Tolerogenic anti-IL-2 mAb prevents graft-versus-host disease while preserving strong graft-versus-leukemia activity.

Donor T cells mediate both graft-versus-leukemia (GVL) activity and graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT). Development of methods that preserve GVL activity while preventing GVHD remains a long-sought goal. Tolerogenic anti-interleukin-2 (IL-2) monoclonal antibody (JES6-1) forms anti-IL-2/IL-2 complexes that block IL-2 binding to IL-2Rß and IL-2Rγ on conventional T cells that have low expression of IL-2Rα. Here, we show that administration of JES6 early after allo-HCT in mice markedly attenuates acute GVHD while preserving GVL activity that is dramatically stronger than observed with tacrolimus (TAC) treatment. The anti-IL-2 treatment downregulated activation of the IL-2-Stat5 pathway and reduced production of granulocyte-macrophage colony-stimulating factor (GM-CSF). In GVHD target tissues, enhanced T-cell programmed cell death protein 1 (PD-1) interaction with tissue-programmed cell death-ligand 1 (PD-L1) led to reduced activation of protein kinase-mammalian target of rapamycin pathway and increased expression of eomesodermin and B-lymphocyte-induced maturation protein-1, increased T-cell anergy/exhaustion, expansion of Foxp3-IL-10-producing type 1 regulatory (Tr1) cells, and depletion of GM-CSF-producing T helper type 1 (Th1)/cytotoxic T cell type 1 (Tc1) cells. In recipient lymphoid tissues, lack of donor T-cell PD-1 interaction with tissue PD-L1 preserved donor PD-1+TCF-1+Ly108+CD8+ T memory progenitors and functional effectors that have strong GVL activity. Anti-IL-2 and TAC treatments have qualitatively distinct effects on donor T cells in the lymphoid tissues, and CD8+ T memory progenitor cells are enriched with anti-IL-2 treatment compared with TAC treatment. We conclude that administration of tolerogenic anti-IL-2 monoclonal antibody early after allo-HCT represents a novel approach for preventing acute GVHD while preserving GVL activity.

A novel prognostic nomogram for adult acute lymphoblastic leukemia: a comprehensive analysis of 321 patients.

The cure rate of acute lymphoblastic leukemia (ALL) in adolescents and adults remains poor. This study aimed to establish a prognostic model for ≥14-year-old patients with ALL to guide treatment decisions. We retrospectively analyzed the data of 321 ALL patients between January 2017 and June 2020. Patients were randomly (2:1 ratio) divided into either the training or validation set. A nomogram was used to construct a prognostic model. Multivariate Cox analysis of the training set showed that age > 50 years, white blood cell count > 28.52×109/L, and MLL rearrangement were independent risk factors for overall survival (OS), while platelet count >37×109/L was an independent protective factor. The nomogram was established according to these independent prognostic factors in the training set, where patients were grouped into two categories: low-risk (≤13.15) and high-risk (>13.15). The survival analysis, for either total patients or sub-group patients, showed that both OS and progression-free survival (PFS) of low-risk patients was significantly better than that of high-risk patients. Moreover, treatment analysis showed that both OS and progression-free survival (PFS) of ALL with stem cell transplantation (SCT) were significantly better than that of ALL without SCT. Further stratified analysis showed that in low-risk patients, the OS and PFS of patients with SCT were significantly better than those of patients without SCT. In contrast, in high-risk patients, compared with non-SCT patients, receiving SCT can only significantly prolong the PFS, but it does not benefit the OS. We established a simple and effective prognostic model for ≥ 14-year-old patients with ALL that can provide accurate risk stratification and determine the clinical strategy.

Curcumin derivative C817 inhibits proliferation of imatinib-resistant chronic myeloid leukemia cells with wild-type or mutant Bcr-Abl in vitro.

