METTL14 facilitates global genome repair and suppresses skin tumorigenesis.

Global genome repair (GGR), a subpathway of nucleotide excision repair, corrects bulky helix-distorting DNA lesions across the whole genome and is essential for preventing mutagenesis and skin cancer. Here, we show that METTL14 (methyltransferase-like 14), a critical component of the N6-methyladenosine (m6A) RNA methyltransferase complex, promotes GGR through regulating m6A mRNA methylation-mediated DDB2 translation and suppresses ultraviolet B (UVB) radiation-induced skin tumorigenesis. UVB irradiation down-regulates METTL14 protein through NBR1-dependent selective autophagy. METTL14 knockdown decreases GGR and DDB2 abundance. Conversely, overexpression of wild-type METTL14 but not its enzymatically inactive mutant increases GGR and DDB2 abundance. METTL14 knockdown decreases m6A methylation and translation of the DDB2 transcripts. Adding DDB2 reverses the GGR repair defect in METTL14 knockdown cells, indicating that METTL14 facilitates GGR through regulating DDB2 m6A methylation and translation. Similarly, knockdown of YTHDF1, an m6A reader promoting translation of m6A-modified transcripts, decreases DDB2 protein levels. Both METTL14 and YTHDF1 bind to the DDB2 transcript. In mice, skin-specific heterozygous METTL14 deletion increases UVB-induced skin tumorigenesis. Furthermore, METTL14 as well as DDB2 is down-regulated in human and mouse skin tumors and by chronic UVB irradiation in mouse skin, and METTL14 level is associated with the DDB2 level, suggesting a tumor-suppressive role of METTL14 in UVB-associated skin tumorigenesis in association with DDB2 regulation. Taken together, these findings demonstrate that METTL14 is a target for selective autophagy and acts as a critical epitranscriptomic mechanism to regulate GGR and suppress UVB-induced skin tumorigenesis.

Human occupation of northern Australia by 65,000 years ago.

The time of arrival of people in Australia is an unresolved question. It is relevant to debates about when modern humans first dispersed out of Africa and when their descendants incorporated genetic material from Neanderthals, Denisovans and possibly other hominins. Humans have also been implicated in the extinction of Australia's megafauna. Here we report the results of new excavations conducted at Madjedbebe, a rock shelter in northern Australia. Artefacts in primary depositional context are concentrated in three dense bands, with the stratigraphic integrity of the deposit demonstrated by artefact refits and by optical dating and other analyses of the sediments. Human occupation began around 65,000 years ago, with a distinctive stone tool assemblage including grinding stones, ground ochres, reflective additives and ground-edge hatchet heads. This evidence sets a new minimum age for the arrival of humans in Australia, the dispersal of modern humans out of Africa, and the subsequent interactions of modern humans with Neanderthals and Denisovans.

Objective Assessment of Perioperative Anxiety using Functional Near-infrared Spectroscopy in Elderly Patients: A Prospective Randomized Observational Pilot Study.

Background: Assessing and managing patient anxiety is essential to reduce postoperative complications in elderly patients. However, monitoring patient anxiety objectively is impossible. This study aimed to investigate the correlation between the level of fNIRS signals and anxiety in patients aged 65 and older undergoing artificial joint replacement surgery. Material and Methods: Sixty patients aged ≥65 years scheduled for elective total knee arthroplasty under spinal anesthesia were included. To differentiate the degree of anxiety, the patients were randomly divided into three groups, each consisting of 20 patients (group 1: administered normal saline as a placebo; groups 2 and 3: administered dexmedetomidine at a rate of 0.2 and 0.5 µg/kg/h, respectively, for 10 min). Functional near-infrared spectroscopy was measured continuously for 10 min in each session (session 1: pre-anesthetic period; session 2: immediately after the spinal anesthesia period; session 3: normal saline or dexmedetomidine receiving period) in all patients. Vital signs were measured thrice at 5-min intervals during each session. State-Trait Anxiety Inventory -S (STAI-S) and Ramsay Sedation Scale (RSS) scores were assessed at the end of each session. Results: The STAI-S score was significantly correlated with power of bandwidth (p = 0.034). In addition, the RSS score was significantly correlated with BW 1, 2, and 3 (p = 0.010, p < 0.001, and p = 0.003, respectively). Conclusion: The STAI-S score and BW 3 were significantly correlated, suggesting that fNIRS might help objectively and directly monitor anxiety levels.

