Associations of granulocyte colony-stimulating factor with toxicities and efficacy of chimeric antigen receptor T-cell therapy in relapsed or refractory B-cell acute lymphoblastic leukemia.

Few studies have reported the associations of granulocyte colony-stimulating factor (G-CSF) with cytokine release syndrome (CRS), neurotoxic events (NEs) and efficacy after chimeric antigen receptor (CAR) T-cell therapy for relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). We present a retrospective study of 67 patients with R/R B-ALL who received anti-CD19 CAR T-cell therapy, 41 (61.2%) patients received G-CSF (G-CSF group), while 26 (38.8%) did not (non-G-CSF group). Patients had similar duration of grade 3-4 neutropenia between the two groups. The incidences of CRS and NEs were higher in G-CSF group, while no differences in severity were found. Further stratified analysis showed that the incidence and severity of CRS were not associated with G-CSF administration in patients with low bone marrow (BM) tumor burden. None of the patients with low BM tumor burden developed NEs. However, there was a significant increase in the incidence of CRS after G-CSF administration in patients with high BM tumor burden. The duration of CRS in patients who used G-CSF was longer. There were no significant differences in response rates at 1 and 3 months after CAR T-cell infusion, as well as overall survival (OS) between the two groups. In conclusion, our results showed that G-CSF administration was not associated with the incidence or severity of CRS in patients with low BM tumor burden, but the incidence of CRS was higher after G-CSF administration in patients with high BM tumor burden. The duration of CRS was prolonged in G-CSF group. G-CSF administration was not associated with the efficacy of CAR T-cell therapy.

A Factor-Free Hydrogel with ROS Scavenging and Responsive Degradation for Enhanced Diabetic Bone Healing.

In view of the increased levels of reactive oxygen species (ROS) that disturb the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), the repair of diabetic bone defects remains a great challenge. Herein, a factor-free hydrogel is reported with ROS scavenging and responsive degradation properties for enhanced diabetic bone healing. These hydrogels contain ROS-cleavable thioketal (TK) linkers and ultraviolet (UV)-responsive norbornene (NB) groups conjugated with 8-arm PEG macromers, which are formed via UV crosslinking-mediated gelation. Upon reacting with high levels of ROS in the bone defect microenvironment, ROS-cleavable TK linkers are destroyed, allowing the responsive degradation of hydrogels, which promotes the migration of BMSCs. Moreover, ROS levels are reduced through hydrogel-mediated ROS scavenging to reverse BMSC differentiation from adipogenic to osteogenic phenotype. As such, a favorable microenvironment is created after simultaneous ROS scavenging and hydrogel degradation, leading to the effective repair of bone defects in diabetic mouse models, even without the addition of growth factors. Thus, this study presents a responsive hydrogel platform that regulates ROS scavenging and stromal degradation in bone engineering.

Drinking in despair: Unintended consequences of automation in China.

The side effects of technological progress on the economy have been discussed frequently, but little is known regarding its health consequences. By combining the national individual-level panel data of alcohol drinking with the prefecture-level robot exposure rate in China, we find that one more robot exposure rate could induce up to 2.2% points increase in the probability of problem drinking. Such a pattern of problem drinking is explained by negative emotions, which can be ascribed to job loss due to substitution, higher income vulnerability, and reduced organization participation. Further, we provide evidence that automation can incur health costs, particularly for easily substituted workers, which would exacerbate health inequality in China. This paper sheds light on the impact of automation and the social incentives of problem drinking, emphasizing the possibly heterogeneous health cost accompanied by the automation process.

Survival strategies and assembly mechanisms of microbial communities in petroleum-contaminated soils.