AIM: To find new kinase inhibitors that overcome the imatinib resistance in treatment of chronic myeloid leukemia (CML), we synthesized C817, a novel derivative of curcumin, and tested its activities against wild-type (WT) and imatinib-resistant mutant Abl kinases, as well as in imatinib-sensitive and resistant CML cells in vitro. METHODS: 32D cells harboring WT or mutant Abl kinases (nucleotide binding P-loop mutants Q252H, Y253F, and imatinib contact residue mutant T315I), as well as K562/G01 cells (with whole Bcr-Abl gene amplication) were tested. Kinase activity was measured using Kinase-Glo Luminescent Kinase Assay Platform in recombinant WT and mutant (Q252H, Y253F, and T315I) Abl kinases. Cell proliferation and apoptosis were examined using MTT assay and flow cytometry, respectively. The phosphorylation levels of Bcr-Abl initiated signaling proteins were analyzed using Western blotting. Colony forming units (CFU) growth and long term culture-initiating cells (LTC-ICs) were used to test the effects of C817 on human leukemia progenitor/stem cells. RESULTS: C817 potently inhibited both WT and mutant (Q252H, Y253F, and T315I) Abl kinase activities in a non-ATP competitive manner with the values of IC50 at low nanomole levels. In consistent with above results, C817 suppressed the growth of both imatinib-sensitive and resistant CML cells, including wild-type K562, K562/G01, 32D-T315I, 32D-Q252H, and 32D-Y253F cells with the values of IC50 at low micromole levels. C817 (0.5 or 1 µmol/L) dose-dependently inhibited the phosphorylation of Bcr-Abl and downstream proteins STAT-5 and CrkL in imatinib-resistant K562/G01 cells. Furthermore, C817 significantly suppressed CFU growth and LTC-ICs, implicating that C817 could eradiate human leukemia progenitor/stem cells. CONCLUSION: C817 is a promising compound for treatment of CML patients with Bcr-Abl kinase domain mutations that confer imatinib resistance.

The effect to IL-3Ralpha, downstream PI3k/Akt signaling of all-trans retinoic acid and arsenic trioxide in NB4 cells.

All-trans retinoic acid (ATRA) and arsenic trioxide (As2O3) are the classic drugs used for induction therapy of acute promyelocytic leukemia (APL). IL-3Ralpha (CD123) is a specific marker of acute myeloid leukemia stem cells (AML-LSCs). The over-expression of IL-3Ralpha in patients with AML is related to high white blood cells counts, high percentages of blast cells, and poor prognosis. Moreover, in some studies, IL-3Ralpha has been considered a new detection marker of minimal residual disease in the bone marrow from patients with APL. In contrast to ATRA, As2O3 reduces both mRNA and protein expression of IL-3Ralpha and inhibits the activity of PI3K/Akt after 24 h, 48 h, and 72 h of exposure. Furthermore, NB4 cells adhered to the human stroma cell line HS-5 cells were used as an in vitro model of APL cells in the bone marrow microenvironment. Our results demonstrate that adhesion to HS-5 cells up-regulated IL-3Ralpha protein expression and activated the downstream PI3K/Akt signaling pathway in NB4 cells. Compared with ATRA, As2O3 more potently inhibits proliferation of NB4 cells adhered to stroma cells.

[The Effect of TCP1 Expression on the Proliferation and the Accumulation of Intracellular Drug of HL60/A and HL60 Cell and Its Mechanism].