Bioresorbable silicon electronic sensors for the brain.

Many procedures in modern clinical medicine rely on the use of electronic implants in treating conditions that range from acute coronary events to traumatic injury. However, standard permanent electronic hardware acts as a nidus for infection: bacteria form biofilms along percutaneous wires, or seed haematogenously, with the potential to migrate within the body and to provoke immune-mediated pathological tissue reactions. The associated surgical retrieval procedures, meanwhile, subject patients to the distress associated with re-operation and expose them to additional complications. Here, we report materials, device architectures, integration strategies, and in vivo demonstrations in rats of implantable, multifunctional silicon sensors for the brain, for which all of the constituent materials naturally resorb via hydrolysis and/or metabolic action, eliminating the need for extraction. Continuous monitoring of intracranial pressure and temperature illustrates functionality essential to the treatment of traumatic brain injury; the measurement performance of our resorbable devices compares favourably with that of non-resorbable clinical standards. In our experiments, insulated percutaneous wires connect to an externally mounted, miniaturized wireless potentiostat for data transmission. In a separate set-up, we connect a sensor to an implanted (but only partially resorbable) data-communication system, proving the principle that there is no need for any percutaneous wiring. The devices can be adapted to sense fluid flow, motion, pH or thermal characteristics, in formats that are compatible with the body's abdomen and extremities, as well as the deep brain, suggesting that the sensors might meet many needs in clinical medicine.

Ectopic Expression of Human Thymosin ß4 Confers Resistance to Legionella pneumophila during Pulmonary and Systemic Infection in Mice.

Thymosin beta-4 (Tß4) is an actin-sequestering peptide that plays important roles in regeneration and remodeling of injured tissues. However, its function in a naturally occurring pathogenic bacterial infection model has remained elusive. We adopted Tß4-overexpressing transgenic (Tg) mice to investigate the role of Tß4 in acute pulmonary infection and systemic sepsis caused by Legionella pneumophila Upon infection, Tß4-Tg mice demonstrated significantly lower bacterial loads in the lung, less hyaline membranes and necrotic abscess, with lower interstitial infiltration of neutrophils, CD4+, and CD8+ T cells. Bronchoalveolar lavage fluid of Tß4-Tg mice possessed higher bactericidal activity against exogenously added L. pneumophila, suggesting that constitutive expression of Tß4 could efficiently control L. pneumophila Furthermore, qPCR analysis of lung homogenates demonstrated significant reduction of interleukin 1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α), which primarily originate from lung macrophages, in Tß4-Tg mice after pulmonary infection. Upon L. pneumophila challenge of bone marrow-derived macrophages (BMDM) in vitro, secretion of IL-1ß and TNF-α proteins was also reduced in Tß4-Tg macrophages, without affecting their survival. The anti-inflammatory effects of BMDM in Tß4-Tg mice on each cytokine were affected when triggering with tlr2, tlr4, tlr5, or tlr9 ligands, suggesting that anti-inflammatory effects of Tß4 are likely mediated by the reduced activation of Toll-like receptors (TLR). Finally, Tß4-Tg mice in a systemic sepsis model were protected from L. pneumophila-induced lethality compared to wild-type controls. Therefore, Tß4 confers effective resistance against L. pneumophila via two pathways, a bactericidal and an anti-inflammatory pathway, which can be harnessed to treat acute pneumonia and septic conditions caused by L. pneumophila in humans.

Induction of protective immune responses against a lethal Zika virus challenge post-vaccination with a dual serotype of recombinant vesicular stomatitis virus carrying the genetically modified Zika virus E protein gene.

