exhibited striking death of niche and hematopoietic cells associated with TNFα over-production. BM from mice with an IFN-I-mediated lupus syndrome induced by pristane showed similar abnormalities. TNFα was produced mainly by BM neutrophils, many with phagocytosed nuclear material (LE cells). TNFα production was abolished in TLR7−/− and µmt mice but was restored in µmt mice by infusing normal plasma. Pristane-treated wild-type- and IFNAR−/− mice developed anemia, BM hypocellularity, and extramedullary hematopoiesis, which were absent in TLR7−/− and TNFα−/− mice. Additionally, CXCL12, which is produced by stromal cells and mediates homing of hematopoietic cells and plasmablasts, was decreased in BM from pristane-treated wild-type mice but normal in TNFα−/− mice. Conclusion Although autoantibodies and glomerulonephritis are IFN-I dependent, lupus-associated BM abnormalities were TLR7- and TNFα-driven, but IFN-I-independent, suggesting that lupus is a disorder of innate immunity in which TLR7 activation by phagocytosed nuclei causes relentless IFN-I and TNFα production mediating glomerulonephritis and hematologic involvement, respectively.

Genetic polymorphisms of IRF5 are associated with an increased risk of lupus in humans. Here, we examined the role of IRF5 in the pathogenesis of pristane-induced lupus in mice. The pathological response to pristane in IRF5−/− mice shared many features with IFN-I receptor (IFNAR) −/− and TLR7−/− mice: production of anti-Sm/RNP autoantibodies, glomerulonephritis, generation of Ly6Chi monocytes, and IFN-I production all were greatly attenuated. Lymphocyte activation following pristane injection was greatly diminished in IRF5−/− mice and helper T cell differentiation was deviated from TH1 in wild type mice toward TH2 in IRF5−/− mice. TH cell development was skewed similarly in TLR7−/− or IFNAR−/− mice, suggesting that IRF5 alters T cell activation and differentiation by affecting cytokine production. Indeed, production of IFN-I, IL-12, and IL-23 in response to pristane was markedly decreased, whereas IL-4 increased. Unexpectedly, plasmacytoid dendritic cells (pDC) were not recruited to the site of inflammation in IRF5−/− or MyD88−/− mice, but were recruited normally in IFNAR−/− and TLR7−/− mice. In striking contrast to wild type mice, pristane did not stimulate local expression of CCL19 and CCL21 in IRF5−/− mice, suggesting that IRF5 regulates chemokine-mediated pDC migration independently of its effects on IFN-I. Collectively, these data indicate that altered production of IFN-I and other cytokines in IRF5−/− mice prevents pristane from inducing lupus pathology by broadly affecting T and B lymphocyte activation/differentiation. Additionally, we uncovered a new, IFN-I independent, role of IRF5 in regulating chemokines involved in the homing of pDCs and certain lymphocyte subsets.

No abstract

This study demonstrates that multiplex detection of mutations in plasma cfDNA is clinically relevant, providing a potential candidate biomarker for prognosis of PDAC.