This study analyzed petroleum-contaminated soils from south and north locations in China to explore the structure, diversity, functional genes and assembly processes of microbial communities' . Compared with soils from south locations, soils from northern regions exhibited elevated pH, total nitrogen (TN), and total petroleum hydrocarbon (TPH) levels. Among these, TN and TPH were the most influential on the microbial community. The dominant phyla for bacteria, archaea, and fungi were Proteobacteria, Thaumarchaeota, and Ascomycota, respectively. Among them, Proteobacteria was strongly correlated with various functional genes including alkB and many aromatics degradation and denitrification genes (r > 0.9, p < 0.01), suggesting that Proteobacteria play an important role in petroleum-contaminated soils. Metabolism in northern regions was more active than that in southern regions. The northern regions showed a pronounced tendency for denitrification, while the southern regions were characterized by acetoclastic methanogenesis. The assembly of microbial communities exhibited regional patterns, the deterministic assembly was more prominent in the northern soils, while the stochastic assembly was evident in the southern soils. Overall, these findings provide a new conceptual framework to understand the biosphere in petroleum-contaminated soil, potentially guiding improved management practices in the environmental remediation.

Shaping Sulfur Precursors to Low Dimensional (0D, 1D and 2D) Sulfur Nanomaterials: Synthesis, Characterization, Mechanism, Functionalization, and Applications.

Low-dimensional sulfur nanomaterials featuring with 0D sulfur nanoparticles (SNPs), sulfur nanodots (SNDs) and sulfur quantum dots (SQDs), 1D sulfur nanorods (SNRs), and 2D sulfur nanosheets (SNSs) have emerged as an environmentally friendly, biocompatible class of metal-free nanomaterials, sparking extensive interest in a wide range application. In this review, various synthetic methods, precise characterization, creative formation mechanism, delicate functionalization, and versatile applications of low dimensional sulfur nanomaterials over the last decades are systematically summarized. Initially, it is striven to summarize the progress of low dimensional sulfur nanomaterials from versatile precursors by using different synthetic approaches and various characterization. Then, a multi-faceted proposed formation mechanism with emphasis on how these different precursors produce corresponding SNPs, SNDs, SQDs, SNRs, and SNSs is highlighted. Besides, it is essential to fine-tune the surface functional groups of low dimensional sulfur nanomaterials to form new complex nanomaterials. Finally, these sulfur nanomaterials are being investigated in bio-sensing, bio-imaging, lithium-sulfur batteries, antibacterial activities, plant growth along with future perspective and challenges in emerging fields. The purpose of this review is to tailor low dimensional nanomaterials through accurately selecting precursors or synthetic approach and provide a foundation for the formation of versatile sulfur nanostructure.

Epstein‒Barr virus-associated cellular immunotherapy.

Epstein‒Barr virus (EBV) is a human herpes virus that is saliva-transmissible and universally asymptomatic. It has been confirmed that more than 90% of the population is latently infected with EBV for life. EBV can cause a variety of related cancers, such as nasopharyngeal carcinoma, diffuse large B-cell lymphoma, and Burkitt lymphoma. Currently, many clinical studies have demonstrated that EBV-specific cytotoxic T lymphocytes and other cell therapies can be safely and effectively transfused to prevent and treat some diseases caused by EBV. This review will mainly focus on discussing EBV-specific cytotoxic T lymphocytes and will touch on therapeutic EBV vaccines and chimeric antigen receptor T-cell therapy briefly.

Functional Validation of the RQR8 Suicide /Marker Gene in CD19 CAR-T Cells and CLL1CAR-T Cells.

Chimeric antigen receptor T cell therapy (CAR-T) is a novel treatment that has produced unprecedented clinical effects in patients with hematological malignancies. Acute adverse events often occur following adoptive immunotherapy. Therefore, a suicide gene is helpful, which is a genetically encoded mechanism that allows selective destruction of adoptively transferred T cells in the face of unacceptable toxicity. RQR8 is a gene that integrates CD34 and CD20 epitopes. In our study, we incorporated the suicide gene RQR8 into CAR-T cells, so it enabled rituximab to eliminate vector/transgene-expressing T cells via antibody-dependent cell-mediated cytotoxicity and complement dependent cytotoxicity. In this work, we explored the functionality of RQR8 CAR-T cells in vitro and in vivo. We believe that RQR8 as a safety switch will make CAR-T cell therapy safer and less costly.

