Application value of metagenomic next-generation sequencing in hematological patients with high-risk febrile neutropenia.

Background: Metagenomic next-generation sequencing (mNGS) is a novel non-invasive and comprehensive technique for etiological diagnosis of infectious diseases. However, its practical significance has been seldom reported in the context of hematological patients with high-risk febrile neutropenia, a unique patient group characterized by neutropenia and compromised immune responses. Methods: This retrospective study evaluated the results of plasma cfDNA sequencing in 164 hematological patients with high-risk febrile neutropenia. We assessed the diagnostic efficacy and clinical impact of mNGS, comparing it with conventional microbiological tests. Results: mNGS identified 68 different pathogens in 111 patients, whereas conventional methods detected only 17 pathogen types in 36 patients. mNGS exhibited a significantly higher positive detection rate than conventional methods (67.7% vs. 22.0%, P < 0.001). This improvement was consistent across bacterial (30.5% vs. 9.1%), fungal (19.5% vs. 4.3%), and viral (37.2% vs. 9.1%) infections (P < 0.001 for all comparisons). The anti-infective treatment strategies were adjusted for 51.2% (84/164) of the patients based on the mNGS results. Conclusions: mNGS of plasma cfDNA offers substantial promise for the early detection of pathogens and the timely optimization of anti-infective therapies in hematological patients with high-risk febrile neutropenia.

FAT1 inhibits the proliferation of DLBCL cells via increasing the m6A modification of YAP1 mRNA.

Emerging evidence shows that FAT atypical cadherin 1 (FAT1) mutations occur in lymphoma and are associated with poorer overall survival. Considering that diffuse large B cell lymphoma (DLBCL) is the category of lymphoma with the highest incidence rate, this study aims to explore the role of FAT1 in DLBCL. The findings demonstrate that FAT1 inhibits the proliferation of DLBCL cell lines by downregulating the expression of YAP1 rather than by altering its cellular localization. Mechanistic analysis via meRIP-qPCR/luciferase reporter assays showed that FAT1 increases the m6A modification of YAP1 mRNA 3'UTR and the subsequent binding of heterogeneous nuclear ribonucleoprotein D (HNRNPD) to the m6A modified YAP1 mRNA, thus decreasing the stability of YAP1 mRNA. Furthermore, FAT1 increases YAP1 mRNA 3'UTR m6A modification by decreasing the activity of the TGFß-Smad2/3 pathway and the subsequent expression of ALKBH5, which is regulated at the transcriptional level by Smad2/3. Collectively, these results reveal that FAT1 inhibits the proliferation of DLBCL cells by increasing the m6A modification of the YAP1 mRNA 3'UTR via the TGFß-Smad2/3-ALKBH5 pathway. The findings of this study therefore indicate that FAT1 exerts anti-tumor effects in DLBCL and may represent a novel target in the treatment of this form of lymphoma.

Splenic marginal zone lymphoma with monoclonal IgG: A case report.

RATIONALE: Splenic marginal zone lymphoma (SMZL), an indolent small B-cell lymphoma, is uncommon, and part of the patients exist plasmocytic differentiation and secrete monoclonal paraproteins including IgM predominantly. SMZL with monoclonal IgG is rarer. PATIENT CONCERNS: We report a case of SMZL (49-year-old, male) with monoclonal IgG, MYD88L265P mutation and hepatitis B virus infection. DIAGNOSES: The patient was presented to our hospital with aggravating complaints of dizziness, fatigue, postprandial abdominal distension, and night sweats. The diagnosis was confirmed by clinical manifestations, immunophenotype, bone marrow pathology. INTERVENTIONS: The patient received rituximab-based chemotherapy and sequential ibrutinib in combination with entecavir. OUTCOMES: After 1 year of follow-up, his blood routine examination had returned to normal with normal level of albumin and significantly lower globulin than before, and the spleen was of normal size. LESSONS: We conclude that rituximab-based chemotherapy is the main treatment option for the patients with SMZL, and Bruton's tyrosine kinase inhibitor has also shown beneficial efficacy.

FAT1 inhibits AML autophagy and proliferation via downregulating ATG4B expression.

