Acute Monocytic Leukemia Patients Research Articles

Clofazimine as a substitute for rifampicin improves efficacy of Mycobacterium avium pulmonary disease treatment in the hollow-fiber model.

Mycobacterium avium complex pulmonary disease is treated with an azithromycin, ethambutol, and rifampicin regimen, with limited efficacy. The role of rifampicin is controversial due to inactivity, adverse effects, and drug interactions. Here, we evaluated the efficacy of clofazimine as a substitute for rifampicin in an intracellular hollow-fiber infection model. THP-1 cells, which are monocytes isolated from peripheral blood from an acute monocytic leukemia patient, were infected with M. avium ATCC 700898 and exposed to a regimen of azithromycin and ethambutol with either rifampicin or clofazimine. Intrapulmonary pharmacokinetic profiles of azithromycin, ethambutol, and rifampicin were simulated. For clofazimine, a steady-state average concentration was targeted. Drug concentrations and bacterial densities were monitored over 21 days. Exposures to azithromycin and ethambutol were 20%-40% lower than targeted but within clinically observed ranges. Clofazimine exposures were 1.7 times higher than targeted. Until day 7, both regimens were able to maintain stasis. Thereafter, regrowth was observed for the rifampicin-containing regimen, while the clofazimine-containing regimen yielded a 2 Log10 colony forming unit (CFU) per mL decrease in bacterial load. The clofazimine regimen also successfully suppressed the emergence of macrolide tolerance. In summary, substitution of rifampicin with clofazimine in the hollow-fiber model improved the antimycobacterial activity of the regimen. Clofazimine-containing regimens merit investigation in clinical trials.

Nanocellulose as Novel Vaccine Adjuvant: Innate Immune Activation and Biocompatibility

Aluminum remains the most widely accepted adjuvant to enhance the immunogenicity of vaccines. However, the clinical use of Aluminum is limited by its neurotoxicity and risk of immunoglobulin E production. Thus, alternative adjuvants with equivalent capability but higher biocompatibility are urgently needed. Among them, nanocellulose is a promising candidate for this purpose. In spite of their biodegradability, their physicochemical properties, including fibrillar nature, crystalline phase, and surface reactivity, affect their activation kinetics, and the relationship remains mostly unknown. Hence, I established a small library of nanocellulose materials using acid hydrolysis to obtain three CNCs with different sizes, aiming to investigate how the size of nanocellulose influences their biocompatibility and immunogenicity. I evaluated their proinflammatory effect on THP-1 cells, a monocyte cell line isolated from the peripheral blood of an acute monocytic leukemia patient, and J774, a murine macrophage cell. Then, I tested their cytotoxicity to both cells and compared their differential effects in IL-1β production. Finally, I correlated their proinflammatory adjuvant effects with their length. In conclusion, I found the production of IL-1β is CNC length-dependent but in a nonlinear relationship. By testing cell viability after stimulation with adjuvants, I found CNCs are a more biocompatible adjuvant than Alum, and the cytotoxicity of CNCs is also size-dependent. This study provides a preliminary demonstration of the optimal length range and crystallinity of CNCs that could induce the most adjuvant effects without posing cytotoxicity.

Adjuvant nanocellulose with different physicochemical property can induce immune adjuvant effects without posing cytotoxicity.

Abstract Objective: To analyze the in vitro effectiveness of cellulose nanocrystals (CNC) in inducing pro-inflammatory and immune adjuvant effects in THP-1 cells, a monocyte cell line isolated from the peripheral blood of an acute monocytic leukemia patient. Methods: THP-1 cells were counted, and their viability was calculated followed by the quantification of IL-1 β production. Results: We observed the following: 1) CNCs were not cytotoxic to the cell lines, and 2) a 280 nm length of CNC induced the strongest immune response within our selected size spectrum. Conclusion: This study provides a preliminary demonstration of the optimal length range CNCs that could induce most adjuvant effects without posing cytotoxicity. This research didn't receive funding support.