Properties and composition of the spent electrolyte for premium circulation mediated by Al-air batteries.

Al-air batteries can serve as a bridge for high-quality cyclic utilization of aluminum resources. However, limited insights into the spent electrolyte are challenging to accurately adjust the recovery process to obtain premium Al-containing products. Herein, the properties and composition of the spent electrolyte were explored through experiments and theoretical calculations. The results demonstrate that the viscosity of the spent electrolyte increased with the rise in discharge current density, time and temperature under highly alkaline conditions, while the ionic conductivity and causticity obviously decreased. Al(OH)4- was the prime and balanced aluminate species when the battery was discharged at 25 °C and coexisted with a bit of [Al2O(OH)6]2-, [Al2O2(OH)6]4- and Al(OH)63- ions. Especially, the characteristics of the spent electrolyte were mainly dominated by the discharge time and temperature when the current density was continuously increased. There was only Al(OH)4- in the electrolyte at a higher discharge temperature. The DFT results also reveal that the polynuclear aluminate ions were produced by the interaction between the mononuclear aluminate ion Al(OH)4- and OH-. This work manifests a profound insight into the spent electrolyte from Al-air batteries for the efficient recycling of aluminum resources.

Developing Strategies to Improve the Efficacy of CAR-T Therapy for Acute Myeloid Leukemia.

OPINION STATEMENT: Acute myeloid leukemia (AML) is a fatal blood malignancy. With the development of immunotherapy, particularly chimeric antigen receptor T cells (CAR-T), the treatment of AML has undergone a significant change. Despite its advantages, CAR-T still faces a number of limitations and challenges while treating AML. Finding novel targets, altering the structure of CAR to increase efficacy while lowering side effects, and using double-target CAR and logic circuits are typical examples of key to answer these problems. With the advancement of gene editing technology, gene editing of tumor cells or normal cells to create therapeutic effects has grown in popularity. Additionally, the combination of multiple drugs is routinely used to address some of the obstacles and difficulties associated with CAR-T therapy. The review's primary goal was to summarize recent strategies and developments of CAR-T therapy for AML.

Small-Molecule Compounds Boost CAR-T Cell Therapy in Hematological Malignancies.

OPINION STATEMENT: Although chimeric antigen receptor T cell immunotherapy has been successfully applied in patients with hematological malignancies, several obstacles still need to be overcome, such as high relapse rates and side effects. Overcoming the limitations of CAR-T cell therapy and boosting the efficacy of CAR-T cell therapy are urgent issues that must be addressed. The exploration of small-molecule compounds in combination with CAR-T cell therapies has achieved promising success in pre-clinical and clinical studies in recent years. Protein kinase inhibitors, demethylating drugs, HDAC inhibitors, PI3K inhibitors, immunomodulatory drugs, Akt inhibitors, mTOR inhibitors, and Bcl-2 inhibitors exhibited potential synergy in combination with CAR-T cell therapy. In this review, we will discuss the recent application of these combination therapies for improved outcomes of CAR-T cell therapy.

"Off-the-Shelf" Allogeneic CAR Cell Therapy-Neglected HvG Effect.

OPINION STATEMENT: Chimeric antigen receptor (CAR) cell therapy offers patients with hematological malignancies a new therapeutic option. Traditionally, autologous T cells are used to generate CAR designed T cells for each patient. However, this method has several drawbacks, the development of allogeneic CAR cell therapy would be a promising breakthrough that could address several of these limitations. From the clinical trials that have published data, the efficacy of allogeneic CAR cell therapy did not meet the expectations. Because of the host-versus-graft (HvG) effect, allogeneic CAR cells are eliminated by the host, resulting in short-term persistence of allogeneic CAR cells and poor efficacy. It is critical to solve the HvG effect of allogeneic CAR cells. The current commonly used methods are suppressing the host's immune system, using HLA-matched homozygous donors, reducing the expression of HLA, targeting alloreactive lymphocytes and eliminating anti-CAR activities. In this review, we will focus on the HvG effect of the "off-the-shelf" allogeneic CAR cell therapy, especially its mechanism and current methods to solve this problem and summarize relevant clinical trial data.