BACKGROUND: Emerging studies have shown that FAT atypical cadherin 1 (FAT1) and autophagy separately inhibits and promotes acute myeloid leukemia (AML) proliferation. However, it is unknown whether FAT1 were associated with autophagy in regulating AML proliferation. METHODS: AML cell lines, 6-week-old male nude mice and AML patient samples were used in this study. qPCR/Western blot and cell viability/3H-TdR incorporation assays were separately used to detect mRNA/protein levels and cell activity/proliferation. Luciferase reporter assay was used to examine gene promoter activity. Co-IP analysis was used to detect the binding of proteins. RESULTS: In this study, we for the first time demonstrated that FAT1 inhibited AML proliferation by decreasing AML autophagy level. Moreover, FAT1 weakened AML autophagy level via decreasing autophagy related 4B (ATG4B) expression. Mechanistically, we found that FAT1 reduced the phosphorylated and intranuclear SMAD family member 2/3 (smad2/3) protein levels, thus decreasing the activity of ATG4B gene promoter. Furthermore, we found that FAT1 competitively bound to TGF-ßR II which decreased the binding of TGF-ßR II to TGF-ßR I and the subsequent phosphorylation of TGF-ßR I, thus reducing the phosphorylation and intranuclear smad2/3. The experiments in nude mice showed that knockdown of FAT1 promoted AML autophagy and proliferation in vivo. CONCLUSIONS: Collectively, these results revealed that FAT1 downregulates ATG4B expression via inhibiting TGFß-smad2/3 signaling activity, thus decreasing the autophagy level and proliferation activity of AML cells. GENERAL SIGNIFICANCE: Our study suggested that the "FAT1-TGFß-smad2/3-ATG4B-autophagy" pathway may be a novel target for developing new targeted drugs to AML treatment.

An acquired BMF with FANCL gene heterozygous mutation: Case report.

RATIONALE: Bone marrow failure (BMF) includes inherited and acquired BMFs. Acquired BMF can be secondary to various factors, such as autoimmune dysfunction, benzene, drugs, radiation, viral infection and so on. Fanconi anemia (FA) complementation group L (FANCL) is an E3 ubiquitin ligase that participates in the repair of DNA damage. Homozygous or compound heterozygous mutations of FANCL can lead to the onset of FA, which is one of the most common inherited BMFs. PATIENT CONCERNS AND DIAGNOSES: Here, we report a case of acquired BMF. This patient had a history of benzene exposure for half a year before the onset of the disease, and presented with progressive pancytopenia, especially the reduction of erythrocytes and megakaryocyte, without malformation. Interestingly, this patient and his brother/father had a heterozygous (non-homozygous/compound heterozygous) mutation (Exon9, c.745C > T, p.H249Y) in the FANCL gene. INTERVENTIONS AND OUTCOMES: The patient successfully underwent unrelated and fully compatible umbilical cord blood hematopoietic stem cell transplantation. LESSONS SUBSECTIONS: We report for the first time an acquired BMF case with FANCL gene heterozygous mutation, and the mutation site (Exon9, c.745C > T, p.H249Y) has never been reported. This case suggests that heterozygous mutations in FANCL gene may be associated with increased susceptibility to acquired BMF. Based on current reports and this case, we speculate that heterozygous mutations in the FA complementation gene may exist in a certain proportion of tumor and acquired BMF patients, but have not been detected. We recommend routine screening for FA complementation gene mutations in tumor and acquired BMF patients in clinical practice. If positive results are found, further screening can be conducted on their families.

Long-Term Molecular Remission after Treatment with Imatinib in a Chronic Myeloid Leukemia Patient with Extreme Thrombocytosis Harboring Rare e14a3 (b3a3) BCR::ABL1 Transcript: A Case Report.

An atypical BCR::ABL1 fusion gene transcript in chronic myeloid leukemia (CML) patients, even those with variant Philadelphia (Ph) chromosome translocation, is very rare. In the present study, we report a case of CML (41 years, female) with extreme thrombocytosis at onset, with the variant Ph chromosome and rare e14a3 (b3a3) BCR::ABL1 transcript. The patient was prescribed imatinib as a first-line therapy and subsequently achieved complete hematologic remission within 2 months and major molecular response (MMR) within 3 months, and the transcript was undetectable within half a year. During up to nine years of follow-up, the quantification of this rare fusion gene was consistently negative with no BCR::ABL1 kinase domain mutations. Furthermore, we collected previously reported CML cases with the e14a3 (b3a3) transcript that indicated that the e14a3 (b3a3) transcripts appeared to have a larger number of thrombocytosis and variant Ph translocations than CML in general. This subgroup of CML might have better responses and outcomes to imatinib than patients with common transcripts.

[Retracted] The inhibitory effect of MEG3/miR-214/AIFM2 axis on the growth of T-cell lymphoblastic lymphoma.

Following the publication of this paper, it was drawn to the authors' attention by an interested reader that a row of the tumour images featured in Fig. 8A of the above paper were strikingly similar to those featured in Fig. 6A of an article appearing in Oncology Reports that had been published by a different research group at a different institution [Zhang L, Liang X and Li Y: Long non­coding RNA MEG3 inhibits cell growth of gliomas by targeting miR­93 and inactivating PI3K/AKT pathway. Oncol Rep 38: 2408­2416, 2017]. The Editor asked the authors for an explanation to account for the appearance of strikingly similar data in their paper independently, although the authors proved to be uncontactable in this regard, and did not respond to various queries. The Editor has therefore taken an executive decision to retract this paper from International Journal of Oncology without the agreement of the authors. The Editor apologizes to the readership for any inconvenience caused. [the original article was published in International Journal of Oncology 51: 316­326, 2017; DOI: 10.3892/ijo.2017.4006].

