Severe Combined Immune Deficiency Mice Research Articles

Therapeutic potential of urine-derived stem cells in renal regeneration following acute kidney injury: A comparative analysis with mesenchymal stem cells

BACKGROUND Acute kidney injury (AKI) is a common clinical syndrome with high morbidity and mortality rates. The use of pluripotent stem cells holds great promise for the treatment of AKI. Urine-derived stem cells (USCs) are a novel and versatile cell source in cell-based therapy and regenerative medicine that provide advantages of a noninvasive, simple, and low-cost approach and are induced with high multidifferentiation potential. Whether these cells could serve as a potential stem cell source for the treatment of AKI has not been determined. AIM To investigate whether USCs can serve as a potential stem cell source to improve renal function and histological structure after experimental AKI. METHODS Stem cell markers with multidifferentiation potential were isolated from human amniotic fluid. AKI severe combined immune deficiency (SCID) mice models were induced by means of an intramuscular injection with glycerol. USCs isolated from human-voided urine were administered via tail veins. The functional changes in the kidney were assessed by the levels of blood urea nitrogen and serum creatinine. The histologic changes were evaluated by hematoxylin and eosin staining and transferase dUTP nick-end labeling staining. Meanwhile, we compared the regenerative potential of USCs with bone marrow-derived mesenchymal stem cells (MSCs). RESULTS Treatment with USCs significantly alleviated histological destruction and functional decline. The renal function was rapidly restored after intravenous injection of 5 × 105 human USCs into SCID mice with glycerol-induced AKI compared with injection of saline. Results from secretion assays conducted in vitro demonstrated that both stem cell varieties released a wide array of cytokines and growth factors. This suggests that a mixture of various mediators closely interacts with their biochemical functions. Two types of stem cells showed enhanced tubular cell proliferation and decreased tubular cell apoptosis, although USC treatment was not more effective than MSC treatment. We found that USC therapy significantly improved renal function and histological damage, inhibited inflammation and apoptosis processes in the kidney, and promoted tubular epithelial proliferation. CONCLUSION Our study demonstrated the potential of USCs for the treatment of AKI, representing a new clinical therapeutic strategy.

Therapeutic interventions on human breast cancer xenografts promote systemic dissemination of oncogenes.

Metastatic dissemination following successful treatment of the primary tumour remains a common cause of death. There is mounting evidence that therapeutic interventions themselves may promote development of metastatic disease. We earlier reported that cell-free chromatin particles (cfChPs) released from dying cancer cells are potentially oncogenic. Based on this observation we hypothesized that therapeutic interventions may lead to the release of cfChPs from therapy induced dying cancer cells which could be carried via the blood stream to distant organs to transform healthy cells into new cancers that would masquerade as metastasis. To test this hypothesis, we generated xenografts of MDA-MB-231 human breast cancer cells in severe combined immune-deficient mice, and using immuno-fluorescence and FISH analysis looked for cfChPs in their brain cells. We detected multiple human DNA signals representing cfChPs in nuclei of brain cells of mice which co-localized with eight human onco-proteins. No intact MDA-MB-231 cells were detected. The number of co-localizing human DNA and human c-Myc signals increased dramatically following treatment with chemotherapy, localized radiotherapy or surgery, which could be prevented by concurrent treatment with three different cfChPs deactivating agents. These results suggest that therapeutic interventions lead to the release cfChPs from therapy induced dying cancer cells carrying oncogenes and are transported via the blood stream to brain cells to potentially transform them to generate new cancers that would appear as metastases. cfChPs induced metastatic spread of cancer is preventable by concurrent treatment with agents that deactivate cfChPs.

Effects of IFN-γ on the immunological microenvironment and TAM polarity in stage IA non-small cell lung cancer and its mechanisms