The development of a vaccine to prevent Zika virus (ZIKV) infection has been one of the priorities in infectious disease research in recent years. There have been numerous attempts to develop an effective vaccine against ZIKV. It is imperative to choose the safest and the most effective ZIKV vaccine from all candidate vaccines to control this infection globally. We have employed a dual serotype of prime-boost recombinant vesicular stomatitis virus (VSV) vaccine strategy, to develop a ZIKV vaccine candidate, using a type 1 IFN-receptor knock-out (Ifnar-/-) mouse model for challenge studies. Prime vaccination with an attenuated recombinant VSV Indiana serotype (rVSVInd) carrying a genetically modified ZIKV envelope (E) protein gene followed by boost vaccination with attenuated recombinant VSV New Jersey serotype (rVSVNJ) carrying the same E gene induced robust adaptive immune responses. In particular, rVSV carrying the ZIKV E gene with the honeybee melittin signal peptide (msp) at the N terminus and VSV G protein transmembrane domain and cytoplasmic tail (Gtc) at the C terminus of the E gene induced strong protective immune responses. This vaccine regimen induced highly potent neutralizing antibodies and T cell responses in the absence of an adjuvant and protected Ifnar-/- mice from a lethal dose of the ZIKV challenge.

The Protective Effects of EMF-LTE against DNA Double-Strand Break Damage In Vitro and In Vivo.

With the rapid growth of the wireless communication industry, humans are extensively exposed to electromagnetic fields (EMF) comprised of radiofrequency (RF). The skin is considered the primary target of EMFs given its outermost location. Recent evidence suggests that extremely low frequency (ELF)-EMF can improve the efficacy of DNA repair in human cell-lines. However, the effects of EMF-RF on DNA damage remain unknown. Here, we investigated the impact of EMF-long term evolution (LTE, 1.762 GHz, 8 W/kg) irradiation on DNA double-strand break (DSB) using the murine melanoma cell line B16 and the human keratinocyte cell line HaCaT. EMF-LTE exposure alone did not affect cell viability or induce apoptosis or necrosis. In addition, DNA DSB damage, as determined by the neutral comet assay, was not induced by EMF-LTE irradiation. Of note, EMF-LTE exposure can attenuate the DNA DSB damage induced by physical and chemical DNA damaging agents (such as ionizing radiation (IR, 10 Gy) in HaCaT and B16 cells and bleomycin (BLM, 3 µM) in HaCaT cells and a human melanoma cell line MNT-1), suggesting that EMF-LTE promotes the repair of DNA DSB damage. The protective effect of EMF-LTE against DNA damage was further confirmed by attenuation of the DNA damage marker γ-H2AX after exposure to EMF-LTE in HaCaT and B16 cells. Most importantly, irradiation of EMF-LTE (1.76 GHz, 6 W/kg, 8 h/day) on mice in vivo for 4 weeks reduced the γ-H2AX level in the skin tissue, further supporting the protective effects of EMF-LTE against DNA DSB damage. Furthermore, p53, the master tumor-suppressor gene, was commonly upregulated by EMF-LTE irradiation in B16 and HaCaT cells. This finding suggests that p53 plays a role in the protective effect of EMF-LTE against DNA DSBs. Collectively, these results demonstrated that EMF-LTE might have a protective effect against DNA DSB damage in the skin, although further studies are necessary to understand its impact on human health.

Explant models for meniscus metabolism, injury, repair, and healing.