Hip circumference has independent association with the risk of hyperuricemia in middle-aged but not in older male patients with type 2 diabetes mellitus.

BACKGROUND: Obesity and type 2 diabetes mellitus (T2DM) are risk factors for hyperuricemia. However, which anthropometric indices can better predict incident hyperuricemia in patients with T2DM remains inconsistent. This study aimed to examine the associations between hyperuricemia and different anthropometric indices in middle-aged and older male patients with T2DM. METHODS: In this retrospective study, a total of 1447 middle-aged (45-65 years, n = 791) and older (≥ 65 years, n = 656) male patients with T2DM were collected from December 2015 to January 2020 at Shanghai Xinhua Hospital. Hyperuricemia was defined as a serum uric acid level above 7.0 mg/dL. Weight, height, waist circumference (WC) and hip circumference (HC) were measured by trained nurses at visit. RESULTS: The median uric acid level of subjects was 5.6 (interquartile ranges: 4.7-6.7) mg/dl, and 279 (19.3%) were hyperuricemia, with 146 (18.5%) in the middle-aged group, and 133 (20.3%) in the older group. After adjusting for age, duration of T2DM, fasting plasma glucose and insulin, homeostasis model assessment-ß, aspartate aminotransferase, triglycerides, high-density lipoprotein cholesterol and estimated glomerular filtration rate, body mass index (BMI), WC, HC, and waist-to-height ratio (WHtR) were associated with a higher risk of hyperuricemia in both middle-aged and older group (P < 0.05). After further adjusting for BMI and WC, HC still showed a positive relationship with the risk of hyperuricemia (Odds Ratio = 1.51, 95% confidence intervals: 1.06-2.14) in the middle-aged group, but such relationship was not found in the older group. Moreover, according to receiver operating characteristic analysis, the optimal cutoff value was 101.3 cm of HC for hyperuricemia screening in the middle-aged male patients with T2DM. CONCLUSION: In middle-aged male patients with T2DM, more attention should be paid to HC with the cutoff value of 101.3 cm in clinical practice for early recognition of individuals with a high risk of hyperuricemia for targeted guidance on disease prevention, such as community screening.

Elevated serum triglyceride levels at first prenatal visit is associated with the development of gestational diabetes mellitus.

AIMS: While several studies have indicated that maternal serum lipid profiles are associated with the development of gestational diabetes mellitus (GDM), the results have been inconsistent. This study aimed to explore the relationship between maternal lipids profiles at first prenatal visit and GDM and determine the optimal cut-off values of possible trimester-specific variables in predicting GDM. MATERIALS AND METHODS: Clinical data of women with singleton pregnancies who delivered in Xinhua Hospital between January 2016 and January 2017 were collected from electronic databases. Multivariate logistic regression was used to determine the potential risk factors of GDM (specific to the trimester at first prenatal visit), including age, body mass index (BMI), and serum lipid profile and fasting plasma glucose (FPG) levels. The receiver operating characteristic (ROC) curve analysis was performed to determine the cut-off values of significant variables. RESULTS: Among the 2191 pregnant women included, 315 (14.38%) were diagnosed with GDM. Of these, 880 (40.16%) had their first prenatal visit before 14 gestational weeks. Univariate and multivariate analyses showed that both FPG and triglyceride (TG) levels in the first and second trimesters were associated with a high risk of GDM (p < 0.05). The ROC curve showed that serum TG levels >1.235 mmol/L and >1.525 mmol/L in the first and second trimesters, respectively, were significantly associated with the development of GDM (p < 0.05). CONCLUSIONS: TG levels at first prenatal visit is associated with GDM risk. Different TG cut-off values should be applied in the different trimesters of pregnancy for GDM screening.