ObjectiveTo investigate the effect of interferon-γ (IFN-γ) on the immune microenvironment and the polarity of tumor-associated macrophages (TAMs) in stage IA non-small cell lung cancer (NSCLC) and its mechanisms.MethodsHuman non-small cell lung cancer A549 cells were treated with a series of IFN-γ concentrations (0, 50, 100, 150, 200, 250, and 300 ng/mL). Tumor tissues from patients with stage IA NSCLC were cultured using the air–liquid interface culture technique to establish a tumor microenvironment (TME) organ model. The NSCLC model was constructed by subcutaneously embedding small tumor pieces into the back of nonobese diabetic severe combined immune deficiency (NOD SCID) mice. The size and weight of the tumors were recorded, and the tumor volume was calculated. CCK-8 assays were used to investigate cell proliferation, flow cytometry and TUNEL staining were used to evaluate cell apoptosis, colony formation was investigated by cloning experiments, and cell invasion and migration were evaluated by Transwell assays and scratch tests. The expression of apoptosis-related proteins (Bax, Bcl-2 and C-caspase 3), M2 polarization-related markers (CD163, CD206 and IDO1), and marker proteins of cytotoxic T cells and helper T cells (CD8 and CD4) was detected by Western blot. The expression of Ki-67 and IDO1 was detected by immunohistochemistry, and the levels of IL-6, IL-10, IL-13 and TNF-α were measured by ELISA. The expression of CD68 was measured by RT‒qPCR, and the phagocytosis of TAMs was evaluated by a Cell Trace CFSE kit and cell probe staining.ResultsThe proliferation activity of A549 cells increased with increasing IFN-γ concentration and peaked when the concentration reached 200 ng/mL, and the proliferation activity of A549 cells was suppressed thereafter. After treatment with 200 ng/mL IFN-γ, the apoptosis rate of cells decreased, the number of cell colonies increased, the invasion and migration of cells were promoted, the expression of Bax and C-caspase 3 was downregulated, and the expression of Bcl-2 was upregulated in cells and the TME model. In the TME model, CD163, CD206, IDO1 and Ki-67 were upregulated, CD8 and CD4 were downregulated, apoptosis was reduced, the levels of IL-6 and TNF-α were decreased, and the levels of IL-10 and IL-13 were increased. IL-4 induced TAMs to express CD163 and CD206, reduced the levels of IL-6 and TNF-α, increased the levels of IL-10 and IL-13, and weakened the phagocytic function of TAMs. IFN-γ treatment further enhanced the effect of IL-4 and enhanced the viability of A549 cells. IDO1 decreased the viability of T cells and NK cells, while suppressing the effect of IFN-γ. In mice, compared with NSCLC mice, the tumor volume and weight of the IFN-γ group were increased, the expression of CD163, CD206, IDO1, Ki-67 and Bcl-2 in tumor tissue was upregulated, the expression of Bax and C-caspase 3 was downregulated, and apoptosis was reduced. The levels of IL-6 and TNF-α were decreased, and the levels of IL-10 and IL-13 were increased in the serum of mice.ConclusionIn stage IA NSCLC, a low concentration of IFN-γ promotes the polarization of TAMs to the M2 phenotype in the TME model by upregulating the expression of IDO1, promoting the viability of cancer cells, inhibiting the viability of T cells and NK cells, and thus establishing an immune microenvironment conducive to tumor progression.

Radioenhancement with the Combination of Docetaxel and Ultrasound Microbubbles: In Vivo Prostate Cancer.

Using an in vitro prostate cancer model, we previously demonstrated the significant enhancement of radiotherapy (XRT) with the combined treatment of docetaxel (Taxotere; TXT) and ultrasound-microbubbles (USMB). Here, we extend these findings to an in vivo cancer model. Severe combined immune-deficient male mice were xenografted with the PC-3 prostate cancer cell line in the hind leg and treated with USMB, TXT, radiotherapy (XRT), and their combinations. The tumors were imaged with ultrasound pre-treatment and 24 h post-treatment, following which they were extracted for the histological analysis of the tumor-cell death (DN; H&E) and apoptosis (DA; TUNEL). The tumors' growths were assessed for up to ~6 weeks and analysed using the exponential Malthusian tumor-growth model. The tumors' doubling time (VT) was characterized as growth (positive) or shrinkage (negative). The cellular death and apoptosis increased ~5-fold with the TXT + USMB + XRT (Dn = 83% and Da = 71%) compared to the XRT alone (Dn = 16% and Da = 14%), and by ~2-3-fold with the TXT + XRT (Dn = 50% and Da = 38%) and USMB + XRT (Dn = 45% and Da = 27%) compared to the XRT. The USMB enhanced the cellular bioeffects of the TXT by ~2-5-fold with the TXT + USMB (Dn = 42% and Da = 50%), compared with the TXT alone (Dn = 19% and Da = 9%). The USMB alone caused cell death (Dn = 17% and Da = 10%) compared to the untreated control (Dn = 0.4% and Da = 0%). The histological cellular bioeffects were correlated with the changes in the ultrasound RF mid-band-fit data, which were associated with the cellular morphology. The linear regression analysis displayed a positive linear correlation between the mid-band fit and the overall cell death (R2 = 0.9164), as well as a positive linear correlation between the mid-band fit and the apoptosis (R2 = 0.8530). These results demonstrate a correlation between the histological and spectral measurements of the tissue microstructure and that cellular morphological changes can be detected by ultrasound scattering analysis. In addition, the tumor volumes from the triple-combination treatment were significantly smaller than those from the control, XRT, USMB + XRT, and TXT + XRT, from day 2 onward. The TXT + USMB + XRT-treated tumors shrank from day 2 and at each subsequent time-point measured (VT ~-6 days). The growth of the XRT-treated tumors was inhibited during the first 16 days, following which the tumors grew (VT ~9 days). The TXT + XRT and USMB + XRT groups displayed an initial decrease in tumor size (day 1-14; TXT + XRT VT ~-12 days; USMB + XRT VT ~-33 days), followed by a growth phase (day 15-37; TXT + XRT VT ~11 days; USMB + XRT VT ~22 days). The triple-combination therapy induced tumor shrinkage to a greater extent than any of the other treatments. This study demonstrates the in vivo radioenhancement potential of chemotherapy combined with therapeutic ultrasound-microbubble treatment in inducing cell death and apoptosis, as well as long-term tumor shrinkage.

Gain-of-Function p53N236S Mutation Drives the Bypassing of HRasV12-Induced Cellular Senescence via PGC-1α.