Purpose/Aim: Knee meniscus is a wedge-shaped fibrocartilaginous tissue, playing important roles in maintaining joint stability and function. Injuries to the meniscus, particularly with the avascular inner third zone, hardly heal and frequently progress into structural breakdown, followed by the initiation of osteoarthritis. As the importance of meniscus in joint function and diseases is being recognized, the field of meniscus research is growing. Not only development, biology, and metabolism but also injury, repair, and healing of meniscus are being actively investigated. As meniscus functions as an integrated unit of a knee joint, in vivo models with various species have been the predominant method for studying meniscus pathophysiology and for testing healing/regeneration strategies. However, in vivo models for meniscus studies suffer from low reproducibility and high cost. To complement the limitations of in vivo animal models, several types of meniscus explants have been applied as highly controlled, standardized in vitro models to investigate meniscus metabolism, pathophysiology, and repair or regeneration process. This review summarizes and compares the existing meniscus explant models. We also discuss the advantages and disadvantages of each explant model.Conclusion: Despite few outstanding challenges, meniscus explant models have potential to serve as an effective tool for investigations of meniscus metabolism, injury, repair and healing.

Crocin suppresses LPS-stimulated expression of inducible nitric oxide synthase by upregulation of heme oxygenase-1 via calcium/calmodulin-dependent protein kinase 4.

Crocin is a water-soluble carotenoid pigment that is primarily used in various cuisines as a seasoning and coloring agent, as well as in traditional medicines for the treatment of edema, fever, and hepatic disorder. In this study, we demonstrated that crocin markedly induces the expression of heme oxygenase-1 (HO-1) which leads to an anti-inflammatory response. Crocin inhibited inducible nitric oxide synthase (iNOS) expression and nitric oxide production via downregulation of nuclear factor kappa B activity in lipopolysaccharide- (LPS-) stimulated RAW 264.7 macrophages. These effects were abrogated by blocking of HO-1 expression or activity. Crocin also induced Ca(2+) mobilization from intracellular pools and phosphorylation of Ca(2+)/calmodulin-dependent protein kinase 4 (CAMK4). CAMK4 knockdown and kinase-dead mutant inhibited crocin-mediated HO-1 expression, Nrf2 activation, and phosphorylation of Akt, indicating that HO-1 expression is mediated by CAMK4 and that Akt is a downstream mediator of CAMK4 in crocin signaling. Moreover, crocin-mediated suppression of iNOS expression was blocked by CAMK4 inhibition. Overall, these results suggest that crocin suppresses LPS-stimulated expression of iNOS by inducing HO-1 expression via Ca(2+)/calmodulin-CAMK4-PI3K/Akt-Nrf2 signaling cascades. Our findings provide a novel molecular mechanism for the inhibitory effects of crocin against endotoxin-mediated inflammation.

Successful treatment of recurrent focal segmental glomerulosclerosis with a low dose rituximab in a kidney transplant recipient.

Recurrence of focal segmental glomerulosclerosis (FSGS) is a major therapeutic challenge in kidney transplantation (KT). Although intensive plasmapheresis and high-dose rituximab have been introduced to treat recurrent FSGS, the most effective dosage and regimen of rituximab have not been determined. Herein we reported the first case of successful treatment of recurrent FSGS with a low-dose rituximab. The patient showed marked proteinuria (3.5 g/d) and oliguria 2 d after KT. Two courses of plasmapheresis and immunoglobulin were applied to the patient, however, nephrotic range proteinuria persisted and creatinine level increased to 3.56 mg/dL. Five months post-transplant, the patient received injection with only one dose of rituximab 100 mg, without further plasmapheresis, which resulted in immediate reduction of serum creatinine and full remission of proteinuria during the following 18 months. This case suggested that recurrent FSGS, which frequently relapses after plasmapheresis, could be treated successfully with a low-dose rituximab even without plasmapheresis.

Predicting personality or prejudice? Facial inference in the age of artificial intelligence.

Facial inference, a cornerstone of person perception, has traditionally been studied through human judgments about personality traits and abilities based on people's faces. Recent advances in artificial intelligence (AI) have introduced new dimensions to this field, employing machine learning algorithms to reveal people's character, capabilities, and social outcomes based just on their faces. This review examines recent research on human and AI-based facial inference across psychology, business, computer science, legal, and policy studies to highlight the need for scientific consensus on whether or not people's faces can reveal their inner traits, and urges researchers to address the critical concerns around epistemic validity, practical relevance, and societal welfare before recommending AI-based facial inference for consequential uses.

