Mitochondrial DNA as a target for analyzing the biodistribution of cell therapy products.

Biodistribution tests are crucial for evaluating the safety of cell therapy (CT) products in order to prevent unwanted organ homing of these products in patients. Quantitative polymerase chain reaction (qPCR) using intronic Alu is a popular method for biodistribution testing owing to its ability to detect donor cells without modifying CT products and low detection limit. However, Alu-qPCR may generate inaccurate information owing to background signals caused by the mixing of human genomic DNA with that of experimental animals. The aim of this study was to develop a test method that is more specific and sensitive than Alu-qPCR, targeting the mitochondrial DNA (mtDNA) sequence that varies substantially between humans and experimental animals. We designed primers for 12S, 16S, and cytochrome B in mtDNA regions, assessed their specificity and sensitivity, and selected primers and probes for the 12S region. Human adipose-derived stem cells, used as CT products, were injected into the tail vein of athymic NCr-nu/nu mice and detected, 7 d after administration, in their lungs at an average concentration of 2.22 ± 0.69 pg/µg mouse DNA, whereas Alu was not detected. Therefore, mtDNA is more specific and sensitive than Alu and is a useful target for evaluating CT product biodistribution.

CAR NK92 Cells Targeting BCMA Can Effectively Kill Multiple Myeloma Cells Both In Vitro and In Vivo.

Multiple myeloma (MM) is a hematological malignancy caused by malignant proliferation of plasma cells in bone marrow. Over the last decade, the survival outcome of patients with multiple myeloma (MM) has been substantially improved with the emergence of novel therapeutic agents. However, MM remains an incurable neoplastic plasma cell disorder. In addition, almost all MM patients inevitably relapse due to drug resistance. Chimeric antigen receptor (CAR)-modified NK cells represent a promising immunotherapeutic modality for cancer treatment. In this study, NK92 cells were engineered to express the third generation of BCMA CAR. In vitro, BCMA CAR-engineered NK92 cells displayed higher cytotoxicity and produced more cytokines such as IFN-γ and granzyme B than NK92 cells when they were co-cultured with MM cell lines. Furthermore, BCMA CAR-engineered NK92 cells released significantly higher amounts of cytokines and showed higher cytotoxicity when they were exposed to primary cells isolated from MM patients. The cytotoxicity of BCMA CAR NK92 cells was enhanced after MM cells were treated with bortezomib. Additionally, BCMA CAR NK92 cells exhibited potent antitumor activities in subcutaneous tumor models of MM. These results demonstrate that regional administration of BCMA CAR NK92 cells is a potentially promising strategy for treating MM.

Single-cell RNA Sequencing Reveals Novel Cellular Factors for Response to Immunosuppressive Therapy in Aplastic Anemia.

Aplastic anemia (AA) is a lethal hematological disorder; however, its pathogenesis is not fully understood. Although immunosuppressive therapy (IST) is a major treatment option for AA, one-third of patients do not respond to IST and its resistance mechanism remains elusive. To understand AA pathogenesis and IST resistance, we performed single-cell RNA sequencing (scRNA-seq) of bone marrow (BM) from healthy controls and patients with AA at diagnosis. We found that CD34+ early-stage erythroid precursor cells and PROM1+ hematopoietic stem cells were significantly depleted in AA, which suggests that the depletion of CD34+ early-stage erythroid precursor cells and PROM1+ hematopoietic stem cells might be one of the major mechanisms for AA pathogenesis related with BM-cell hypoplasia. More importantly, we observed the significant enrichment of CD8+ T cells and T cell-activating intercellular interactions in IST responders, indicating the association between the expansion and activation of T cells and the positive response of IST in AA. Taken together, our findings represent a valuable resource offering novel insights into the cellular heterogeneity in the BM of AA and reveal potential biomarkers for IST, building the foundation for future precision therapies in AA.

A Novel Therapeutic Reagent, KA-1002 for Alleviating Lysophosphatidic Acid-Mediated Inflammation Related Gene Expression in Swine Macrophages.