One of the key steps in tumorigenic transformation is immortalization in which cells bypass cancer-initiating barriers such as senescence. Senescence can be triggered by either telomere erosion or oncogenic stress (oncogene-induced senescence, OIS) and undergo p53- or Rb-dependent cell cycle arrest. The tumor suppressor p53 is mutated in 50% of human cancers. In this study, we generated p53N236S (p53S) mutant knock-in mice and observed that p53S heterozygous mouse embryonic fibroblasts (p53S/+) escaped HRasV12-induced senescence after subculture in vitro and formed tumors after subcutaneous injection into severe combined immune deficiency (SCID) mice. We found that p53S increased the level and nuclear translocation of PGC-1α in late-stage p53S/++Ras cells (LS cells, which bypassed the OIS). The increase in PGC-1α promoted the biosynthesis and function of mitochondria in LS cells by inhibiting senescence-associated reactive oxygen species (ROS) and ROS-induced autophagy. In addition, p53S regulated the interaction between PGC-1α and PPARγ and promoted lipid synthesis, which may indicate an auxiliary pathway for facilitating cell escape from aging. Our results illuminate the mechanisms underlying p53S mutant-regulated senescence bypass and demonstrate the role played by PGC-1α in this process.

Rapamycin Inhibits Cytotoxic T Lymphocytes-Mediated Platelet Destruction in Immune Thrombocytopenia

Primary immune thrombocytopenia (ITP) is an autoimmune disorder characterized with increased risk of bleeding. CD8+ cytotoxic T lymphocytes (CTLs) of ITP patients express high level of lethal molecules, leading to the lysis and apoptosis of their own platelets, which is an important mechanism of platelet destruction in ITP patients. Accumulating evidence have highlighted that the activation of mTOR signaling pathway promotes the differentiation of CD8+ T cells to CTLs, which leads to the up-regulation of cell-mediated immunity. Several studies have showed that rapamycin, a mTOR inhibitor, was effective in the treatment of relapsed/refractory ITP. However, the endogenous mechanism remains to be explored. The activation of mTOR signaling pathway in CD8+ T cells was determined by flow cytometry. We found that the phosphorylation level of p-mTOR, p-Akt, p-4E-BP1 and p-P70S6K in ITP patients were significantly higher than that in healthy controls. Meanwhile, we found that the levels of perforin and granzyme B in CD8+ T cells in ITP patients were significantly higher than that in healthy controls. In the in vitro experiments, CTLs were isolated from PBMCs of ITP patients or healthy controls and cultured with different concentrations of rapamycin (0-1 uM) for 3 days, and we chose an optimal concentration of 20 nM. There was no significant difference in the expression levels of mTOR, Akt, 4E-BP1, and P70S6K after rapamycin treatment, but the phosphorylation level of p-mTOR, p-Akt, p-4E-BP1, p-P70S6K was significantly inhibited by rapamycin treatment. Likewise, the ratios of p-mTOR/mTOR, p-AKT/AKT, p-4E-BP1/4E-BP1, p-P70S6K/P70S6K were also significantly down-regulated. Flow cytometry analysis found that rapamycin significantly inhibited the expression of perforin and granzyme B (Figure 1) in CD8+ T cells of ITP patients. We further established the ITP murine model by transferring splenocytes of C57BL/6 CD61 knockout mice, immunized against platelets from wild-type syngeneic C57BL/6 mice, into severe combined immune deficient (SCID) mice. Rapamycin was injected intraperitoneally daily at a final concentration 2.0 mg/kg. Our data showed that the rapamycin group had significantly higher platelet counts compared with control group. In summary, our findings suggest that the activation of mTOR signaling pathway-related molecules of CD8+ T cells in patients with ITP are abnormal. These data shed new light on the molecular mechanism of rapamycin action by inhibiting CTLs cytotoxicity. This novel mechanism of rapamycin underlies the potential therapeutic strategy for ITP. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Adipose-Derived Stem Cell Extracellular Vesicles Improve Wound Closure and Angiogenesis in Diabetic Mice.

Currently, there is a lack in therapy that promotes the reepithelialization of diabetic wounds as an alternative to skin grafting. Here, the authors hypothesized that extracellular vesicles from adipose-derived stem cells (ADSC-EVs) could accelerate wound closure through rescuing the function of keratinocytes in diabetic mice. The effect of ADSC-EVs on the biological function of human keratinocyte cells was assayed in vitro. In vivo, 81 male severe combined immune deficiency mice aged 8 weeks were divided randomly into the extracellular vesicle-treated diabetes group (n = 27), the phosphate-buffered saline-treated diabetes group (n = 27), and the phosphate-buffered saline-treated normal group (n = 27). A round, 8-mm-diameter, full-skin defect was performed on the back skin of each mouse. The wound closure kinetics, average healing time, reepithelialization rate, and neovascularization were evaluated by histological staining. In vitro, ADSC-EVs improved proliferation, migration, and proangiogenic potential, and inhibited the apoptosis of human keratinocyte cells by suppressing Fasl expression with the optimal dose of 40 μg/mL. In vivo, postoperative dripping of ADSC-EVs at the dose of 40 μg/mL accelerated diabetic wound healing, with a 15.8% increase in closure rate and a 3.3-day decrease in average healing time. ADSC-EVs improved reepithelialization (18.2%) with enhanced epithelial proliferation and filaggrin expression, and suppressed epithelial apoptosis and Fasl expression. A 2.7-fold increase in the number of CD31-positive cells was also observed. ADSC-EVs improve diabetic wound closure and angiogenesis by enhancing keratinocyte-mediated reepithelialization and vascularization. ADSC-EVs could be developed as a regenerative medicine for diabetic wound care.