Low-dose decitabine plus venetoclax is safe and effective as post-transplant maintenance therapy for high-risk acute myeloid leukemia and myelodysplastic syndrome.

Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) are usually associated with poor outcomes, especially in high-risk AML/MDS. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative option for patients suffering from high-risk AML/MDS. However, many patients relapse after allo-HSCT. Novel therapy to prevent relapse is urgently needed. Both the BCL-2 inhibitor venetoclax (VEN) and the hypomethylating agent decitabine (DEC) possess significant antitumor activity effects against AML/MDS. Administration of DEC has been shown to ameliorate graft-versus-host disease (GVHD) and boost the graft-versus-leukemia (GVL) effect post-transplantation. We therefore conducted a prospective study (ChiCTR1900025374) to examine the tolerability and efficacy of a maintenance therapy of low-dose decitabine (LDEC) plus VEN to prevent relapse after allo-HSCT for high-risk AML/MDS patients. Twenty patients with high-risk AML (n = 17) or high-risk MDS (n = 3) post-transplantation were recruited. Approximately day 100 post-transplantation, all patients received LDEC (15 mg/m2 for 3 d) followed by VEN (200 mg) on d 1-21. The cycle interval was 2 mo, and there was 10 cycles. The primary end points of this study were rates of overall survival (OS) and event-free survival (EFS). The secondary endpoints included adverse events (AEs), cumulative incidence of relapse (CIR), nonrelapse mortality (NRM), incidences of acute GVHD (aGVHD) and chronic GVHD (cGVHD), and incidences of viral infection after allo-HSCT. Survival outcomes were assessed using Kaplan-Meier analysis. The median follow-up was 598 (149-1072) d. Two patients relapsed, 1 died, and 1 is still alive after the second transplant. The 2-y OS and EFS rates were 85.2% and 84.7%, respectively. The median 2-y EFS time was 525 (149-1072) d, and 17 patients still had EFS and were alive at the time of this writing. The most common AEs were neutropenia, anemia, thrombocytopenia, neutropenic fever, and fatigue. Grade 2 or 3 AEs were observed in 35% (7/20) and 20% (4/20) of the patients, respectively. No grade >3 AEs were observed. aGVHD (any grade) and cGVHD (limited or extensive) occurred in 55% and 20% of patients, respectively. We conclude that LDEC + VEN can be administered safely after allo-HSCT with no evidence of an increased incidence of GVHD, and this combination decreases the relapse rate in high-risk AML/MDS patients. This novel maintenance therapy may be a promising way to prevent relapse in high-risk AML/MDS patients.

CD19 CAR-T cell treatment conferred sustained remission in B-ALL patients with minimal residual disease.

The persistence or recurrence of minimal residual disease (MRD) after chemotherapy predicts relapse of B-cell acute lymphoblastic leukemia (B-ALL). CD19-directed chimeric antigen receptor T (CD19 CAR-T) cells have shown promising responses in B-ALL. However, their role in chemotherapy-refractory MRD-positive B-ALL remains unclear. Here we aimed to assess the effectiveness and safety of CD19 CAR-T cells in MRD-positive B-ALL patients. From January 2018, a total of 14 MRD-positive B-ALL patients received one or more infusions of autogenous CD19 CAR-T cells. Among them, 12 patients achieved MRD-negative remission after one cycle of CAR-T infusion. At a median follow-up time of 647 days (range 172-945 days), the 2-year event-free survival rate in MRD-positive patients was 61.2% ± 14.0% and the 2-year overall survival was 78.6 ± 11.0%, which were significantly higher than patients with active disease (blasts ≥ 5% or with extramedullary disease). Moreover, patients with MRD had a lower grade of cytokine release syndrome (CRS) than patients with active disease. However, the peak expansion of CAR-T cells in MRD positive patients showed no statistical difference compared to patients with active disease. Five patients received two or more CAR-T cell infusions and these patients showed a decreased peak expansion of CAR-T cell in subsequent infusions. In conclusion, pre-emptive CD19 CAR-T cell treatment is an effective and safe approach and may confer sustained remission in B-ALL patients with chemotherapy-refractory MRD. The trials were registered at www.chictr.org.cn as ChiCTR-ONN-16009862 (November 14, 2016) and ChiCTR1800015164 (March 11, 2018).