OBJECTIVE: To investigate the effect of TCP1 expression on the proliferation and the accumulation of intracellular drug of HL60/A and HL60 cells and its possible molecular mechanism. METHODS: Lentiviral transfection technology was used to construct HL60/A and HL60 cells with knocked down or overexpressed TCP1 and their control cells. The efficiency of knockdown and overexpression was evaluated by Western blot. The cell proliferation was detected by CCK-8 assay. The intracellular drug accumulation was detected by laser confocal detection and flow cytometry. The expression levels of MRP1, P-gP and p-AKT were evaluated by flow cytometry and Western blot. RESULTS: After TCP1 was knocked down,the proliferation ability of HL60/A cells was significantly reduced, the accumulation of intracellular drug was significantly increased and the expression of MRP1 and P-gP protein were decreased. After TCP1 was overexpressed, the proliferation ability of HL60 was significantly increased, the accumulation of intracellular drug was significantly decreased and the expression of MRP1 and P-gP protein were increased. Intervention of LY294002 significantly antagonized the promotion on cell proliferation, the inhibition on intracellular drug accumulation and the expression of MRP1 and P-gP mediated by TCP1 overexpressing in HL60 cells. CONCLUSION: TCP1 can promote cell proliferation, improve the expression of MRP1 and P-gP by activating PI3K/AKT signal, and reduce intracellular drug accumulation.

Bovine serum albumin-based probe carrier platform for electrochemical DNA biosensing.

A bovine serum albumin (BSA)-monolayer-based probe carrier platform is shown to improve the performance of a conventional thiolated single-stranded DNA probe self-assembled-monolayer-based electrochemical DNA hybridization biosensor. A detection limit of 0.5 fM can be obtained in a very reproducible manner (relative standard deviation <5%), along with high specificity. The performance of the biosensor can be attributed primarily to the enhanced spatial positioning range and accessibility of the probes on the BSA-based platform. Furthermore, the novel biosensor shows high resistance to nonspecific adsorption of nucleic acid and protein and can be directly employed in detection in biological fluids. These advantages give this simple developed methodology great promise for a wide range of nucleic acid testing.

Sequence-specific electrochemical detection of double-strand PCR amplicons of PML/RARα fusion gene in acute promyelocytic leukemia.

A novel electrochemical method for the sequence-specific detection of double-stranded polymerase chain reaction (PCR) products of PML/RARα fusion gene in acute promyelocytic leukemia (APL) was described in detail. Based on a "sandwich" sensing mode involving a pair of locked nucleic acids probes (capture probe and reporter probe), this DNA sensor exhibited excellent selectivity and specificity. The direct and quantitative analysis of double-stranded complementary was firstly performed by our sensor without the use of alkali, helicase enzymes, or denaturants. Finally, combining PCR technique with electrochemical detection scheme, PCR amplicons (191 bp) of the PML/RARα fusion gene were obtained and rapidly identified with a low detection limit of 79 fmol in the 100-µL hybridization system. The results clearly showed the power of sensor as a promising tool for the sensitive, specific, and portable detection of APL and other diseases.

IL-22-dependent dysbiosis and mononuclear phagocyte depletion contribute to steroid-resistant gut graft-versus-host disease in mice.

Efforts to improve the prognosis of steroid-resistant gut acute graft-versus-host-disease (SR-Gut-aGVHD) have suffered from poor understanding of its pathogenesis. Here we show that the pathogenesis of SR-Gut-aGVHD is associated with reduction of IFN-γ+ Th/Tc1 cells and preferential expansion of IL-17-IL-22+ Th/Tc22 cells. The IL-22 from Th/Tc22 cells causes dysbiosis in a Reg3γ-dependent manner. Transplantation of IFN-γ-deficient donor CD8+ T cells in the absence of CD4+ T cells produces a phenocopy of SR-Gut-aGVHD. IFN-γ deficiency in donor CD8+ T cells also leads to a PD-1-dependent depletion of intestinal protective CX3CR1hi mononuclear phagocytes (MNP), which also augments expansion of Tc22 cells. Supporting the dual regulation, simultaneous dysbiosis induction and depletion of CX3CR1hi MNP results in full-blown Gut-aGVHD. Our results thus provide insights into SR-Gut-aGVHD pathogenesis and suggest the potential efficacy of IL-22 antagonists and IFN-γ agonists in SR-Gut-aGVHD therapy.

N-terminal and C-terminal heparin-binding domain polypeptides derived from fibronectin reduce adhesion and invasion of liver cancer cells.