Stresses and various infectious reagents caused multiple inflammatory diseases in swine in a livestock industrial environment. Therefore, there is a need for an effective therapeutic or preventive agent that could alleviate chronic and acute inflammation. We found that lysophosphatidic acid (LPA), a stress-induced potent endogenous inflammatory molecule, causes a broad range-regulation of inflammation related genes inflammation in swine macrophages. We further investigated the genome scaled transcriptional regulatory effect of a novel LPA-signaling antagonist, KA-1002 on swine macrophages, inducing the alleviated LPA-mediated inflammation related gene expression. Therefore, KA-1002 could potentially serve as a novel therapeutic or preventive agent to maintain physiologically healthy and balanced conditions of pigs.

IL-32γ suppresses lung cancer stem cell growth via inhibition of ITGAV-mediated STAT5 pathway.

The cancer stem cells (CSCs) are thought to be responsible for cancer initiation, recurrence, and metastasis via a multifactorial process. IL-32γ has been known to inhibit several tumor developments. However, the role of IL-32γ in CSCs is unknown. The role of IL-32γ on tumor development was assessed in IL-32γ transgenic (Tg) mice allograft and xenograft model. In the in vitro assay, we analyzed CSC growth and apoptosis in cells with IL-32γ overexpression by cell viability assay and tumor-sphere formation assay. In addition, expression of cell proliferation, apoptosis markers, and signaling molecules was determined by western blot analysis. IL-32γ suppressed CD133+ CSC-induced allograft model in IL-32γ Tg mice and xenograft model. Tumor-sphere formation and cell viability assay revealed a greater inhibition of CSC proliferation and antineoplastic activity of IL-32γ in CD133+ CSCs as compared with normal cancer cells. The inhibitory effects of IL-32γ on tumor development were associated with inhibition of the STAT5 pathway. In addition, inhibition of STAT5 increased cleavage of caspase-3, but suppressed CD133 expression and colony formation. Web-based gene network analysis showed that IL-32 is correlated with ITGAV, an integrin gene. Our result revealed that knockdown of ITGAV by siRNA inhibited the phosphorylation of STAT5. Moreover, we identified that ITGAV overexpression reversed the effect of IL-32γ on phosphorylation of STAT5 and the expression of CD133. Our results demonstrate that IL-32γ negatively regulates CD133+ CSC proliferation and tumor development and suggest that IL-32γ has great potential for use in the treatment of cancer progression.

Tolerogenic dendritic cells are efficiently generated using minocycline and dexamethasone.

Tolerogenic dendritic cells (tDCs) represent a promising tool for cellular therapy against autoimmune diseases, allergies, and transplantation rejection. Numerous pharmacological agents are known to induce tDC generation. Minocycline, which has long been used as a broad-spectrum antibiotic, was recently shown to significantly increase the generation of DCs with regulatory properties. Here, we examined the effect of the combination of minocycline with dexamethasone, rapamycin, vitamin D3, and interleukin (IL)-10, which are all known inducers of tDC generation. The highest number of tDCs was generated when minocycline and dexamethasone were used together with granulocyte colony-stimulating factor (GM-SCF) and IL-4. The tolerogenicity of the minocycline/dexamethasone-conditioned tDCs was much better than or at least equal to those of the tDCs generated with either one of these agents, as assessed through in vitro phenotypic and functional assays. In addition, pretreatment with MOG35-55 peptide-pulsed minocycline/dexamethasone-conditioned tDCs significantly ameliorated the clinical signs of experimental autoimmune encephalitis induced by MOG peptide injection in a murine model. These results confirmed that tDCs with potent tolerogenic properties could be efficiently generated by the combined use of minocycline and dexamethasone, along with GM-CSF and IL-4. Our results would help in the development of ex vivo tDC-based immunotherapies.

Minocycline promotes the generation of dendritic cells with regulatory properties.