LILRB4 Is a Novel Target for KMT2A Rearranged Acute Leukemia

Background: KMT2a gene rearrangement (KMT2Ar) is a recurrent mutation in acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), occurring both in children and adults. Patients with KMT2Ar acute leukemia have poor outcomes related to increased rates of relapsed and refractory disease. Novel therapies including BITE and CAR-T cells has proven efficacious for subgroups of patients with acute leukemia, however those with KMT2Ar are at risk for lineage switch relapse following immunotherapy. In a recent analysis, there have been no reported survivors following lineage switch relapse. Therefore, it is critical to identify novel targets and treatment strategies for KMT2Ar acute leukemia. We have previously demonstrated leukocyte immunoglobulin like receptor B4 (LILRB4) is a novel tumor associated antigen with complete expression on AML with monocytic differentiation (FAB M5), which can be effectively targeted by a novel anti-LILRB4 CAR-T cell we developed and validated in preclinical models. A majority of acute monocytic leukemia cell lines and patient samples contain the KTMT2Ar, which led us to hypothesize LILRB4 is expressed on other KMT2Ar acute leukemias including B-ALL, and may retain expression on lineage switch relapsed disease. In the current study, we evaluate the anti-leukemia function of anti-LILRB4 CAR-T cells against KMT2Ar ALL and lineage switch disease. Methods and results: We first analyzed the expression of LILRB4 on a panel of B-ALL cell lines by flow cytometry. The KMT2Ar cell lines, RS411 [t(4;11)] and KOPN8 [t(11;19)] demonstrated complete expression of LILRB4 regardless of fusion gene partner, while cell lines without KMT2Ar including 697 and NALM6, were negative for LILRB4. We next analyzed expression of LILRB4 on primary patient samples of de novo, relapsed or refractory acute leukemia identified as KMT2Ar from our institution biospecimen repository. In five cases of B-ALL with KMT2Ar, LILRB4 was co-expressed with CD19 on leukemia blasts as demonstrated by flow cytometry. In one patient with multiply relapsed AML with KMT2Ar, expression of LILRB4 was retained on each relapse following stem cell transplant. Lastly, in an infant with refractory KMTAr B-ALL who developed lineage switch AML following treatment with Blinatumomab, LILRB4 was strongly expressed on the complete population of leukemia blasts. We next evaluated the effector function of anti-LILRB4 CAR-T cells against LILRB4-positive, KMT2Ar B-ALL cell lines. Human T-cells were activated with CD3/CD28 beads, then transduced with lentiviral vector encoding the second generation anti-LILRB4 CAR transgene, incorporating a 4-1BB co-stimulatory and CD3-zeta activation domain, and were expanded for 7-10 days with IL7 and IL15. In vitro, anti-LILRB4 CAR-T cells demonstrated potent cytotoxicity in mixed culture against KOPN8, and similarly displayed significantly increased cytokine release of IL2, TNF-alpha and Interferon-gamma compared to control-T cells. To evaluate the function of anti-LILRB4 CAR-T cell in vivo, we engrafted KOPN8 cells in NSG immunocompromised mice, followed by treatment with control or CAR-T cells. Anti-LILRB4 CAR-T cell treated mice demonstrated significantly decreased leukemia burden and prolonged survival compared to control treated mice. Patient derived mouse xenografts (PDX) of KMT2Ar relapsed AML (PDX1) and lineage switch leukemia (PDX2) were also established. Significantly, when utilizing autologous anti-LILRB4 CAR-T cells, leukemia was eliminated in the PDX1 model. And strikingly in PDX2, we demonstrate elimination of KMT2Ar lineage switch leukemia in mice treated with anti-LILRB4 CAR-T cells. Conclusion: In this preclinical work, we demonstrate LILRB4 is expressed on KMT2Ar acute leukemias including B-ALL, and importantly displays a retained pattern of expression on fatal lineage switch relapsed AML. Furthermore, we demonstrate potent anti-leukemia activity of anti-LILRB4 CAR-T cells against LILRB4 expressing KMT2Ar acute leukemia, broadening the application of this cell therapy. This work offers a new treatment strategy for patients with dismal outcomes and will be tested in a Phase I clinical trial for children and adults with LILRB4 expressing acute leukemia. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Efficacy and Prognosis of Allogeneic Hematopoietic Stem Cell Transplantation for Acute Monocytic Leukemia Patients

To investigate the efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in the treatment of acute monocytic leukemia (AML-M5) and the related factors that affecting the prognosis of the patients. The clinical data of 71 patients with AML-M5 treated with allo-HSCT in Zhujiang Hospital Affiliated to Southern Medical University from April 2009 to October 2019 were collected and retrospectively analyzed. The incidence of graft-versus-host disease (GVHD), cumulative overall survival (OS) rate, cumulative progression-free survival (PFS) rate, transplantation-related mortality (TRM), relapse rate and the risk factors affecting prognosis in the patients were analyzed. 66 patients obtained hematopoietic reconstruction after transplantation, the median time of granulocyte implantation was 12 (9-26) d, and the median time of megakaryocytic implantation was 13 (8-72) d. The incidence of acute GVHD and chronic GVHD was 33.8% (24/71) and 36.6% (26/71), respectively. The median follow-up time was 13.81 (0.16 to 112.54) months; the median OS and PFS was 31.27 and 26.07 months, respectively. The cumulative OS of the patients in 1 and 3 years after transplantation was 64.9% and 48.6%, respectively, and the cumulative PFS of the patients in 1 and 3 years was 55.0% and 39.5%, respectively. The cumulative relapse rate of the patients in 1 and 3 years was 24% and 40%, respectively. Multivariate analysis showed that pre-transplantation relapse was the independent risk factor affecting OS (HR=2.32, 95%CI:1.17-4.62, P=0.02) and PFS (HR=3.08, 95%CI:1.61-5.90, P=0.001) of the patients; invasive fungal disease after transplantation was the independent risk factor affecting OS (HR=2.71, 95% CI:1.32-5.56, P=0.007) and PFS (HR=2.87, 95%CI=1.40-5.86, P=0.004) of the patients; FLT3 mutation was the independent risk factor affecting PFS (HR=2.13, 95%CI=1.07-4.24, P=0.03) of the patients. AML-M5 is the intermediate or high-risk leukemia, and allo-HSCT can improve the survival prognosis of the patients. Pre-transplantation relapse and invasive fungal disease after transplantation are the important factors affecting the efficacy of allo-HSCT in patients with AML-M5.