BACKGROUND: Fibronectin (FN) is known to be a large multifunction glycoprotein with binding sites for many substances, including N-terminal and C-terminal heparin-binding domains. We investigated the effects of highly purified rhFNHN29 and rhFNHC36 polypeptides originally cloned from the two heparin-binding domains on the adhesion and invasion of highly metastatic human hepatocellular carcinoma cells (MHCC97H) and analyzed the underlying mechanism involved. METHODS: The MHCC97H cells that adhered to FN in the presence of various concentrations of rhFNHN29 and rhFNHC36 polypeptides were stained with crystal violet and measured, and the effects of rhFNHN29 and rhFNHC36 on the invasion of the MHCC97H cells were then detected using the Matrigel invasion assay as well as a lung-metastasis mouse model. The expression level of integrins and focal adhesion kinase (FAK) phosphotyrosyl protein was examined by Western blot, and the activity of matrix metalloproteinases (MMPs) and activator protein 1 (AP-1) was analyzed by gelatin zymography and the electrophoretic mobility band-shift assay (EMSA), respectively. RESULTS: Both of the polypeptides rhFNHN29 and rhFNHC36 inhibited adhesion and invasion of MHCC97H cells; however, rhFNHC36 exhibited inhibition at a lower dose than rhFNHN29. These inhibitory effects were mediated by integrin αvß3 and reversed by a protein tyrosine phosphatase inhibitor. Polypeptides rhFNHN29 and rhFNHC36 abrogated the tyrosine phosphorylation of focal adhesion kinase (p-FAK) and activation of activator protein 1 (AP-1), resulting in the decrease of integrin αv, ß3 and ß1 expression as well as the reduction of MMP-9 activity. CONCLUSIONS: Polypeptides rhFNHN29 and rhFNHC36 could potentially be applicable to human liver cancer as anti-adhesive and anti-invasive agents.

Sepsis progression monitoring via human serum fibronectin detection based on sandwich-type electrochemical immunosensor.

Sepsis has always been a severe clinical problem in critical care medicine due to its rather high mortality and poor prognosis. The current study reported for the first time a practical immunosensor for fibronectin (FN) detection in human serum by electrochemistry. A simple but robust sandwich-type strategy was employed without any complex design or material modifications, which exhibited a linear calibration plot over the 15.625-500 ng/mL concentration range and a detection limit of 15 ng/mL. The results showed that the proposed strategy displayed an excellent selectivity against other non-target substances in human serum, a higher accuracy and a better stability when compared with the traditional enzyme-linked immunosorbent assay (ELISA) in detecting the same or mixed serum from 21 healthy subjects. Furthermore, the proposed electrochemical immunosensor successfully monitored the level of serum FN at various time points in five septic patients during the treatment. These findings demonstrate that the proposed strategy is highly sensitive and accurate in monitoring sepsis progress and has significant clinical improvements over the ELISA methodology, signifying a great potential of a commercial kit.

[Recombinant polypeptide of N-terminal heparin-binding domain of fibronectin antagonizes hepatic failure induced by endotoxin in mice].