EID3 Promotes Glioma Cell Proliferation and Survival by Inactivating AMPKα1.

EID3 (EP300-interacting inhibitor of differentiation) was identified as a novel member of EID family and plays a pivotal role in colorectal cancer development. However, its role in glioma remained elusive. In current study, we identified EID3 as a novel oncogenic molecule in human glioma and is critical for glioma cell survival, proliferation and invasion. A total of five patients with glioma were recruited in present study and fresh glioma samples were removed from patients. Four weeks old male non-obese diabetic severe combined immune deficiency (NOD/SCID) mice were used as transplant recipient models. The subcutaneous tumor size was calculated and recorded every week with vernier caliper. EID3 and AMP-activated protein kinase α1 (AMPKα1) expression levels were confirmed by real-time polymerase chain reaction and Western blot assays. Colony formation assays were performed to evaluate cell proliferation. Methyl thiazolyl tetrazolium (MTT) assays were performed for cell viability assessment. Trypan blue staining approach was applied for cell death assessment. Cell Apoptosis DNA ELISA Detection Kit was used for apoptosis assessment. EID3 was preferentially expressed in glioma tissues/cells, while undetectable in astrocytes, neuronal cells, or normal brain tissues. EID3 knocking down significantly hindered glioma cell proliferation and invasion, as well as induced reduction of cell viability, apoptosis and cell death. EID3 knocking down also greatly inhibited tumor growth in SCID mice. Knocking down of AMPKα1 could effectively rescue glioma cells from apoptosis and cell death caused by EID3 absence, indicating that AMPKα1 acted as a key downstream regulator of EID3 and mediated suppression effects caused by EID3 knocking down inhibition. These findings were confirmed in glioma cells generated patient-derived xenograft models. AMPKα1 protein levels were affected by MG132 treatment in glioma, which suggested EID3 might down regulate AMPKα1 through protein degradation. Collectively, our study demonstrated that EID3 promoted glioma cell proliferation and survival by inhibiting AMPKα1 expression. Targeting EID3 might represent a promising strategy for treating glioma.

Glucose restriction induces ROS-AMPK-mediated CTR1 expression and increases cisplatin efficiency in NSCLC

Cisplatin is one of the principal platinum-based chemotherapeutic agents for many types of cancer, including non-small-cell lung cancer (NSCLC). Copper transporter 1 (CTR1) plays a significant role in increasing cellular cisplatin uptake and sensitivity. The current study found that glucose restriction upregulated AMPK (AMP-activated protein kinase) through reactive oxygen species (ROS) to induce CTR1 expression in NSCLC cells. Direct upregulation of ROS levels also activated AMPK expression. The changes in CTR1 expression were consistent with glucose concentrations and AMPK expression. Feeding a low-carbohydrate ketogenic diet (a glucose restriction diet) to a severe combined immune deficiency (SCID) mouse xenograft model significantly enhanced the efficacy of cisplatin. The tumor size was significantly smaller in the group treated with cisplatin plus the low-carbohydrate ketogenic diet than in the group treated with cisplatin alone. Survival was longer in mice treated with the low-carbohydrate ketogenic diet than in the controls. Mice fed the low-carbohydrate ketogenic diet showed increased expression of CTR1 and AMPK in tumor tissues. These results suggest a novel mechanism whereby glucose restriction induces ROS-AMPK-mediated CTR1 expression in NSCLC, indicating glucose restriction as an effective adjuvant NSCLC therapy.

A general toxicity and biodistribution study of human natural killer cells by single or repeated intravenous dose in severe combined immune deficient mice

Natural killer (NK) cells are a part of the innate immune system and represent the first line of defense against infections and tumors. NK cells can eliminate tumor cells without major histocompatibility restriction and are independent of the expression of tumor-associated antigens. Therefore, they are considered an emerging tool for cancer immunotherapy. However, the general toxicity and biodistribution of NK cells after transplantation remain to be understood. This study was conducted to evaluate the general toxicity and biodistribution of human NK cells after single or repeated intravenous dosing in severely combined immunodeficient (SCID) mice. There were no test item-related toxicological changes in single and repeated administration groups. The no observed adverse effect level of human NK cells was 2 × 107 cells/head for both male and female SCID mice. Results from the biodistribution study showed that human NK cells were mainly distributed in the lungs, and a small number of the cells were detected in the liver, heart, spleen, and kidney of SCID mice, in both the single and repeated dose groups. Additionally, human NK cells were completely eliminated from all organs of the mice in the single dose group on day 7, while the cells persisted in mice in the repeated dose group until day 64. In conclusion, transplantation of human NK cells in SCID mice had no toxic effects. The cells were mainly distributed in the lungs and completely disappeared from the body over time after single or repeated intravenous administration.

Coxiella burnetii Plasmid Effector B Promotes LC3-II Accumulation and Contributes To Bacterial Virulence in a SCID Mouse Model.