Umbilical cord blood-derived mesenchymal stromal cells promote myeloid-derived suppressor cell proliferation by secreting HLA-G to reduce acute graft-versus-host disease after hematopoietic stem cell transplantation.

BACKGROUND AIMS: Mesenchymal stem cells (MSCs) use multiple mechanisms to constrain both innate and adaptive immune responses to prevent graft-versus-host disease (GVHD). Myeloid-derived suppressor cells (MDSCs), as a heterogeneous population of early myeloid progenitor cells originating from bone marrow, are a naturally occurring immune regulatory population associated with inhibition of ongoing inflammatory responses, indicating their potential for GVHD therapy. There is accumulating evidence that MSCs and MDSCs do not act independently, but rather establish crosstalk. However, the role of MSCs in MDSC expansion and activation in GVHD remains unexplored. METHODS: In vitro experiments included 2 groups: peripheral blood mononuclear cells (PBMCs) after mobilization and human umbilical cord blood-derived MSCs (UCB-MSCs) co-cultured with PBMCs. The number and functional difference of MDSCs in PBMCs were determined by flow cytometry. The culture supernatants of co-cultured cells were analyzed to identify cytokines involved in MDSC proliferation. The relationship between MSCs and MDSCs was clarified in GVHD and graft-versus-leukemia (GVL) animal models. RESULTS: In vitro experiments confirmed that UCB-MSCs secreted HLA-G protein to promote and maintain the proliferation of MDSCs in peripheral blood after granulocyte colony-stimulating factor mobilization, and UCB-MSCs mediated the function of MDSCs to inhibit the proliferation of T cells and promote the proliferation of regulatory T cells. UCB-MSCs overexpressing HLA-G induced MDSC production in recipient mice, improved the ability of MDSCs to suppress T cells and further reduced acute GVHD (aGVHD) symptoms and survival time without influencing GVL effects. CONCLUSIONS: UCB-MSCs expanded MDSCs via HLA-G/Ig-like transcript 4, reducing the severity of aGVHD without affecting GVL. The immunosuppressive potential of MSCs for the treatment of aGVHD significantly affects the development of MDSCs, thereby consolidating the position of MSCs in the prevention and treatment of aGVHD.

PEDF promotes the repair of bone marrow endothelial cell injury and accelerates hematopoietic reconstruction after bone marrow transplantation.

BACKGROUND: Preconditioning before bone marrow transplantation such as irradiation causes vascular endothelial cells damage and promoting the repair of damaged endothelial cells is beneficial for hematopoietic reconstitution. Pigment epithelium-derived factor (PEDF) regulates vascular permeability. However, PEDF's role in the repair of damaged endothelial cells during preconditioning remains unclear. The purpose of our study is to investigate PEDF's effect on preconditioning-induced damage of endothelial cells and hematopoietic reconstitution. METHODS: Damaged endothelial cells induced by irradiation was co-cultured with hematopoietic stem cells (HSC) in the absence or presence of PEDF followed by analysis of HSC number, cell cycle, colony formation and differentiation. In addition, PEDF was injected into mice model of bone marrow transplantation followed by analysis of bone marrow injury, HSC number and peripheral hematopoietic reconstitution as well as the secretion of cytokines (SCF, TGF-ß, IL-6 and TNF-α). Comparisons between two groups were performed by student t-test and multiple groups by one-way or two-way ANOVA. RESULTS: Damaged endothelial cells reduced HSC expansion and colony formation, induced HSC cell cycle arrest and apoptosis and promoted HSC differentiation as well as decreased PEDF expression. Addition of PEDF increased CD144 expression in damaged endothelial cells and inhibited the increase of endothelial permeability, which were abolished after addition of PEDF receptor inhibitor Atglistatin. Additionally, PEDF ameliorated the inhibitory effect of damaged endothelial cells on HSC expansion in vitro. Finally, PEDF accelerated hematopoietic reconstitution after bone marrow transplantation in mice and promoted the secretion of SCF, TGF-ß and IL-6. CONCLUSIONS: PEDF inhibits the increased endothelial permeability induced by irradiation and reverse the inhibitory effect of injured endothelial cells on hematopoietic stem cells and promote hematopoietic reconstruction.