Nationwide assessment of illicit drug consumption patterns in South Korea using wastewater-based epidemiology during the COVID-19 pandemic (2020-2022).

Illicit drugs have become a crucial global social issue, with South Korea experiencing a continuous increase in the number of offenders and drug smuggling. This study employed wastewater-based epidemiology to investigate consumption patterns of 8 illicit drugs and their 7 metabolites during the COVID-19 pandemic (2020-2022) in South Korea. Ten compouds were detected in the wastewater influent. Methamphetamine (METH) was prevalent in samples, followed by amphetamine and ecstasy (MDMA). Interestingly, MDMA and ketamine (KET), which were not detected in previous Korean studies conducted before COVID-19 pandemic, were detected in this study. METH exhibited the highest consumption rates, decreasing from 16.6 to 12.4 mg/day/1000 people between 2020 and 2022, while MDMA increased over the three years (mean: 1.16, 1.24, and 1.62 mg/day/1000 people in 2020, 2021, and 2022, respectively) (p < 0.05). Significant correlations were identified between regional income levels and the consumption rates of METH (p < 0.01), MDMA (p < 0.01), and KET (p < 0.05). Furthermore, METH and MDMA consumption rates in cities were positively correlated with the number of drug offenders arrested and local clubs in those cities. The findings of this study provide valuable insights into shaping regulatory policies related to illicit drugs and future studies.

Context-dependent roles for autophagy in myeloid cells in tumor progression.

Autophagy is known to suppress tumor initiation by removing genotoxic stresses in normal cells. Conversely, autophagy is also known to support tumor progression by alleviating metabolic stresses in neoplastic cells. Centered on this pro-tumor role of autophagy, there have been many clinical trials to treat cancers through systemic blocking of autophagy. Such systemic inhibition affects both tumor cells and non-tumor cells, and the consequence of blocked autophagy in non-tumor cells in the context of tumor microenvironment is relatively understudied. Here, we examined the effect of autophagy-deficient myeloid cells on the progression of autophagy-competent tumors. We found that blocking autophagy only in myeloid cells modulated tumor progression markedly but such effects were context dependent. In a tumor implantation model, the growth of implanted tumor cells was substantially reduced in mice with autophagy-deficient myeloid cells; T cells infiltrated deeper into the tumors and were responsible for the reduced growth of the implanted tumor cells. In an oncogene-driven tumor induction model, however, tumors grew faster and metastasized more in mice with autophagy-deficient myeloid cells. These data demonstrate that the autophagy status of myeloid cells plays a critical role in tumor progression, promoting or suppressing tumor growth depending on the context of tumor-myeloid cell interactions. This study indicates that systemic use of autophagy inhibitors in cancer therapy may have differential effects on rates of tumor progression in patients due to effects on myeloid cells and that this warrants more targeted use of selective autophagy inhibitors in a cancer therapy in a clinical setting.

A possible usage of a CDK4 inhibitor for breast cancer stem cell-targeted therapy.

Cancer stem cells (CSCs) are one of the main reasons behind cancer recurrence due to their resistance to conventional anti-cancer therapies. Thus, many efforts are being devoted to developing CSC-targeted therapies to overcome the resistance of CSCs to conventional anti-cancer therapies and decrease cancer recurrence. Differentiation therapy is one potential approach to achieve CSC-targeted therapies. This method involves inducing immature cancer cells with stem cell characteristics into more mature or differentiated cancer cells. In this study, we found that a CDK4 inhibitor sensitized MDA-MB-231 cells but not MCF7 cells to irradiation. This difference appeared to be associated with the relative percentage of CSC-population between the two breast cancer cells. The CDK4 inhibitor induced differentiation and reduced the cancer stem cell activity of MDA-MB-231 cells, which are shown by multiple marker or phenotypes of CSCs. Thus, these results suggest that radiosensitization effects may be caused by reducing the CSC-population of MDA-MB-231 through the use of the CDK4 inhibitor. Thus, further investigations into the possible application of the CDK4 inhibitor for CSC-targeted therapy should be performed to enhance the efficacy of radiotherapy for breast cancer.