Minocycline, which has long been used as a broad-spectrum antibiotic, also exhibits non-antibiotic properties such as inhibition of inflammation and angiogenesis. In this study, we show that minocycline significantly enhances the generation of dendritic cells (DCs) from mouse bone marrow (BM) cells when used together with GM-CSF and IL-4. DCs generated from BM cells in the presence of minocycline (Mino-DCs) demonstrate the characteristics of regulatory DCs. Compared with control DCs, Mino-DCs are resistant to subsequent maturation stimuli, impaired in MHC class II-restricted exogenous Ag presentation, and show decreased cytokine secretion. Mino-DCs also show decreased ability to prime allogeneic-specific T cells, while increasing the expansion of CD4+CD25+Foxp3+ T regulatory cells both in vitro and in vivo. In addition, pretreatment with MOG35-55 peptide-pulsed Mino-DCs ameliorates clinical signs of experimental autoimmune encephalitis induced by MOG peptide injection. Our study identifies minocycline as a new pharmacological agent that could be potentially used to increase the production of regulatory DCs for cell therapy to treat autoimmune disorders, allergy, and transplant rejection.

Decreased severity of collagen antibody and lipopolysaccharide-induced arthritis in human IL-32ß overexpressed transgenic mice.

Interleukin (IL)-32, mainly produced by T-lymphocytes, natural killer cells, epithelial cells, and blood monocytes, is dominantly known as a pro-inflammatory cytokine. However, the role of IL-32 on inflammatory disease has been doubtful according to diverse conflicting results. This study was designed to examine the role of IL-32ß on the development of collagen antibody (CAIA) and lipopolysaccharide (LPS)-induced inflammatory arthritis. Our data showed that the paw swelling volume and clinical score were significantly reduced in the CAIA and LPS-treated IL-32ß transgenic mice compared with non-transgenic mice. The populations of cytotoxic T, NK and dendritic cells was inhibited and NF-κB and STAT3 activities were significantly lowered in the CAIA and LPS-treated IL-32ß transgenic mice. The expression of pro-inflammatory proteins was prevented in the paw tissues of CAIA and LPS-treated IL-32ß transgenic mice. In addition, IL-32ß altered several cytokine levels in the blood, spleen and paw joint. Our data indicates that IL-32ß comprehensively inhibits the inflammation responses in the CAIA and LPS-induced inflammatory arthritis model.

Interleukin-32γ attenuates ethanol-induced liver injury by the inhibition of cytochrome P450 2E1 expression and inflammatory responses.

Alcohol abuse and alcoholism lead to alcoholic liver disease (ALD), which is a major type of chronic liver disease worldwide. Interleukin-32 (IL-32) is a novel cytokine involved in inflammation and cancer development. However, the role of IL-32 in chronic liver disease has not been reported. In the present paper, we tested the effect of IL-32γ on ethanol-induced liver injury in IL-32γ-overexpressing transgenic mice (IL-32γ mice) after chronic ethanol feeding. Male C57BL/6 and IL-32γ mice (10-12 weeks old) were fed on a Lieber-DeCarli diet containing 6.6% ethanol for 6 weeks. IL-32γ-transfected HepG2 and Huh7 cells, as well as primary hepatocytes from IL-32γ mice, were treated with or without ethanol. The hepatic steatosis and damage induced by ethanol administration were attenuated in IL-32γ mice. Ethanol-induced cytochrome P450 2E1 expression and hydrogen peroxide levels were decreased in the livers of IL-32γ mice, primary hepatocytes from IL-32γ mice and IL-32γ-overexpressing human hepatic cells. The ethanol-induced expression levels of cyclo-oxygenase-2 (COX-2) and IL-6 were reduced in the livers of IL-32γ mice. Because nuclear transcription factor κB (NF-κB) is a key redox transcription factor of inflammatory responses, we examined NF-κB activity. Ethanol-induced NF-κB activities were significantly lower in the livers of IL-32γ mice than in wild-type (WT) mice. Furthermore, reduced infiltration of natural killer cells, cytotoxic T-cells and macrophages in the liver after ethanol administration was observed in IL-32γ mice. These data suggest that IL-32γ prevents ethanol-induced hepatic injury via the inhibition of oxidative damage and inflammatory responses.