OBJECTIVE: To study the preventive effect of recombinant polypeptide of N-terminal heparin-binding domain of fibronectin on hepatic failure induced by endotoxin in mice. METHODS: The 40 hepatic failure Balb/C mice were established by intraperitoneal injection of lipopolysaccharide (LPS) and d-galactosamine (GalN). The mice were randomly divided into two groups, one for polypeptide treatment, the othe for saline treatment.Another 20 mice were used as normal control. Half hour prior to, 1, 2, and 3 hours after injection of LPS and GalN, the rhFNHN-29 polypeptide (10 mg/kg) was injected through the tail vein of mice. The same volume of saline was given to the saline treated group and the normal control group.Six hours after the injection of LPS and GalN, 250 microl blood was taken from the eye vein of each mouse for plasma TNFalpha testing, and 72 hours after the injection, mortality rates of the mice of different groups were observated. The liver, lung, heart, kidney, and brain tissues of the survival mice were examined for histopathology after 72 hours. The Liver tissue was also examined for electron micrograph and for mRNA expression of TNFalpha, IL-1beta, IL-6 by RT-PCR. RESULTS: The 72 hours mortality rates in saline-treated and polypeptide treated-mice were 70% and 15% respectively (P < 0.01). The histopathology showed that necrosis occurred less on the hepatocytes of polypeptide treated mice than on the saline treated ones. The ultrastructure of hepatocyte under the electron microscope showed that cell apparatus of saline treated mice were destroyed and cytoplasm become loose. The expression level of TNFalpha, IL-1beta, IL-6 mRNA on hepatocytes in polypeptide treated mice was significantly lower (1.26 +/- 0.37, 0.98 +/- 0.21, 0.43 +/- 0.17, 87.43 +/- 16.7 respectively) than that in the saline treated ones (1.98 +/- 0.56, 1.24 +/- 0.35, 0.64 +/- 0.25 and 236.11 +/- 32.7, respectively) (P < 0.01). Similarly, the plasm TNFalpha level (87.43 +/- 16.7) in polypeptide treated group was significantly lower than that (236.11 +/- 32.7) in the saline treated group (P < 0.01). CONCLUSION: The rhFNHN-29 polypeptide can prevent and treat hepatic failure induced by endotoxin. The mechanism by which the polypeptide takes the effect may involve its ability to down-regulate expression of those inflammation factors such as TNFalpha, IL-1beta, IL-6.

[Selective effect of nispex in inhibiting human cancer cell proliferation and inducing cell apoptosis].

OBJECTIVE: To observe the in vitro anticancer effect of Nispex, the total flavonoids extract from Scurrula parasitic L. METHODS: The cell proliferation inhibitory effects of Nispex on various kinds of tumor cells or non-tumor cells in human and rats were detected with MTT assay and colony forming assay respectively, the cell apoptosis induced by Nispex was detected by AO/EB fluorescence staining, TUNEL assay and AnnexinV-FITC/PI flow cytometry. RESULTS: Nispex could significantly inhibit human cancer cell proliferation and induce human cancer cell apoptosis, especially to the proliferative cell group, but its inhibition on human non-tumor cell was insignificant, and showed no effect on murine cancer cells in the tested scope. CONCLUSION: Nispex is a nature plant extract which shows good selectivity for killing human cancer cell.

[Study on cytotoxic activities on human leukemia cell line HL-60 by flavonoids extracts of Scurrula parasitica from four different host trees].

OBJECTIVE: To compare the anticancer effects of flavonoids extracts of Scurrula parasitica from different host trees in vitro. METHOD: 80% ethanol extracts of S. parasitica parasitizing on Nernium indicum, Morus alba, Opsmanthus fragrans, and Sapindus mulorossi were purified by polyamides column chromatography, and the eluates of 30%, 50%, 70% and 90% ethanol were mixed as flavonoids extracts. Human acute myeloid leukemia cell line HL-60 was used to evaluate the cytotoxicity induced by flavonoids extracts of S. parasitica L with MTT assay. Apoptosis was detected by AO/EB fluorescence staining and DNA fragmentation analysis, apoptosis rates and cell cycle distribution were detected by flow cytometry analysis. RESULT: Extract of S. parasitica parasitizing on N. indicum (NISPEX) was the most sensitive to HL-60 cells of the 4 different host trees, the IC50 value being 0.60 mg x L(-1); and extract of S. parasitica parasitizing on M. alba took the second place, the IC50 value, being 2.49 mg x L(-1); extract of S. parasitica parasitizing on O. fragrans had no effectiveness as high as 50 mg x L(-1) concentration. NISPEX induced HL-60 cell apoptosis and inhibited the cell proliferation in dose and time-dependent manner. Cell cycles were arrested at G0-G1 phase after treated with NISPEX. CONCLUSION: Anticancer effects of S. parasitica correlated with the host trees. Flavonoids extracts of S. parasitica parasitizing on N. indicum exhibited comparatively better anticancer activity in vitro among the host trees studied. NISPEX is found to be a good candidate for anticancer.