Role of toll-like receptor 4 on tissue factor regulation in human monocytes

Abstract Background Inflammation and coagulation play a pivotal role in the pathogenesis of acute coronary events and an extensive cross-talk links the two systems, whereby inflammation activates coagulation and coagulation affects inflammatory activity. Infact, pro-inflammatory stimuli can induce tissue factor (TF) expression, the principal initiator of the clotting cascade, in circulating monocytes and activate pathways leading to thrombin generation.In turn, TF may bind cellular receptors which may affect the production and release of inflammatory mediators. According to our recent results, proprotein convertase subtilisin/kexin9 (PCSK9) and Gamma-Glutamyltransferase (GGT), two molecules involved in the pathogenesis of cardiovascular disease, are able to up-regulate TF expression in monocytes by activating NFkB pathway but the mechanism and the receptor involved in this biological response is unknown. One plausible possibility is that both molecules bind a Toll-like Receptor (TLR)4 located on membranes of human and cultured monocytes,activating the TLR4/MyD88-NFkB pathway and eventually leading to stimulation of TF expression. Aim To assess whether both molecules are able to bind to TLR4 located on the surface of human monocytes and whether this specific binding involves the TLR4/MyD88-NFkB pathway on TF modulation. Methods THP-1 cells, a human monocytic cell line derived from an acute monocytic leukemia patient, was used as in vitro model. The cells were stimulated with human (h) recombinant (r) PCSK9 (5 μg/ml) and hrGGT (1 μg/ml) or pre-incubated with BAY-117082 (BAY, 10–5M) a NFκB inhibitor, CLI-095 (3x10–6M), a highly Myd88/TLR4 signaling specific inhibitor and LPS-RS (1 μg/ml) a TLR4 antagonist. TF procoagulant activity (PCA), was assessed by 1-stage clotting assay and the results expressed by ρg/mL of active protein. Experimental series were carried out in endotoxin-free conditions, in order to exclude lipopolysaccharide (LPS)-dependent immune responses. Results hrPCSK9 and hrGGT stimulated TF expression (PCA: from 50±20 to 120±20, n=10, p<0.01) and (PCA: from 190±140 to 460±360, n=15, p<0.001) respectively, an effect down-regulated by BAY,a NFκB inhibitor (PCA by hrPCSK9: −71±23%, n=5, p<0.01; PCA by hrGGT: −90±21%, n=7, p<0.001). CLI-095, a TLR4 inhibitor (PCA by hrPCSK9: −86±26%, n=3, p<0.05; and PCA by GGT: −89±10%, n=5, p<0.001).LPS-RS, a TLR4 antagonist, (PCA by hrPCSK9: −74±25%, n=3, p<0.05; PCA by hrGGT: −70±17%, n=5, p<0.001) abolished both PCSK9 and GGT-induced TF expression. Conclusions These data are the first demonstration of a direct role of PCSK9 and GGT as active mediators of inflammatory-based thrombotic diseases. The possible mechanism of action involves recognition of two proteins by TLR4 on monocytes membrane surface, lead to activation of the transcription factor NFκB. Further studies will be needed to better understand the regulatory mechanisms underlying this complex set of biological responses that bind TLR4 modulation and TF expression. Funding Acknowledgement Type of funding source: None

Plasmacytoid Dendritic Cell Infiltration in Acute Myeloid Leukemia.

IntroductionIncreasing evidence has demonstrated that plasmacytoid dendritic cells (PDCs) in the tumor microenvironment (TME) play an important role in tumorigenesis and progression. PDC infiltration has been found in certain malignancies such as classic Hodgkin’s lymphoma and chronic myelomonocytic leukemia. Our previous work reported that PDC infiltration could occur in acute myeloid leukemia (AML), but the clinical significance of PDC in AML has not been thoroughly investigated.Patients and MethodsHere, we evaluated the clinical significance of PDC to AML transition in a leukemia microenvironment. The frequency of PDCs in 80 acute myelomonocytic leukemia (AML-M4) and 83 acute monocytic leukemia (AML-M5) patients was determined by flow cytometry.ResultsWe found 62 cases with PDC infiltration. These patients showed higher numbers of bone marrow blasts, higher mean Hb concentration, and required more cycles of chemotherapy before achieving complete remission (CR), but had lower white blood cell and platelet counts compared to patients without PDC infiltration. Drug sensitivity analysis showed that patients with PDC infiltration had lower sensitivity to standard chemotherapy regimens. Kaplan–Meier survival curves demonstrated that patients with PDC infiltration had a shorter overall survival (OS) time and progression-free survival time.DiscussionThese results suggested that PDC infiltration can be used for risk stratification of AML-M4/M5, and PDCs may transdifferentiate into leukemia in an AML microenvironment.