Suberoylanilide hydroxamic acid induces ROS-mediated cleavage of HSP90 in leukemia cells.

Heat shock protein 90 (HSP90) is a molecular chaperone that supports stability of client proteins. We found that HSP90 was cleaved to 55 kDa protein after treatment with histone deacetylase (HDAC) inhibitors including suberoylanilide hydroxamic acid (SAHA) in several leukemia cell lines. We further analyzed molecular changes induced by SAHA in K562 cells. The SAHA-induced cleavage of HSP90 was blocked by a pan-caspase inhibitor, z-VAD-fmk, implying that the process is dependent on caspase activity. However, the experiments using antagonistic and agonistic Fas antibodies revealed that the cleavage of HSP90 was not dependent on Fas signaling. SAHA induced generation of reactive oxygen species (ROS), and the cleavage of HSP90 was blocked by a ROS scavenger N-acetylcystein (NAC). We also confirmed that hydrogen peroxide (H2O2) induced cleavage of HSP90 in a similar manner. Caspase 2, 3, 4, 6, 8, and 10 were activated by treatment with SAHA, and the activities were reduced by the pretreatment of NAC. Treatment of the cells with caspase 10 inhihitor, but not other inhibitors of caspases activated by SAHA, prevented cleavage of HSP90 by SAHA. SAHA-induced ROS generation and HSP90 cleavage were dependent on newly synthesized unknown proteins. Taken together, our results suggest that the cleavage of HSP90 by SAHA is mediated by ROS generation and caspase 10 activation. HSP90 cleavage may provide an additional mechanism involved in anti-cancer effects of HDAC inhibitors.

Identification of HLA-A2-restricted immunogenic peptides derived from a xenogenic porcine major histocompatibility complex.

BACKGROUND: Little information is available regarding the precise swine leukocyte antigen (SLA)-derived immunogenic peptides that are presented in the context of human HLA molecules. Here, we identified SLA-derived immunogenic peptides that are presented in association with human HLA-A2 molecule. METHODS: The SLA-derived peptides that bind to HLA-A*0201, a representative of the A2 supertype, were predicted using a computer-assisted algorithm. The candidate peptides were synthesized, and the stabilities of complexes formed between peptides and HLA-A*0201 were compared using major histocompatibility complex (MHC) stabilization assays. The cytotoxic T lymphocyte (CTL)-inducing activity of the selected peptides was examined in HLA-A*0201-transgenic mice. RESULTS: Among 15 candidate peptides synthesized, two peptides, peptide-35 (YLGPDGLLL) and peptide-43 (TLICHVDSI), were selected to have high affinity and stability with HLA-A*0201. Examination of the CTL-inducing activity of the two peptides in HLA-A*0201-transgenic mice showed that immunization with peptide-35, but not peptide-43, elicited potent CD8-specific CTL responses. The Peptide-35 is present in non-polymorphic α2 domains of 34 SLA-1 alleles, 18 SLA-2 alleles, and 1 SLA-3 allele. CONCLUSION: This study identifies an immunogenic HLA-A*0201-restricted epitope derived from the SLA, which may be valuable for the development of epitope-specific immunoregulation strategies.

Baccatin III, a precursor for the semisynthesis of paclitaxel, inhibits the accumulation and suppressive activity of myeloid-derived suppressor cells in tumor-bearing mice.