Expression and role of granulocyte macrophage colony-stimulating factor receptor (GM-CSFR) and granulocyte colony-stimulating factor receptor (G-CSFR) on Ph-positive acute B lymphoblastic leukemia.

OBJECTIVE: We observed that ph + ALL patients administrated with recombinant human G-CSF (rhG-CSF) after intense chemotherapy have presented a trend of disease relapse. Thus, we aim to thoroughly investigate the expression and role of GM-CSFR and G-CSFR on ph + ALL patients. METHOD: SUP-B15, BALL-1 and primary leukemia cells were used in this study. Transcript levels were analyzed by quantitative PCR while cell viability was measured using a CCK-8 assay. Flow cytometry was used to assess the different stages of cell cycle. RESULTS: We found that the mRNA expression levels of GM-CSFR and G-CSFR were higher in patients with ph + ALL, as well as in SUP-B15 cells. rhG-CSF was also observed to promote the viability of SUP-B15 cells while inversely inhibiting BALL-1 cell viability. In addition, we also determined that rhG-CSF (100 ng/ml) decreased the sensitivity of SUP-B15 cells to imatinib and nilotinib, while the results were exactly the contrary for dasatinib. CONCLUSION: We demonstrated high expression levels of GM-CSFR and G-CSFR, as well as their promotable role for viability in ph + ALL cells. We further found that rhG-CSF influenced the sensitivity of SUP-B15 cells to TKIs.

[Expression and Clinical Significances of HGFA, Matriptase, HAI-1 and HAI-2 in Acute Myeloid Leukemia].

OBJECTIVE: To investigate the expression and clinical significances of HGFA, Matriptase, HAI-1 and HAI-2 in patients with acute myeloid leukemia (AML). METHODS: The bone marrow samples from 91 AML patients, 41 AML patients in complete remission, and 32 normal controls were collected. Real time fluorescence quantitative RT-PCR (qRT-PCR) was used to detect the mRNA expressions levels of HGFA, Matriptase, HAI-1, HAI-2 . The expressions of these genes were compared among AML untreated group, the complete remission group and the healthy control group. The correlation of their expression with clinical characteristics was analyzed. RESULTS: The level of HGFA in the AML untreated group was higher than that in the healthy control group（P<0.05）, while the HAI-2 mRNA level was lower than that in the healthy control group（P<0.05）. The mRNA levels of HAI-1 and Matriptase were not changed significantly in all groups. The HAI-2 mRNA expression level was significantly lower in the high white blood cell group (P<0.05). CONCLUSION: The abnormal activation of HGF/c-Met signaling system in AML may result from the increase of HGFA expression and the decrease of HAI-2 expression of the upstream regulatory factors.

2'-Fluoro ribonucleic acid modified DNA dual-probe sensing strategy for enzyme-amplified electrochemical detection of double-strand DNA of PML/RARα related fusion gene.

In the study, a novel sensing strategy based on dual-probe mode, which involved two groups of 2'-fluoro ribonucleic acid (2'-F RNA) modified probes, was designed for the detection of synthetic target double-strand DNA (dsDNA) of PML/RARα fusion genes in APL. And each pair of probes contained a thiolated capture probe (C1 or C2) immobilized on one of electrode surfaces in the dual-channel electrochemical biosensor and a biotinylated reporter probe (R1 or R2). The two groups of 2'-F RNA modified probes were separately complementary with the corresponding strand (Sa or Sb) from target dsDNA in order to prevent renaturation of target dsDNA. Through flanking target dsDNA, two "sandwitch" complexes (C1/Sa/R1 and C2/Sb/R2) were separately shaped by capture probes (C1 and C2) and free reporter probes (R1 and R2) in hybridization solution on the surfaces of different electrodes after the thermal denaturation. The biotin-modified enzyme which produced the measurable electrochemical current signal was localized to the surface by affinity binding between biotin with streptavidin. Under the optimal condition, the biosensor was able to detect 84 fM target dsDNA and showed a good specificity in PBS hybridization solution. Otherwise, the investigations of the specificity and sensitivity of the biosensor were carried out further in the mixed hybridization solution containing different kinds of mismatch sequences as interference background. It can be seen that under a certain interference background, the method still exhibited excellent selectivity and specificity for the discrimination between the fully-complementary and the mismatch sequences. The results of our research laid a good basis of further detection research in practical samples.