Coxiella burnetii, the causative agent of zoonotic Q fever, is characterized by replicating inside the lysosome-derived Coxiella-containing vacuole (CCV) in host cells. Some effector proteins secreted by C. burnetii have been reported to be involved in the manipulation of autophagy to facilitate the development of CCVs and bacterial replication. Here, we found that the Coxiella plasmid effector B (CpeB) localizes on vacuole membrane targeted by LC3 and LAMP1 and promotes LC3-II accumulation. Meanwhile, the C. burnetii strain lacking the QpH1 plasmid induced less LC3-II accumulation, which was accompanied by smaller CCVs and lower bacterial loads in THP-1 cells. Expression of CpeB in the strain lacking QpH1 led to restoration in LC3-II accumulation but had no effect on the smaller CCV phenotype. In the severe combined immune deficiency (SCID) mouse model, infections with the strain expressing CpeB led to significantly higher bacterial burdens in the spleen and liver than its parent strain devoid of QpH1. We also found that CpeB targets Rab11a to promote LC3-II accumulation. Intratracheally inoculated C. burnetii resulted in lower bacterial burdens and milder lung lesions in Rab11a conditional knockout (Rab11a-/- CKO) mice. Collectively, these results suggest that CpeB promotes C. burnetii virulence by inducing LC3-II accumulation via a pathway involving Rab11a.

Pre-Clinical Proof of Concept: Intra-Carotid Injection of Autologous CD34-Positive Cells for Chronic Ischemic Stroke.

This study was conducted to evaluate the safety and efficacy of human peripheral blood CD34 positive (CD34+) cells transplanted into a murine chronic stroke model to obtain pre-clinical proof of concept, prior to clinical testing. Granulocyte colony stimulating factor (G-CSF) mobilized human CD34+ cells [1 × 104 cells in 50 μl phosphate-buffered saline (PBS)] were intravenously (iv) or intra-carotid arterially (ia) transplanted 4 weeks after the induction of stroke (chronic stage), and neurological function was evaluated. In this study, severe combined immune deficiency (SCID) mice were used to prevent excessive immune response after cell therapy. Two weeks post cell therapy, the ia CD34+ cells group demonstrated a significant improvement in neurological functions compared to the PBS control. The therapeutic effect was maintained 8 weeks after the treatment. Even after a single administration, ia transplantation of CD34+ cells had a significant therapeutic effect on chronic stroke. Based on the result of this pre-clinical proof of concept study, a future clinical trial of autologous peripheral blood CD34+ cells administration in the intra-carotid artery for chronic stroke patients is planned.

Molecular Mechanism of Malignant Transformation of Balb/c-3T3 Cells Induced by Long-Term Exposure to 1800 MHz Radiofrequency Electromagnetic Radiation (RF-EMR).

Purpose: We aimed to investigate RF-EMR-induced cell malignant transformation. Methods: We divided Balb/c-3T3 cells into sham and expo groups. The expo groups were exposed to a 1800 MHz RF continuous wave for 40 and 60 days, for 4 h per day. The sham group was sham-exposed. Cells were harvested for a cell transformation assay, transplantation in severe combined immune deficient (SCID) mice, soft agar clone formation detection, and a transwell assay. The mRNA microarray assay was used to declare key genes and pathways. Results: The exposed Balb/c-3T3 cells showed a strong increase in cell proliferation and migration. Malignant transformation was observed in expo Balb/c-3T3 cells exposed for 40 days and 60 days, which was symbolized with visible foci and clone formation. Expo Balb/c-3T3 cells that were exposed for 40 days and 60 days produced visible tumors in the SCID mice. Lipid metabolism was the key biological process and pathway involved. The mevalonate (MVA) pathway was the key metabolic pathway. The interacted miRNAs could be further research targets to examine the molecular mechanism of the carcinogenic effects of long-term exposure. Conclusion: Exposure for 40 and 60 days to 1800 MHz RF-EMR induced malignant transformation in Balb/c-3T3 cells at the SAR of 8.0 W/kg. We declared that lipid metabolism was the pivotal biological process and pathway. The MVA pathway was the key metabolic pathway.

Adaptive Immune Deficiency Impairs Neural Activity After Training and Retrieval.

Neuroimmune interactions have been studied for decades. Several neurodevelopmental disorders have been associated with immune dysfunction. However, the effects of immune system on neuronal function remain unknown. Herein, based on c-Fos protein expression, we characterized the brain areas that are activated after contextual fear conditioning (CFC) training or retrieval in severe combined immune deficiency (SCID) and wild-type mice. Further, we analyzed the interregional correlations of c-Fos activity that are affected by deficiency in adaptive immunity. Results showed significantly lower c-Fos density in learning and memory-associated brain regions of SCID mice after memory retrieval, but not during the CFC training. Moreover, SCID mice exhibited remarkably discordant interregional neuronal activities of learning neuron circuits after CFC training, which could be the cause of inefficient activation of the memory circuit after retrieval. These results provide a new perspective on how adaptive immunity affects neuronal function. Adaptive immune deficiency impairs the coordination of neural activity after training and retrieval, which might be a potential therapeutic target for neurodevelopmental disorders.