Single step isolation and activation of primary CD3+ T lymphocytes using alcohol-dispersed electrospun magnetic nanofibers.

Electrospun polymer nanofibers with entrapped magnetic nanoparticles (magnetic NP-NF) represent a novel scaffold substrate that can be functionalized for single-step isolation and activation of specific lymphocyte subsets. Using a surface-embedded T cell receptor ligand/trigger (anti-CD3 monoclonal antibody), we demonstrate, as proof of principle, the use of magnetic NP-NF to specifically isolate, enrich, and activate CD3(+) T cells from a heterogeneous cell mixture, leading to preferential expansion of CD8(+)CD3(+) T cells. The large surface area, adjustable antibody density, and embedded paramagnetic properties of the NP-NF permitted enhanced activation and expansion; its use represents a strategy that is amenable to an efficient selection process for adoptive cellular therapy as well as for the isolation of other cellular subsets for downstream translational applications.

The Effects of a Tailored Dietary Education Program for Older Adult Patients on Hemodialysis: A Preliminary Study.

This study aimed to investigate the overall effects of a tailored dietary education program for older adult patients on hemodialysis (HD) based on self-efficacy theory, dietary knowledge and habits, nutritional intake, and biochemical parameters. A nonequivalent control group pre-test-post-test design was conducted for 8 weeks. The experimental and control groups received a weekly nutritional program and standard nursing care with an additional educational session, respectively. A clinical survey was conducted before and after the intervention. After the intervention, self-efficacy, dietary knowledge, and dietary habits were higher in the experimental group than in the control group. Moreover, carbohydrate, phosphorus, and sodium intake significantly decreased post-intervention in the experimental group but not in the control group. The dietary education program for older HD patients showed positive effects on boosting their self-efficacy, increasing dietary knowledge, improving dietary habits, and decreasing carbohydrate, calcium, phosphorus, and sodium intake.

KAS-Analyzer: a novel computational framework for exploring KAS-seq data.

Motivation: Kethoxal-assisted ssDNA sequencing (KAS-seq) is rapidly gaining popularity as a robust and effective approach to study the nascent dynamics of transcriptionally engaged RNA polymerases through profiling of genome-wide single-stranded DNA (ssDNA). Its latest variant, spKAS-seq, a strand-specific version of KAS-seq, has been developed to map genome-wide R-loop structures by detecting imbalances of ssDNA on two strands. However, user-friendly, open-source computational tools tailored for KAS-seq data are still lacking. Results: Here, we introduce KAS-Analyzer, the first comprehensive computational framework aimed at streamlining and enhancing the analysis and interpretation of KAS-seq and spKAS-seq data. In addition to standard analyses, KAS-Analyzer offers many novel tools specifically designed for KAS-seq data, including, but not limited to: calculation of transcription-related metrics, identification of single-stranded transcribing (SST) enhancers, high-resolution mapping of R-loops, and differential RNA polymerase activity analysis. We provided a detailed overview of KAS-seq data and its diverse applications through the implementation of KAS-Analyzer. Using the example time-course KAS-seq datasets, we further showcase the robust capabilities of KAS-Analyzer for investigating dynamic transcriptional regulatory programs in response to UVB radiation. Availability and implementation: KAS-Analyzer is available at https://github.com/Ruitulyu/KAS-Analyzer.

WNT7A suppresses adipogenesis of skeletal muscle mesenchymal stem cells and fatty infiltration through the alternative Wnt-Rho-YAP/TAZ signaling axis.