Efficacy of ZnPcS2P2 photodynamic therapy solely or with tumor vaccines on mouse tumor models.

BACKGROUND AND OBJECTIVES: Granulocyte-macrophage colony-stimulating factor (GM-CSF) and B7.1 transduced tumor vaccine cells could induce efficient anti-tumor immune response. It is interesting to study whether they could be an adjuvant to photodynamic therapy (PDT). Recent in vitro study proved that novel photosensitizer ZnPcS2P2 has capability of effective photodynamic killing of leukemic cells. In this preliminary study, we evaluated the photodynamic efficacy of ZnPcS2P2 on two tumor models and the improving anti-tumor efficacy of PDT in combination with GM-CSF gene-transduced vaccine and B7.1 gene-transduced vaccine. METHODS: Nude mice bearing human leukemia xenograft and C57BL/6 mice bearing EL-4 thymic lymphoma were used to evaluate photodynamic efficacy of ZnPcS2P2. The EL-4 thymic lymphoma was used to test the improving anti-tumor efficacy of ZnPcS2P2-PDT in combination with GM-CSF gene-transduced vaccine and B7.1 gene-transduced vaccine. Each vaccine was administered near the tumor bed three times: 2 days before PDT, 0 and 2 days after PDT. RESULTS: ZnPcS2P2-PDT could significantly reduce tumor growth and prolong the survival time in both tumor models. Improving anti-tumor efficacy of ZnPcS2P2-PDT was demonstrated when utilizing GM-CSF-transduced vaccine and B7.1-transduced vaccine prior to and after ZnPcS2P2-PDT in lymphoma-bearing mice. Twenty-five percent of lymphoma-bearing mice were completely cured with a combination of PDT and vaccine cells. CONCLUSIONS: ZnPcS2P2-PDT may be a beneficial treatment for hemotopoietic malignance. GM-CSF-transduced vaccine and B7.1-transduced vaccine could strengthen ZnPcS2P2-PDT-elicited anti-lymphoma potency.

PD-L1 interacts with CD80 to regulate graft-versus-leukemia activity of donor CD8+ T cells.

Programmed death ligand-1 (PD-L1) interacts with programmed death-1 (PD-1) and the immunostimulatory molecule CD80 and functions as a checkpoint to regulate immune responses. The interaction of PD-L1 with CD80 alone has been shown to exacerbate the severity of graft-versus-host disease (GVHD), whereas costimulation of CD80 and PD-1 ameliorates GVHD. Here we have demonstrated that temporary depletion of donor CD4+ T cells early after hematopoietic cell transplantation effectively prevents GVHD while preserving strong graft-versus-leukemia (GVL) effects in allogeneic and xenogeneic murine GVHD models. Depletion of donor CD4+ T cells increased serum IFN-γ but reduced IL-2 concentrations, leading to upregulation of PD-L1 expression by recipient tissues and donor CD8+ T cells. In GVHD target tissues, the interactions of PD-L1 with PD-1 on donor CD8+ T cells cause anergy, exhaustion, and apoptosis, thereby preventing GVHD. In lymphoid tissues, the interactions of PD-L1 with CD80 augment CD8+ T cell expansion without increasing anergy, exhaustion, or apoptosis, resulting in strong GVL effects. These results indicate that the outcome of PD-L1-mediated signaling in CD8+ T cells depends on the presence or absence of CD4+ T cells, the nature of the interacting receptor expressed by CD8+ T cells, and the tissue environment in which the signaling occurs.