Myeloid-derived suppressor cells (MDSCs) mediate tumor-associated immune suppression in both cancer patients and tumor-bearing animals. Reduction or elimination of MDSCs reduces the rate of tumor progression and improves cancer therapies that employ mechanisms of immunity. Here we show that baccatin III, which is the precursor for the semisynthesis of paclitaxel, exerts anti-tumor immunomodulatory activity in very low doses (0.05-0.5mg/kg), although it is regarded as an inactive derivative of paclitaxel. Oral administration of baccatin III significantly reduced the growth of tumors induced by engrafting BALB/c mice with either 4 T1 mammary carcinoma or CT26 colon cancer cells. Baccatin III (0.5mg/kg) did not exert anti-tumor activity in athymic nude mice. Baccatin III decreased the accumulation of MDSCs in the spleens of the tumor-bearing mice. Furthermore, MDSCs isolated from baccatin III-treated mice, compared with those isolated from vehicle-treated mice, had a significantly reduced suppressive effect on T cells treated with the anti-CD3 and anti-CD28 monoclonal antibodies. Moreover, these cells produced significantly reduced amounts of reactive oxygen species and nitric oxide. These results suggest that baccatin III reduced tumor progression by inhibiting the accumulation and suppressive function of MDSCs.

Metallothionein-III increases ADAM10 activity in association with furin, PC7, and PKCα during non-amyloidogenic processing.

A-disintegrin and metalloproteinase 10 (ADAM10) is involved in the generation of amyloid-ß (Aß) during amyloid precursor protein (APP) processing, and has a protective effect against Aß neurotoxicity. We explored how metallothionein-III (MT-III) is regulated in the non-amyloidogenic pathway to generate soluble APPα (sAPPα). MT-ІІІ increased sAPPα levels and reduced Aß peptide levels, but did not affect ADAM10 expression. However, MT-III increased the activity of ADAM10. MT-ІІІ-induced sAPPα secretion, and Aß peptide formation was blocked by specific inhibitors of furin, proprotein convertase7 (PC7), and PKCα. These results demonstrate that MT-ІІІ increases the amount of active ADAM10 in association with furin, PC7 and PKCα.

Resiquimod, a TLR7/8 agonist, promotes differentiation of myeloid-derived suppressor cells into macrophages and dendritic cells.

Myeloid-derived suppressor cells (MDSCs) accumulate in cancer patients and tumor-bearing mice, subsequently suppressing the host immune system. MDSCs represent a group of immature myeloid cells expressing CD11b and Gr-1. Here, we show that a Toll-like receptor (TLR) agonist, resiquimod, which binds to TLR7 and TLR8, induces the differentiation of MDSCs into mature myeloid cells. MDSCs were isolated from mice bearing mammary carcinoma 4T1 cells, and the purified MDSCs were cultured in the presence of resiquimod for 5 days. Phenotypic analysis showed that the resiquimod-treated MDSCs differentiated into F4/80⁺ macrophages and CD11c⁺/I-A(d⁺) dendritic cells. Functional analysis showed that the MDSCs also lost their suppressive activity on T cells. Resiquimod-treated MDSCs significantly enhanced the proliferation of T cells that were treated with anti-CD3 and anti-CD28 monoclonal antibodies. These results show that resiquimod induces the differentiation of MDSCs into macrophages and dendritic cells, and also suggest that resiquimod may improve cancer immunotherapy by reducing immunosuppressive MDSCs.

Lung tumor growth-promoting function of peroxiredoxin 6.