Low-dose decitabine modulates myeloid-derived suppressor cell function and restores immune tolerance in immune thrombocytopenia

Low-dose decitabine modulates myeloid-derived suppressor cell function and restores immune tolerance in immune thrombocytopeniaPrimary immune thrombocytopenia (ITP) is an autoimmune disorder characterized with increased risk of bleeding. Myeloid-derived suppressor cells (MDSCs) are heterogeneous immature cells and natural inhibitors of adaptive immunity. Metabolic changes within MDSCs elucidate a direct influence on immunologic consequences of their suppressive activity. Liver kinase B1 (LKB1) is a tumor suppressor gene of STK11/LKB1 coding serine/Sue, and LKB1 signaling pathway plays an important role as a “bridge” between metabolic balance and functional homeostasis of immune cells. Our previous studies demonstrated that low dose decitabine, a hypomethylating agent, significantly increased the number of mature polyploidy megakaryocytes and exhibited long-term clinical efficacy. Besides, it also increased the production of Treg and enhanced their immunosuppressive function in ITP. However, whether decitabine could regulate the metabolic and suppressive activity of MDSCs in ITP is unknown.The percentage of MDSCs in peripheral blood mononuclear cells (PBMCs) was determined by flow cytometry, which was shown to be significantly lower in ITP compared with that in healthy controls. We then investigated the effect of low-dose decitabine in patients with active ITP, where decitabine induced a significant expansion of MDSCs in line with an impressive platelet response.In the in vitro experiments, MDSCs were isolated from PBMCs of ITP patients or healthy controls and cultured with different concentrations of decitabine (0/10nM/50nM/100 nM/1uM/10μM) for 7 days. A concentration gradient from 50nM to 1uM stimulated MDSCs amplification in a dose-dependent manner, and we chose an optimal concentration of 100 nM. Moreover, we found the mRNA expression level of LKB1, AMPKα1, AMPKα2, AMPKβ1, AMPKβ2, AMPKγ1, and AMPKγ2 was significantly lower in ITP patients than that in healthy control subjects. After incubation with decitabine (100nM), the relative expression of the above molecules were significantly increased compared to untreated levels.We also analyzed oxygen consumption rate (OCR) and key parameters of mitochondrial function within MDSCs. Overall, the OCR curve of ITP patients was lower than that of the healthy control subjects, and the OCR curve of ITP patients significantly improved after treatment with decitabine. We sorted the cultured MDSCs and co-cultured them with CFSE-labeled CD4 +CD25 - T cells to evaluate the suppressive activity of MDSCs. Results indicated that the inhibitory function of decitabine-modulated MDSCs was corrected in line with metabolic rewriting.We further established the ITP murine model by transferring splenocytes of C57BL/6 CD61 knockout mice, immunized against platelets from wild-type syngeneic C57BL/6 mice, into severe combined immune deficient (SCID) mice. MDSCs were sorted from the bone marrow of wild-type mice and incubated with PBS or decitabine, respectively. SCID mice were divided into three groups and received the same numbers of splenocyte transfer, two groups were given additional transfer of PBS-treated or decitabine-treated MDSCs. Our data showed that the decitaine-treated MDSCs group had significantly higher platelet counts compared with control group and PBS-treated MDSCs group.In summary, our findings suggest that the immune function and metabolic characteristics of MDSCs in ITP patients are impaired. These data shed new light on the molecular mechanism of decitabine action by regulating immune function and aerobic metabolism via LKB1, which supervises the immunosuppressive functions of MDSCs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

GABAergic neuronal IL-4R mediates T cell effect on memory

Mechanisms governing how immune cells and their derived molecules impact homeostatic brain function are still poorly understood. Here, we elucidate neuronal mechanisms underlying Tcell effects on synaptic function and episodic memory. Depletion of CD4 Tcells led to memory deficits and impaired long-term potentiation. Severe combined immune-deficient mice exhibited amnesia, which was reversible by repopulation with Tcells from wild-type but not from IL-4-knockout mice. Behaviors impacted by Tcells were mediated via IL-4 receptors expressed on neurons. Exploration of snRNA-seq of neurons participating in memory processing provided insights into synaptic organization and plasticity-associated pathways regulated by immune cells. IL-4Rα knockout in inhibitory (but not in excitatory) neurons was sufficient to impair contextual fear memory, and snRNA-seq from these mice pointed to IL-4-driven regulation of synaptic function in promoting memory. These findings provide new insights into complex neuroimmune interactions at the transcriptional and functional levels in neurons under physiological conditions.

A recombinant selective drug-resistant M. bovis BCG enhances the bactericidal activity of a second-line anti-tuberculosis regimen

Drug-resistant tuberculosis (DR-TB) poses a new threat to global health; to improve the treatment outcome, therapeutic vaccines are considered the best chemotherapy adjuvants. Unfortunately, there is no therapeutic vaccine approved against DR-TB. Our study assessed the therapeutic efficacy of a recombinant drug-resistant BCG (RdrBCG) vaccine in DR-TB. We constructed the RdrBCG overexpressing Ag85B and Rv2628 by selecting drug-resistant BCG strains and transformed them with plasmid pEBCG or pIBCG to create RdrBCG-E and RdrBCG-I respectively. Following successful stability testing, we tested the vaccine’s safety in severe combined immune deficient (SCID) mice that lack both T and B lymphocytes plus immunoglobulins. Finally, we evaluated the RdrBCG’s therapeutic efficacy in BALB/c mice infected with rifampin-resistant M. tuberculosis and treated with a second-line anti-TB regimen. We obtained M. bovis strains which were resistant to several second-line drugs and M. tuberculosis resistant to rifampin. Notably, the exogenously inserted genes were lost in RdrBCG-E but remained stable in the RdrBCG-I both in vitro and in vivo. When administered adjunct to a second-line anti-TB regimen in a murine model of DR-TB, the RdrBCG-I lowered lung M. tuberculosis burden by 1 log10. Furthermore, vaccination with RdrBCG-I adjunct to chemotherapy minimized lung tissue pathology in mice. Most importantly, the RdrBCG-I showed almost the same virulence as its parent BCG Tice strain in SCID mice. Our findings suggested that the RdrBCG-I was stable, safe and effective as a therapeutic vaccine. Hence, the “recombinant” plus “drug-resistant” BCG strategy could be a useful concept for developing therapeutic vaccines against DR-TB.