Reference gene selection and validation for mRNA expression analysis by RT-qPCR in murine M1- and M2-polarized macrophage.

Murine bone marrow-derived macrophages (M0) and M1- and M2-polarized macrophages are being widely used as a laboratory model for polarized macrophages related molecular mechanism analysis. Gene expression analysis based on reference gene normalization using RT-qPCR was a powerful way to explore the molecular mechanism. But little is known about reference genes in these cell models. So, the goal of this study was to identify reference genes in these types of macrophages. Candidate reference genes in murine bone marrow-derived and polarized macrophages were selected from microarray data using Limma linear model method and evaluated by determining the stability value using five algorithms: BestKeeper, NormFinder, GeNorm, Delta CT method, and RefFinder. Finally, the selected stable reference genes were validated by testing three important immune and inflammatory genes (NLRP1, IL-1ß, and TNF-α) in the cell lines. Our study has clearly shown that Ubc followed by Eef1a1 and B2m respectively were recognized as the three ideal reference genes for gene expression analysis in murine bone marrow-derived and polarized macrophages. When three reference genes with strong different stability were used for validation, a large variation of a gene expression level of IL-1ß, TNF-α and NLRP1 were obtained which provides clear evidence of the need for careful selection of reference genes for RT-qPCR analysis. Normalization of mRNA expression level with Ubc rather than Actb or Gusb by qPCR in macrophages and polarized macrophages is required to ensure the accuracy of the qPCR analysis.

S100A16 suppresses the growth and survival of leukaemia cells and correlates with relapse and relapse free survival in adults with Philadelphia chromosome-negative B-cell acute lymphoblastic leukaemia.

Refinement of risk stratification in Philadelphia chromosome (Ph)-negative B-cell acute lymphoblastic leukaemia (ALL) might aid the identification of patients who are likely to relapse. Abnormal S100 calcium binding protein A16 (S100A16) has been implicated in various cancers, but its function remains unclear. We found S100A16 transcript levels were higher in 130 adults with newly-diagnosed Ph-negative B-cell ALL compared with 33 healthy controls. In 115 of 130 patients who achieved first complete remission, those with high S100A16 transcript levels displayed a lower 3-year cumulative incidence of relapse (CIR; 34% [21, 47%] vs. 40% [48, 72%]; P = 0·012) and higher 3-year relapse-free survival (RFS; 65% [53, 78%] vs. 35% [23, 46%]; P = 0·012), especially when receiving chemotherapy only. In multivariate analysis a low S100A16 transcript level was independently-associated with a higher CIR (Hazard ratio [HR] = 3·74 [1·01-13·82]; P = 0·048) and inferior RFS (HR = 5·78 [1·91, 17·84]; P < 0·001). Function analysis indicated that knockdown of S100A16 promoted proliferation and anti-apoptosis and reduced chemosensitivity. S100A16 over-expression revealed an opposite trend, especially in a xeno-transplant mouse model. Western blotting analysis showed upregulation of PI3K/AKT and ERK1/2 in S100A16-knockdown and S100A16-overexpression B-cell ALL cell lines respectively. Inhibition assays suggested these two signalling pathways participated in the S100A16-mediated proliferation and survival effects in B-cell ALL cell lines. Trial Registration: Registered in the Chinese Clinical Trial Registry [ChiCTR-OCH-10000940]; http://www.chictr.org.cn.