Hepatocyte growth factor promotes proliferation, invasion, and metastasis of myeloid leukemia cells through PI3K-AKT and MAPK/ERK signaling pathway.

This study aims to investigate effects of HGF expression on biological behaviors of Kasumi-1 and HL60. Expression of HGF and c-Met gene were detected using qRT-PCR. Short hairpin RNA (shRNA) was used to reduce HGF expression. Silencing effect of shRNA was verified by qRT-PCR and western blot. Cell reproductive capacity, cell clonality and cell cycle (apoptosis) were detected by CCK-8, clone formation, flow cytometry (FCM), respectively. Cell adhesion, cell invasion ability and cell proliferation were also examined. Changes of PI3K-AKT, MAPK/ERK signaling factors were detected by western blot. HGF and c-Met expression in first-vist AML group was significantly higher than in AML-relief and normal control group. HGF shRNA can inhibit cell proliferation, inhibit cloning ability. Compared with control group, apoptosis ratios of Kasumi-1 and HL60 cell in interference groups were significantly higher. After shRNA interference, the number of adherent cells and transmembrane cells were significantly decreased compared with control group. Meanwhile, shRNA also down-regulated Bad, Bcl-XL, Bcl-2, CDK1, Cyclin B, MMP2, MMP9, and up-regulated cleaved caspase9, cleaved caspase3, cleaved PARP, Bax, and P21. Moreover, phosphorylated c-Met, AKT, Erk, and mTOR were also reduced. In conclusion, HGF and c-Met gene highly expressed among first-visit AML patients, but decreased after relief treatment. HGF may promote proliferation, invasion, and metastasis of AML cells through PI3K-AKT and MAPK/ERK signaling pathway. Therefore, proliferation and invasion ability of AML cell can be inhibited by down-regulating HGF gene to retardate cell in G2/M stage.

Experimental studies of the effects of ZnPcS2P2-based-photodynamic therapy on bone marrow purging.

BACKGROUND: An effective purging technique plays an important role in autologous hematopoietic stem cells transplantation. Photodynamic therapy (PDT) provides a novel approach for this purpose. This study dealt with the purging effects of di-sulfo-di-phthalimidomethyl phthalolcyanine zinc (ZnPcS2P2)-based photodynamic therapy (ZnPc-PDT). METHODS: Fluorescence intensity of cell extracts was measured using a fluorescence spectrophotometry. The proliferative potency of K562 cells and HL60 cells was detected using MTT colorimetric assay, Typan blue dye exclusion method, colony formation test. The proliferative potency of normal hematopoietic cells was evaluated using mixture colony-forming unit (CFU-Mix), granulocyte-macrophage colony-forming unit (CFU-GM), and erythrocyte colony-forming unit (CFU-E) assays. K562 cells were mixed with normal mononuclear cells (MNCs) at ratios of 1:100 and 1:1000 for creating the model of simulated remission bone marrow. Colony formation test and nested-RT-PCR were carried out to detect the residual K562 cells in cell mixture. RESULTS: After a 5-hour incubation with ZnPcS2P2, the content of ZnPcS2P2 in normal MNCs was the lowest value. At the same time, the content in K562 cells and HL60 cells was very high. Therefore, the time point was selected as the optimal one for irradiating the cell suspensions. ZnPc-PDT could significantly kill proliferative K562 cells and HL60 cells in a dose-dependent manner. At the concentration of 1.0 microg/ml, the inhibitory rate of ZnPc-PDT on the colony formation was 100% for K562 cells, 89.7% for HL60 cells. 0.25 microg/ml ZnPc-PDT could completely photoinactivate residual K562 cells in the simulated remission bone marrow. Under an identical condition, the inhibitory rates of CFU-Mix, CFU-GM, CFU-E were 18.0%, 18.6%, and 17.8% respectively. CONCLUSION: ZnPc-PDT appears to be a promising approach for bone marrow purging.