Intramuscular fatty infiltration in muscle injuries and diseases, caused by aberrant adipogenesis of fibro-adipogenic progenitors, negatively impacts function. Intramuscular delivery of wingless-type MMTV integration site family 7a (WNT7A) offers a promising strategy to stimulate muscle regeneration, but its effects on adipogenic conversion of fibro-adipogenic progenitors remain unknown. Here, we show that WNT7A decreases adipogenesis of fibro-adipogenic progenitors (FAPs) by inducing nuclear localization of Yes-associated protein (YAP) through Rho in a ß-CATENIN-independent manner and by promoting nuclear retention of YAP and transcriptional co-activator with PDZ-binding motif (TAZ) in differentiating FAPs. Furthermore, intramuscular injection of WNT7A in vivo effectively suppresses fatty infiltration in mice following glycerol-induced injury. Our results collectively suggest WNT7A as a potential protein-based therapeutic for diminishing adipogenesis of FAPs and intramuscular fatty infiltration in pathological muscle injuries or diseases.

Antitumor effects of NK cells expanded by activation pre­processing of autologous feeder cells before irradiation in colorectal cancer.

Natural killer (NK) cells play a crucial role in early immune defenses against transformed cells and are used in the therapeutic management of cancer. However, it is difficult to sufficiently obtain high purity activated NK cells for clinical application. The function of NK cells is dependent on the balance of activating and inhibitory signals. Strong and diverse stimuli are required to increase the function of NK cells. Radiotherapy modulates the expression of various immunomodulatory molecules that recruit and activate NK cells. NK cell-mediated antibody-dependent cellular cytotoxicity is one of the most potent cytotoxic effects of NK cells against target cancer cells. To generate activated and irradiated autologous peripheral blood mononuclear cells (PBMCs), cytokine and monoclonal antibody stimulation followed by ionizing radiation was performed in the present study. The expanded NK cells were cultured for 21 days using activated/irradiated autologous PBMCs. Colorectal cancer cells (SW480 and HT-29) were used to analyze the expression of NK group 2D ligands and EGFR by radiation. The cytotoxicity of radiation plus NK cell-based targeted therapy against colorectal cancer cell lines was analyzed using flow cytometry. Activated and irradiated PBMCs exhibited significantly increased expression of various activating ligands that stimulated NK cells. In total, >10,000-fold high-purity activated NK cells were obtained, with negligible T-cell contamination. To confirm the antitumor activity of the NK cells expanded by this method, the expanded NK cells were treated with cetuximab, radiotherapy, or a combination of cetuximab and radiotherapy in the presence of human colorectal cancer cells. Expanded NK cells were effective at targeting human colorectal cancer cells, particularly when combined with cetuximab and radiotherapy. Thus, in the present study, a novel method for high-purity activated NK cell expansion was developed using activated and irradiated PBMCs. In addition, combined radiotherapy and antibody-based immunotherapy with expanded NK cells may be an effective strategy to enhance the efficiency of treatment against colorectal cancer.

Combinatorial immunotherapy with gemcitabine and ex vivo-expanded NK cells induces anti-tumor effects in pancreatic cancer.

Pancreatic cancer is difficult to diagnose at the initial stage and is often discovered after metastasis to nearby organs. Gemcitabine is currently used as a standard treatment for pancreatic cancer. However, since chemotherapy for pancreatic cancer has not yet reached satisfactory therapeutic results, adjuvant chemotherapy methods are attempted. It can be expected that combining immune cell therapy with existing anticancer drug combination treatment will prevent cancer recurrence and increase survival rates. We isolated natural killer (NK) cells and co-cultured them with strongly activated autologous peripheral blood mononuclear cells (PBMCs) as feeder cells, activated using CD3 antibody, IFN-r, IL-2, and γ-radiation. NK cells expanded in this method showed greater cytotoxicity than resting NK cells, when co-cultured with pancreatic cancer cell lines. Tumor growth was effectively inhibited in a pancreatic cancer mouse xenograft model. Therapeutic efficacy was increased by using gemcitabine and erlotinib in combination. These findings suggest that NK cells cultured by the method proposed here have excellent anti-tumor activity. We demonstrate that activated NK cells can efficiently inhibit pancreatic tumors when used in combination with gemcitabine-based therapy.