MLAA-34 knockdown shows enhanced antitumor activity via JAK2/STAT3 signaling pathway in acute monocytic leukemia.

MLAA-34 is a novel leukemia-associated gene closely related to the carcinogenesis of acute monocytic leukemia (AML). MLAA-34 over expression has been observed to inhibit apoptosis in vitro. JAK2/STAT3 pathway plays an important role in cell proliferation, differentiation and inhibition of apoptosis in number of cancers. However, the relationship and interaction between MLAA-34 and JAK2/STAT3 has never been investigated in AML.This study investigates and reports a novel relationship between MLAA-34 and JAK2/STAT3 pathway in AML both in vitro and in vivo. We constructed MLAA-34 knockdown vector and transfected U937 cells to observe its apoptotic activities in relation to JAK2/STAT3 signaling pathway in vitro and then in vivo in mouse model. Levels of expression of MLAA-34 and JAK2/STAT3 and its downstream targets were also measured in AML patients and a few volunteers.We found that MLAA-34 knockdown increased U937 apoptosis in vitro and inhibited tumor growth in vivo. Components of the canonical JAK2/STAT3 pathway or its downstream targets, including c-myc, bcl-2, Bax, and caspase-3, were shown to be involved in the carcinogenesis of AML. We also found that the JAK2/STAT3 pathway positively regulated MLAA-34 expression. We additionally identified a STAT3 binding site in the MLAA-34 promoter where STAT3 binds directly and activates MLAA-34 expression. In addition, MLAA-34 was found to form a complex with JAK2 and was enhanced by JAK2 activation. Correlation of MLAA-34 and JAK2/STAT3 was further confirmed in AML patients.In conclusion, MLAA-34 is a novel regulator for JAK2/STAT3 signaling, and in turn, is regulated by this interaction in a positive feedback loop. Thus we report a novel model of interaction mechanism between MLAA-34 and JAK2/STAT3 which can be utilized as a potential target for a novel therapeutic approach in AML.

Methylation status of p15 and E-cadherin gene in a cohort of Egyptian acute monocytic leukemia patients

BACKGROUND: Acute monocytic leukemia (AML) is a common hematological malignancy with different subtypes. AML with monocytic involvement (AML M5) is the most frequent subtype among adults. Many genetic and epigenetic abnormalities were described in adult AML. Methylation status in AML is studied for classification, as prognostic marker and even for therapeutic implications. p15 and E-cadherin promotor region methylation are frequently implicated in different kinds of hematological and nonhematological malignancies. AIM OF THE WORK: The aim is to detect methylation status of p15 and E-cadherin in a group of adult Egyptian monocytic leukemia patients and relate it to further events; relapse and central nervous system (CNS) infiltration. Patients and methods: The study was conducted on 76 acute monocytic leukemia patients and 50 healthy controls. PCR specific methylation for p15 and E cadherin was done for both groups. RESULTS: p15 and E-cadherin aberrant methylation are very frequent in adult acute monocytic leukemia. p15 methylation is related to CNS infiltration, while p15 and E-cadherin methylation status is related to relapse. CONCLUSIONS: Methylation of p15 and E-cadherin can be added to prognostic markers of acute monocytic leukemias.

Characterizing the molecular cytogenetics in acute monocytic leukemia

To characterize the molecular genetics of 81 patients with acute monocytic leukemia (AML). Fluorescence in situ hybridization (FISH) was employed to detect MLL gene rearrangements. Combined mutations of 17 genes were detected by DNA-based PCR and Sanger sequencing. Sixty seven patients were found to harbor at least one mutation. The most commonly mutated gene was NPM1 (n=18), which was followed by FLT3-ITD (n=16), NRAS (n=16), DNMT3A (n=15), TET2 (n=12), RUNX1 (n=11) and KRAS (n=9). Based on the functions of mutated genes, the most frequently involved genes were those involved in DNA methylation (38.27%), tyrosine kinase receptor signaling (32.1%), transcription regulation (28.4%), and RAS pathway (24.7%). Single gene mutation predominated in patient with cytogenetic abnormalities, while coexistence of 2 mutations have predominated in patient with normal cytogenetic findings. Stratified by cytogenetic findings, patients with single gene mutations (intermediate-risk group) had significantly higher complete remission (CR) rates than those with ≥2 gene mutations (unfavorable-risk group) (91.7% vs. 57.6% , 87.5% vs. 25.0%, P =0.0319, 0.0117, respectively). Over 80% of AML patients were found to harbor at least one mutation. Their clinical phenotype and prognosis may be impacted by the synergy of MLL gene rearrangement and multiple mutations. For patients under the same risk stratification, the number of mutations is reversely correlated with the CR rate.