The requirement of mitochondrial RNA polymerase for non-small cell lung cancer cell growth

POLRMT (RNA polymerase mitochondrial) is responsible for the transcription of mitochondrial genome encoding key components of oxidative phosphorylation. This process is important for cancer cell growth. The current study tested expression and potential functions of POLRMT in non-small cell lung cancer (NSCLC). TCGA cohorts and the results from the local lung cancer tissues showed that POLRMT is overexpressed in human lung cancer tissues. In both primary human NSCLC cells and A549 cells, POLRMT silencing (by targeted lentiviral shRNAs) or knockout (through CRSIPR/Cas9 gene editing method) potently inhibited cell viability, proliferation, migration, and invasion, and induced apoptosis activation. On the contrast, ectopic overexpression of POLRMT using a lentiviral construct accelerated cell proliferation and migration in NSCLC cells. The mtDNA contents, mRNA levels of mitochondrial transcripts, and subunits of respiratory chain complexes, as well as S6 phosphorylation, were decreased in POLRMT-silenced or -knockout NSCLC cells, but increased after ectopic POLRMT overexpression. In vivo, intratumoral injection of POLRMT shRNA adeno-associated virus (AAV) potently inhibited NSCLC xenograft growth in severe combined immune deficiency mice. The mtDNA contents, mRNA levels of mitochondria respiratory chain complex subunits, and S6 phosphorylation were decreased in POLRMT shRNA AAV-injected NSCLC xenograft tissues. These results show that POLRMT is a novel and important oncogene required for NSCLC cell growth in vitro and in vivo.

Anti-Myeloma Effects of the Selective JAK1 Inhibitor INCB052793 Using a Platform Study Utilizing the Lagκ-1A Multiple Myeloma Xenograft Model

Introduction: We have previously shown that severe combined immune deficiency (SCID)mice bearing the human multiple myeloma (MM) LAGk-1A xenograft develop significantly smaller tumors when treated with the JAK1 inhibitor INCB052793 (INCB; Incyte, Wilmington, DE) alone. Additionally, mice receiving the doublets of this agent and dexamethasone (DEX), lenalidomide (LEN) or pomalidomide (POM) demonstrated enhanced anti-MM effects on LAGk-1A growth in vivo that was superior to the doublets of DEX with LEN or POM. Mice receiving the triple combination of INCB + DEX with LEN or POM demonstrated the most pronounced anti-MM effect on tumor growth in vivo compared to all other combinations tested. Herein, we evaluated the response and toxicity of animals treated in combination or sequentially with INCB as well as with LEN plus DEX. For example, is there enhanced anti-MM activity when INCB + LEN are administered first, and DEX is added after disease progression? Or is there enhanced anti-MM activity when INCB + LEN is administered first and then substituted with DEX? This same treatment strategy was evaluated with INCB + DEX administered first, followed by the various permutations of LEN. Additionally, given that in the clinical setting DEX plus LEN is commonly administered to patients with MM, we evaluated these combinations with the addition of INCB or substitution of one of these drugs with INCB. Similarly, we evaluated the anti-MM effects of mice progressing from DEX therapy and added in INCB, substituted DEX with INCB, continued DEX and added in INCB + LEN.Materials and Methods: For the in vivo studies, each SCID mouse was implanted into the hind limb with a piece of the human MM tumor LAGk-1A. All animal studies were approved by the Institutional Animal Care and Use Committee. Fourteen days post-implantation mice were randomized into various treatment groups, and tumor size was measured on a weekly basis following treatment with various drugs following progression from their initial treatment regimen. Progression is defined as an increase in human B-cell maturation antigen (BCMA) equal to or above 25% when compared to the prior assessment (plasma BCMA levels were assessed on a weekly basis). After the mouse meets the criteria for disease progression, the mouse was placed into their respective treatment group.Results: When each individual mouse progressed, their dosing regimen was changed according to the treatment group to which they were randomized. We observed pronounced anti-MM effects when DEX was administered, followed by additions, or substitutions, of INCB and/or LEN. No significant anti-MM effects were observed when comparing the vehicle control group to mice treated in the LEN, INCB or DEX + LEN cohorts. However, mice treated with the doublets INCB + DEX or INCB + LEN, or triplet of INCB + LEN + DEX showed smaller tumors compared to the vehicle control group, (P= 0.0420; P= 0.0388; P= 0.0079, respectively). Furthermore, mice treated with the triplet, INCB + LEN + DEX, had markedly smaller tumors when compared to those treated with LEN (P= 0.0005), DEX + LEN(P= 0.0244), or INCB + DEX(P= 0.0357).Conclusions: These in vivo studies should provide important data in support of the optimal clinical sequencing of INCB, as they clearly show that the sequencing of drug combinations involving the novel JAK1 inhibitor INCB052793 + anti-MM agents have a profound impact on the anti-MM effects of this new treatment regimen, and provide further support for the clinical evaluation of these drug combinations to treat relapsed/refractory MM patients. DisclosuresBerenson:Incyte: Consultancy, Research Funding; OncoTracker, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