This study compared lung tumor growth in PRDX6-overexpressing transgenic (Tg) mice and normal mice. These mice expressed elevated levels of PRDX6 mRNA and protein in multiple tissues. In vivo, Tg mice displayed a greater increase in the growth of lung tumor compared with normal mice. Glutathione peroxidase and calcium-independent phospholipase 2 (iPLA2) activities in tumor tissues of Tg mice were much higher than in tumor tissues of normal mice. Higher tumor growth in PRDX6-overexpressing Tg mice was associated with an increase in activating protein-1 (AP-1) DNA-binding activity. Moreover, expression of proliferating cell nuclear antigen, Ki67, vascular endothelial growth factor, c-Jun, c-Fos, metalloproteinase-9, cyclin-dependent kinases, and cyclins was much higher in the tumor tissues of PRDX6-overexpressing Tg mice than in tumor tissues of normal mice. However, the expression of apoptotic regulatory proteins including caspase-3 and Bax was slightly less in the tumor tissues of normal mice. In tumor tissues of PRDX6-overexpressing Tg mice, activation of mitogen-activated protein kinases (MAPKs) was much higher than in normal mice. In cultured lung cancer cells, PRDX6 siRNA suppressed glutathione peroxidase and iPLA2 activities and cancer cell growth, but the enforced overexpression of PRDX6 increased cancer cell growth associated with their increased activities. In vitro, among the tested MAPK inhibitors, c-Jun NH2-terminal kinase (JNK) inhibitor clearly suppressed the growth of lung cancer cells and AP-1 DNA binding, glutathione peroxidase activity, and iPLA2 activity in normal and PRDX6-overexpressing lung cancer cells. These data indicate that overexpression of PRDX6 promotes lung tumor growth via increased glutathione peroxidase and iPLA2 activities through the upregulation of the AP-1 and JNK pathways.

Prediction of microbial infection of cultured cells using DNA microarray gene-expression profiles of host responses.

Infection by microorganisms may cause fatally erroneous interpretations in the biologic researches based on cell culture. The contamination by microorganism in the cell culture is quite frequent (5% to 35%). However, current approaches to identify the presence of contamination have many limitations such as high cost of time and labor, and difficulty in interpreting the result. In this paper, we propose a model to predict cell infection, using a microarray technique which gives an overview of the whole genome profile. By analysis of 62 microarray expression profiles under various experimental conditions altering cell type, source of infection and collection time, we discovered 5 marker genes, NM_005298, NM_016408, NM_014588, S76389, and NM_001853. In addition, we discovered two of these genes, S76389, and NM_001853, are involved in a Mycolplasma-specific infection process. We also suggest models to predict the source of infection, cell type or time after infection. We implemented a web based prediction tool in microarray data, named Prediction of Microbial Infection (http://www.snubi.org/software/PMI).

Identification of HLA-DR4-restricted immunogenic peptide derived from xenogenic porcine major histocompatibility complex class I molecule.

Indirect recognition of xenoantigens has been implicated as the major mechanism underlying xenospecific CD4+ T-cell activation in chronic rejection. We identified swine leukocyte antigen (SLA)-derived immunogenic peptides that are presented in the context of human HLA-DR4 molecules. The SLA class I-derived peptides that bind HLA-DRB1*0401, a representative of the DR4 supertype, were predicted using a computer-assisted algorithm. The candidate peptides were synthesized, and their binding capacities to HLA-DRB1*0401 were compared in a competitive ELISA using biotinylated hemagglutinin reporter peptides [HA(307-319)]. Peptide-11 (LRSWTAADTAAQISK) was determined to exhibit the most potent binding capacity to HLA-DRB1*0401 in vitro and thus selected for in vivo immunization. Immunization of HLA-DRB1*0401-transgenic mice with peptide-11 elicited potent CD4+ Th1 responses. Peptide-11 shares homology to α2 domains of three SLA-1 alleles, six SLA-2 alleles, and 14 SLA-3 alleles. Thus, this study has important implications not only for the identification of an immunogenic indirect epitope shared by diverse SLA class I alleles, but also for the development of epitope-specific immunoregulation strategies.

Design and synthesis of 4-O-methylhonokiol analogs as inhibitors of cyclooxygenase-2 (COX-2) and PGF1 production.

A series of novel 4-O-methylhonokiol analogs were synthesized in light of revealing structure-activity relationship for inhibitory effect of COX-2 enzyme. The key strategy of the molecular design was oriented towards modification of the potential metabolic soft spots (e.g., phenol and olefin) or by altering the polar surface area via incorporating heterocycles such as isoxazole and triazole. Most of all exhibited the inhibitory effects on COX-2 and PGF(1) production but not macrophage NO production. Especially, aryl carbamates 10 and 11 exhibited more potent inhibitory activity against COX-2 and PGF(1) production.