Determination of electrophysiological properties of human monocytes and THP-1 cells by dielectrophoresis

Introduction: Dielectrophoresis (DEP) is based on polarization and bioparticle movement in applied electric fields. Each type of cells has its own electrical properties within the DEP spectra and undergoes significant changes in direction of frequency following an increase of an applied nonuniform alternating current (AC) electric field. Therefore, DEP can be an effective technique for characterization and separation of different cells. The goal of the current study was to determine the electrophysiological properties of human monocytes and a human monocytic cell line originated from an acute monocytic leukemia patient (THP-1), using a lab on a chip (LOC) device that utilised microarray dot electrodes.
 Methods: Ten microliters of human monocytes isolated from peripheral blood mononuclear cells and highly confluent THP-1 cells were diluted in DEP medium and added to the spacer of the LOC device. Subsequently, the AC signal in the range of 10 kHz to 2 MHz was supplied to the LOC device, and the dynamic behaviours of monocytes and THP-1 cells due to the DEP force were observed. Images were captured and analyzed using MATLAB software.
 Results: Microscopic visualization showed that THP-1 cells were physically larger than human monocytes. The dielectric parameters (radius, diameter, and conductivity and permittivity of the cytoplasm and membrane) were greater for THP-1 cells compared to monocytes. The cross-over frequencies for THP-1 cells and monocytes were respectively 66 kHz and 280 kHz.
 Conclusion: In conclusion, the DEP spectra reflected the morphological differences between monocytes and THP-1 cells. The dielectric properties for each type of cells can be used as the criteria in the development of DEP-based characterization assays.

Establishment of Human Acute Monocyte Leukemia Model with Systemic Leukemia in Npg Mice

Although tumor cells are easily to growth in the bodies of immunodeficicent animals such as nude mice and NOD-SCID mice, it's hard for acute leukemia cells to grow in the bone marrow of nude mice or NOD-SCID mice even when mice receive extra immunosuppressive treatment such as splenectomy, cyclophosphamide and irradiation. This study aimed to establish a mice model with systemic leukemia using another highly immunodeficicent NPG mice without immunosuppressive treatment before inoculation. 5-week NPG mice were inoculated with 1x107(Group A) or 5x107 (Group B) SHI-1 cells (a cell line derived from a refractory acute monocytic leukemia patient) via tail vein. One NPG mice in each group was killed by ether randomly at the day 14, 21, 28 after inoculation, other NPG mice were observed the survival time. The leukemic cells engrafted in the NPG mice were detected by the following methods: the blast cells were detected by the blood smear and flow cytometer, the MLL-AF6 fuse gene of SHI-1 cells were detected by PCR amplification, the human CD45 positive cells infiltrated in the organs of NPG mice were detected by histopathological examination and immunohistochemistry. At the day 14 after inoculation with SHI-1 cells, fewer blasts cells were found in the smear of peripheral blood of group B; MLL-AF6 fuse gene could be amplified in the spleen of NPG mice in group A and in spleen and bone marrow in group B (Fig A); histopathological examination had shown that CD45 positive leukemia cell just infiltrated in spleen. At the day 21 after inoculation, more blasts were found in the smear of peripheral blood both in group A and B; MLL-AF6 fuse gene were amplified in the organs of NPG mice such as Spleen, liver, kidney, stomach, lung, heart and bone marrow(Fig A); 5.16% and 0.82% of CD45 and CD33 positive cells were detected in the peripheral blood of NPG mice in group A and B respectively; a green solid neoplasm were found in the kidney of NPG mice in group B, leukemia cells were found in the organ of heart, liver, spleen, stomach, kidney and lung in the NPG mice of both groups by histopathological examination. From the third week, the NPG mice presented anorexia, hunched posture, lethargy and weight loss. For the mice sacrificed in the day 28 after inoculation, the proportion of CD45 and CD33 positive cells in peripheral blood, bone marrow and spleen were 9.60%, 11.4% and 23.20% in group A and were 11.0%,37.80% and 60.5% in group B (Fig B). Green solid tumors were grown in many organs such as kidney, liver, spleen, stomach, heart, lymph node and the soft tissues in the NPG mice killed in day 28 after inoculation and the mice which were dead spontaneous (Fig C); When NPG mice were dead, the weight of spleen in group B is significantly higher than the weight of spleen in group A(P<0.05) (Fig D). The median survival time of NPG in group A and group B is 33 and 30 days respectively. Pathological examination and immunohistochemical staining had shown that leukemic cells could infiltrated to many of the organs of NPG mice and the grade of leukemia infiltration was positively correlated with cells numbers of inoculation and the survival time of NPG mice (Fig E). Altogether, SHI-1 cell could growth in the NPG mice without any pre-immunosuppressive treatment and formed a systemic leukemia in NPG mice as like in acute leukemia patients. This efficient and reproducible model may be a useful tool for the studies of the pathogenesis in acute monocytic leukemia. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Expression of triggering receptors expressed by myeloid cells-1 in macrophages stimulated by Porphyromonas gingivalis-lipopolysaccharide