CD74 Is Expressed in Relapsed and Refractory Multiple Myeloma and Can be Targeted with a Novel Anti-CD74 Antibody Drug Conjugate, SΤRO-001

BACKGROUNDCD74 is a transmembrane glycoprotein involved in MHC II protein formation and transport. STRO-001 is a novel antibody drug conjugate (ADC) comprised of a human aglycosylated anti-CD74 IgG1 antibody (SP7219) genetically incorporating the non-natural amino acid para-azidomethy-L-phenyalanine (pAMF) to enable the site-specific conjugation of a non-cleavable maytansinoid linker-warhead. Highly efficient and precise site-specific conjugation enabled by Sutro's cell-free antibody synthesis technology produced a well-defined homogeneous ADC with a drug-antibody ratio (DAR) of 2. Conjugation sites were selected based on the highest in vitro and in vivo stability. METHODSBiotinylated SP7219 was used for immunohistochemistry (IHC). Bone marrow (BM) biopsy samples from patients with multiple myeloma (MM) from the UCSF Dept. of Laboratory Medicine were used after appropriate permissions were obtained. CD74 expression in plasma cells was measured as follows: 0, no staining; 1+, weak staining; 2+, moderate staining; 3+, strong staining (Figure 1A). Plasma cells were identified based on morphology and paired CD138+ staining. Two pathologists blinded to the patient's clinical history and pathology results independently reviewed IHC specimens. SP7219 conjugated to a fluorescent dye (DBCO-Alexa647) was used for detection and quantitation of CD74 expression on MM cell lines. STRO-001 was used to determine the EC50 and the proportion of MM cells killed (span killing) in an in vitro viability assay. The anti-tumor activity of STRO-001 was evaluated in the disseminated ARP-1 and MM.1S MM models. In vivo bioluminescence imaging (BLI) for animals bearing luciferase-expressing MM.1s (MM.1s-luc) cells was performed using an IVIS Spectrum. BLI images were collected 7, 14, 21, and 28 days post-tumor cell inoculation. RESULTSOverall, CD74 expression levels were not significantly different between newly diagnosed MM (n=13) and relapsed or refractory MM (n=24) (Figure 1B). Of 37 total samples analyzed, only 1 sample lacked detectable CD74 and the majority (67%) of relapsed/refractory patients expressed CD74 levels higher than 1+ by IHC (Figure 1C). In vitro cytotoxicity assays show nanomolar potency of STRO-001 in five MM cell lines: MC/CAR (EC50 0.8 nM), ARD (EC50 ~7.0 nM), MM.1S (EC50 10-11 nM), U266B1 (EC50 8.5-9.3 nM), and ARP-1 (EC50 4.3-22 nM). CD74 cell surface expression is required for STRO-001 cytotoxic activity but expression level, measured by antibody-binding capacity, does not correlate strongly with in vitro potency (R2=0.5837 for MM cell lines). STRO-001 inhibits the growth of CD138+ plasma cells in BM and formation of visceral tumors (p=0.002 for kidney; p<0.0001 for ovary) after 4 weekly doses of 3 mg/kg in the ARP-1 disseminated MM xenograft model in severe combined immune deficient (SCID) mice. STRO-001 dosed at 3 mg/kg and 10 mg/kg weekly x 3 also eradicates malignant BM plasma cells by day 32 post-inoculation (p<0.0001) and prolongs survival in the MM.1S disseminated model in NOD SCID gamma (NSG) mice. At termination of the study, 129 days post-inoculation, 100% of the STRO-001 treated animals survived and showed no evidence of disease with no CD138+ cells in their BM, while mean survival of control animals was 35 days with almost 50% of their BM containing myeloma cells. Additionally, BLI enabled noninvasive quantitation of tumor burden in MM.1S-luc-bearing NSG mice. Single doses of 1, 3, and 10 mg/kg STRO-001 (administered on day 7 post-inoculation) resulted in eradication of myeloma by day 28 based on BLI (Figure 2A and 2B) and quantification of CD138+ cells in BM (p<.0001) (Figure 2C). CONCLUSIONCD74 expression was detected in most MM samples examined in this study. Expression is heterogeneous and observed in treatment-naïve and heavily pre-treated patients. STRO-001 demonstrates potent in vitro cytotoxicity in MM cell lines and reduces tumor burden in MM xenograft models, including prolongation of survival in the MM.1S model. Based on these encouraging observations, STRO-001 is advancing to the clinic for the treatment of myeloma and other B-cell malignancies. [Display omitted] DisclosuresEmbry:Sutro Biopharma: Employment. Li:Sutro Biopharma: Employment. Yu:Sutro Biopharma: Employment. Abrahams:Sutro Biopharma: Employment. DeAlmeida:Sutro Biopharma: Employment. Krimm:Sutro Biopharma: Employment. Matheny:Sutro Biopharma: Employment. Kline:Sutro Biopharma: Employment. Yam:Sutro Biopharma: Employment. Stafford:Sutro Biopharma: Employment. Wiita:Sutro Biopharma: Research Funding; TeneoBio, Inc.: Research Funding. Hallam:Sutro Biopharma: Employment. Lupher:Sutro Biopharma: Employment. Molina:Sutro Biopharma: Employment.