Soluble triggering receptors expressed by myeloid cells-1 (sTREM-1) and inflammatory cytokine tumor necrosis factor-α (TNF-α) in macrophage cells were stimulated by Porphyromonas gingivalis-lipopolysaccharide (Pg-LPS) to investigate the expression of triggering receptors expressed by myeloid cells-1 (TREM-1) and further explore the correlation between TREM-1 and the pathogenesis of periodontitis. THP-1 cells (a human monocytic cell line derived from an acute monocytic leukemia patient) were induced to differentiate THP-1 macrophages by phorbol-12-myristate-13-acetate and were injected with 0 (blank control), 0.5, or 1.0 μg·mL⁻¹ Pg-LPS. The THP-1 cells were then grouped in accordance with incubation time, and each group was incubated for 4, 6, 12, or 24 h. The expression of the TREM-1 mRNA in macrophages was detected by real-time quantitative polymerase chain reaction, while the expression of TREM-1 protein was detected by Western blot; the site where TREM-1 protein expression was observed in macrophages was detected by immunofluorescence staining, and the expression of soluble sTREM-1 and TNF-α in cell culture medium was detected by enzyme-linked immunosorbent assay. Compared with the blank control group, the expression of TREM-1 mRNA, TREM-1 protein, and sTREM-1 in Pg-LPS-stimulated macrophages was significantly upregulated (P<0.05). The expression of TREM-1 mRNA, TREM-1 protein, and sTREM-1 in the supernatant of cell culture was higher in the 1.0 μg·mL⁻¹ Pg-LPS group than in the 0.5 μg·mL⁻¹ group; this expression was statistically significant since the 6, 4, and 4 h time point (P<0.05). Cell immunofluorescence staining showed that TREM-1 protein was positive when the THP-1 macrophages was stimulated by Pg-LPS (1.0 μg·mL⁻¹) for 24 h, and the staining sites of TREM-1 were mainly located in the cell membrane of the macrophages (P<0.05). The expression level of TNF-α increased in groups stimulated by Pg-LPS, and the expression level of TNF-α was significantly higher in 1.0 μg·mL⁻¹ Pg-LPS stimulated groups than in 0.5 μg·mL⁻¹ Pg-LPS-stimulated groups since the 6 h time point (P<0.05). The expressions of TREM-1 mRNA, TREM-1 protein, and sTREM-1 in 0.5 μg·mL⁻¹ Pg-LPS-stimulated macrophages were positively correlated with one another (r=1, P<0.05), but no statistically significant correlation was found in the expression of TNF-α. The positive correlation between sTREM-1 and TNF-α expressions was detected when macrophages were stimulated by 1.0 μg·mL⁻¹ Pg-LPS (r=1, P<0.05). The expression of TREM-1 mRNA, TREM-1 protein, and sTREM-1 in the culture supernatant in Pg-LPS-stimulated macrophages was significantly upregulated on the basis of the concentration of Pg-LPS; moreover, their upregulation was positively correlated with one another. The expression of TNF-α in the supernatant of cell culture was also upregulated and was positively correlated with the expression of sTREM-1 at the group of high Pg-LPS concentration (1.0 μg·mL⁻¹). Results reveal that TREM-1, which has been realized as a proinflammatory receptor protein, can promote the development of periodontitis by regulating the expression of TNF-α in macrophages.

Haemophagocytic lymphohistiocytosis occurred during induction chemotherapy in an acute monocytic leukemia patient with FLT3-ITD and DNMT3A mutations

BackgroundHaemophagocytic lymphohistiocytosis (HLH) is considered to be a large challenge for clinicians due to the variable overlaps of symptoms with other severe diseases and a high rate of mortality. Prompt diagnosis and treatment are crucial to avoid a fatal outcome. However, very limited reports have focused on HLH during chemotherapy (Ch-HLH) due to a low incidence and insufficient knowledge.Case presentationA 22-year-old male was diagnosed with acute monocytic leukemia with FLT3-ITD and DNMT3A mutations and pulmonary infection. He received IA regimen (Idarubicin, 8 mg/m2/d for 3 days and cytarabine, 100 mg/m2/d for 7 days) chemotherapy, anti-infection drugs and blood components transfusions. During the stage of bone marrow suppression, he presented with a fever, cytopenia (WBC, 0.43 × 109/L; Hb, 73 g/L and PLT, 1 × 109/L), refractory coagulation dysfunction (APTT, 104.0 s; PT, 30.5 s and Fbg, 0.87 g/L), splenomegaly (3 cm below the costal margin), hyperferritinemia (SF > 3000 μg/L), increased soluble interleukin-II receptors (sIL-2R > 7500 u/mL) and haemophagocytosis in the bone marrow and was diagnosed with HLH. After he was treated with methylprednisolone at 500 mg/d for 3 days, 120 mg/d for 3 days and 80 mg/d for 3 days, followed by a gradually reduced dose combined with powerful anti-infection drugs, his symptoms subsided and his abnormal parameters recovered to normal levels.ConclusionPatients with HLH in acute leukemia have a high rate of mortality. This case report provides helpful clinical experiences relative to the recognition and treatment of Ch-HLH for clinicians.

Lentivirus-mediated RNA interference targeting FAMLF-1 inhibits cell growth and enhances cell differentiation of acute myeloid leukemia partially differentiated cells via inhibition of AKT and c-MYC.

Genetic heterogeneity is the basis of clinical heterogeneity among different subtypes of AML. We have successfully cloned a gene related to AML termed FAMLF from a FAB-M2 patient's sample of a second largest AML pedigree. Then we revealed at least three splice variants, named as FAMLF-1, FAMLF-2 and FAMLF-3, and found miR181a1/b1 in the second intron of FAMLF gene family. Higher expression of FAMLF-1 was related to a higher complete remission (CR) rate, but shorter relapse free survival (RFS) in AML. We further found that the FAMLF-1 single nucleotide polymorphism (SNP) haplotype and its expression were positively correlated to clinical parameters of acute myeloid leukemia partially differentiated (FAB-M2) patients, but not FAB non-M2 patients or Acute Monocytic Leukemia (FAB-M5) patients. GTAGG SNP haplotype of FAMLF gene might increase FAB-M2 susceptibility in Han population and act as a useful candidate biomarker for FAB-M2 screening. We also demonstrated that FAMLF-1 gene silencing in FAB-M2 cells could lead to proliferation inhibition, cell cycle G0/G1 phase arrest, and differentiation promotion independent of its intronic miR-181a1, which might be related to Akt/c-Myc pathway. These findings reveal a role of FAMLF-1 as a potential pathogenic gene for FAB-M2.

Evaluation of Cytotoxic, Oxidative, and Pro-Inflammatory Effects of Aqueous Cigarette Smoke Extract on Human Monocytes: A Potential Model System for Assessment of Next-Generation Tobacco and Nicotine Products

Tobacco smoking is a risk factor for the development of atherosclerosis. One strategy to reduce the harmful side effects of tobacco smoking is the development of next-generation tobacco and nicotine products. A crucial step in the development of atherosclerosis is the activation and adhesion of monocytes. Remarkably, little is known about the direct effects of cigarette smoking on monocytes. In this study, we developed methods to determine the detrimental effects of aqueous cigarette smoke extract (CSEaq) from 3R4F reference cigarettes on human monocytes in vitro. First, we tested the cytotoxic effects of CSEaq on human peripheral blood monocyte cell line derived from an acute monocytic leukemia patient (THP-1) and primary monocytes. Treatment of THP-1 cells with CSEaq reduced cell viability at dosages ≥40% in a dose-dependent manner. Cell viability of primary monocytes was reduced by CSEaq at dosages ≥10%. CSEaq activated the antioxidative NRF2 system and its target gene NAD(P)H dehydrogenase, quinone 1 in a dose-dependent manner in both cell types. Expression of target gene HMOX1 was increased only in THP-1 cells. In addition, we showed an increased expression of NADPH oxidase subunit NOX2 in response to CSEaq in THP-1 cells, but not in primary monocytes. Furthermore, CSEaq strongly induced adhesion molecule intercellular adhesion molecule 1 in THP-1 cells, but not in primary monocytes. Pro-inflammatory markers CCL2 and CD31 showed a strong induction in response to CSEaq in THP-1 cells, but they remained unchanged in primary monocytes. In conclusion, we have developed in vitro test systems allowing the evaluation of cytotoxic, oxidative, and pro-inflammatory effects of CSEaq on monocytes. These test systems can be considered potential model systems for assessment of next-generation tobacco and nicotine products. Although THP-1 cells were more susceptible to changes of gene expression, primary monocytes showed a stronger reduction of cell viability in response to CSEaq.

Scanning Electrochemical Microscopy Imaging during Respiratory Burst in Human Cell.

Phagocytic cells, such as neutrophils and monocytes, consume oxygen and generate reactive oxygen species (ROS) in response to external stimuli. Among the various ROS, the superoxide anion radical is known to be primarily produced by nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase. In the current study, we attempt to evaluate the respiratory burst by monitoring the rapid consumption of oxygen by using scanning electrochemical microscopy (SECM) imaging. The respiratory burst was measured in a human monocytic cell line (THP-1 cells) derived from an acute monocytic leukemia patient under the effect of the exogenous addition of phorbol 12-myristate 13-acetate, which acts as a differentiation inducer. SECM imaging composed of a microelectrode was used to compare oxygen consumption between normal cellular respiration and during respiratory burst in THP-1 cells. Two-dimensional respiratory activity imaging was performed using XY-scan. In addition, the quantitative evaluation of oxygen consumption in THP-1 cells was performed using a Z-scan. The results obtained show higher consumption of oxygen in cells undergoing respiratory burst. SECM imaging is thus claimed to be a highly sensitive and appropriate technique compared to other existing techniques available for evaluating oxidative stress in human cells, making it potentially useful for widespread applications in biomedical research and clinical trials.

Changes in expression of WT1 during induced differentiation of the acute myeloid leukemia cell lines by treatment with 5-aza-2'-deoxycytidine and all-trans retinoic acid.

The aim of the present study was to investigate the effect of 5-aza-2'-deoxycytidine (decitabine; DAC) and all-trans retinoic acid (ATRA) on Wilms' tumor 1 (WT1) in acute myeloid leukemia (AML) in vitro. The methylation status of the WT1 promoter was analyzed using methylation-specific polymerase chain reaction (MSP). The expression level of WT1 was detected by reverse transcription-quantitative polymerase chain reaction. The effect of DAC and ATRA on cell differentiation was evaluated by flow cytometry. The WT1 gene was methylated in U937 cells, but unmethylated in SHI-1 and K562 cells; the U937 cells did not express the WT1 gene, but the SHI-1 and K562 cells highly expressed the WT1 gene. DAC and ATRA, alone or in combination, exhibited no effect on the expression level of WT1 in the U937 cells and on the differentiation of the K562 cells. The combined treatment of DAC and ATRA markedly decreased the WT1 expression levels of the SHI-1 and K562 cells, and induced the differentiation of the SHI-1 and U937 cells. In the SHI-1 cells, WT1 expression changed inversely to the dynamic changes of cluster of differentiation 11b-positive rates. In conclusion, the combined treatment of DAC and ATRA has clinical therapeutic potential in acute monocytic leukemia patients with high WT1 expression and a poor response to standard induction chemotherapy.

Hyperleptinemia suppresses aggravation of arthritis of collagen-antibody-induced arthritis in mice

BackgroundLeptin is an adipocytokine produced by adipocytes and controlling body weight. It is unclear whether leptin works as a proinflammatory or an anti-inflammatory cytokine. We investigated the effects of hyperleptinemia on leptin transgenic (LepTg) mice in terms of cartilage destruction, bone destruction, joint synovitis, and serum cytokine levels by using a mouse model of collagen-antibody-induced arthritis (CAIA). MethodsCAIA was induced for female age-matched 6- to 8-week-old C57BL/6J control mice and LepTg mice. Mice were injected intraperitoneally with 5mg of a combination of monoclonal antibody specific for type II collagen on day 0 and 12.5mg of lipopolysaccharide (LPS) on day 3. Clinical evaluation of arthritis was monitored for 14days, and hind paws were examined clinically and histologically. Serum cytokine levels of interleukin (IL)-1β, IL-6, IL-10, and IL-17 and tumor necrosis factor alpha (TNF-α) were also analyzed on days 0 and 5. Moreover, THP-1 cells, which are human monocytic cell line derived from an acute monocytic leukemia patient, were cultured and differentiated into macrophages. The effects of leptin on messenger RNA (mRNA) expression of IL-6 were examined by real-time quantitative polymerase chain reaction (RT-PCR). ResultsSerum leptin concentrations were approximately ninefold higher in LepTg mice (62.0±20.7ng/ml) than in control mice (7.2±0.5ng/ml). Severity of clinical paw swelling, arthritis score, synovial hyperplasia, and cartilage damage were suppressed in LepTg mice with CAIA. Although serum cytokine levels of IL-1β, IL-17, and IL-10 and TNF-α showed no significant changes in two mice, serum levels of IL-6 in LepTg mice were suppressed at day 5. Moreover, in vitro study showed that IL-6 elevation following LPS exposure in THP-1 cells was suppressed with high leptin concentrations. ConclusionOur finding suggests that hyperleptinemia suppress IL-6 responses and progression of joint inflammation. Leptin may play an anti-inflammatory role under hyperleptinemia.

Treating cancer stem cells and cancer metastasis using glucose-coated gold nanoparticles.

Cancer ranks among the leading causes of human mortality. Cancer becomes intractable when it spreads from the primary tumor site to various organs (such as bone, lung, liver, and then brain). Unlike solid tumor cells, cancer stem cells and metastatic cancer cells grow in a non-attached (suspension) form when moving from their source to other locations in the body. Due to the non-attached growth nature, metastasis is often first detected in the circulatory systems, for instance in a lymph node near the primary tumor. Cancer research over the past several decades has primarily focused on treating solid tumors, but targeted therapy to treat cancer stem cells and cancer metastasis has yet to be developed. Because cancers undergo faster metabolism and consume more glucose than normal cells, glucose was chosen in this study as a reagent to target cancer cells. In particular, by covalently binding gold nanoparticles (GNPs) with thio-PEG (polyethylene glycol) and thio-glucose, the resulting functionalized GNPs (Glu-GNPs) were created for targeted treatment of cancer metastasis and cancer stem cells. Suspension cancer cell THP-1 (human monocytic cell line derived from acute monocytic leukemia patients) was selected because it has properties similar to cancer stem cells and has been used as a metastatic cancer cell model for in vitro studies. To take advantage of cancer cells’ elevated glucose consumption over normal cells, different starvation periods were screened in order to achieve optimal treatment effects. Cancer cells were then fed using Glu-GNPs followed by X-ray irradiation treatment. For comparison, solid tumor MCF-7 cells (breast cancer cell line) were studied as well. Our irradiation experimental results show that Glu-GNPs are better irradiation sensitizers to treat THP-1 cells than MCF-7 cells, or Glu-GNPs enhance the cancer killing of THP-1 cells 20% more than X-ray irradiation alone and GNP treatment alone. This finding can help oncologists to design therapeutic strategies to target cancer stem cells and cancer metastasis.