NK cell expression of Tim-3: First impressions matter.

Given the heightened interest in manipulation of co-signaling cascades for cancer immunotherapy, we sought to determine how/whether tumors decorated with therapeutic monoclonal antibodies (mAbs) impact the expression of co-signaling molecules on human NK cells. Stimulation of NK cells with aggregated IgG1 resulted in the upregulation of HAVCR2 - the gene encoding T-cell immunoglobulin and mucin-containing domain (Tim)-3 - known to be involved in the induction of peripheral T cell tolerance. This upregulation of HAVCR2 was recapitulated at the protein level, following NK cell stimulation by either mAb opsonized tumors, recombinant human IgG1 Fc multimer, and/or non-Fc stimuli e.g. IL-12/IL-18. The patterns of Tim-3 expression were temporally distinct from the FcR mediated induction of the co-signaling molecule, 4-1BB (CD137), with Tim-3 increases observed twenty minutes following exposure to Fc multimers and remaining at high levels for at least six hours, while increases in CD137 expression were first observed at the four-hour time point. Importantly, these Tim-3+ NK cells were functionally diverse, as evidenced by the fact that their ability to produce IFN-γ in response to an NK cell responsive tumor was strictly dependent upon the stimuli employed for Tim-3 induction. These data suggest that Tim-3 upregulation is the common end-result of NK cell activation by a variety of unique and overlapping stimuli and is not an independent marker of NK cell exhaustion. Furthermore, our observations potentially explain the diverse functionality attributed to Tim-3+ NK cells and should be considered prior to use of anti-Tim-3 inhibitory mAbs for cancer immunotherapy.

MEG3 affects the progression and chemoresistance of T-cell lymphoblastic lymphoma by suppressing epithelial-mesenchymal transition via the PI3K/mTOR pathway.

Long noncoding RNAs (lncRNA) are emerging as integral functional and regulatory components in the development of different diseases including cancer. Maternally expressed gene 3 (MEG3), is a lncRNA, that has a depressed expression in multiple tumor types, including T-cell lymphoblastic lymphoma (T-LBL). However, the molecular mechanisms that regulate the tumorigenic functions of MEG3 in T-LBL remain largely unknown. In this study, we aimed to discover and identify the function of MEG3 in T-LBL tumorigenesis, epithelial-mesenchymal transition (EMT) and drug resistance, and explore their mechanisms of action. Knockdown MEG3 promoted the proliferation, migration, invasion, and drug resistance of T-LBL cells while overexpression of MEG3 gets the opposite results. The mechanism study showed that decreased MEG3 expression in T-LBL cells could activate PI3K/mTOR signaling pathways, increase the expression of p-glycoprotein and affect the expression of EMT markers for transforming to mesenchymal cells in vitro and in vivo. Together, these results indicate that MEG3 could inhibit the migration, invasion, and drug resistance in T-LBL cells by suppression of the PI3K/mTOR pathway. MEG3 might be a potential target, through which poor prognosis with high recurrence and drug resistance of T-LBL in a clinical setting could be reversed.

PD-1 blockade potentially enhances adoptive cytotoxic T cell potency in a human acute myeloid leukaemia animal model.

OBJECTIVES: Acute myeloid leukaemia (AML) is a malignant haematological disease that remains difficult to cure. Cytotoxic T cell (CTL) adoptive infusion therapy may be conducive to tumour remission by boosting physical immunity. Furthermore, programmed death receptor-1 (PD-1) blockade immunotherapy has shown tremendous success in many cancer therapies. METHOD: We attempted to combine these two immunotherapy strategies to intervene in AML by generating AML cellspecific cytotoxic T lymphocytes in vitro and in vivo with an AML cell strain expressing specific antigens. RESULTS: First, we observed that peripheral blood mononuclear cells (PBMCs) could be induced to generate large numbers of CD8+ CTL cells through immune stimulation. In addition, these CD8+ cells could effectively recognize a human AML cell line and exert cytotoxicity. In animal tests, PD-1 blockade combined with CTL infusion could induce significantly more AML tumour reduction than either treatment alone. This synergistic effect was thought to be connected to immune modulation enhancement, as regulatory T cells (Tregs) in the peripheral blood (PB) were found to be suppressed. CONCLUSIONS: This finding suggested the potential application of PD-1 blockade in AML. The present work demonstrated an excellent synergistic tumour therapeutic effect of PD-1 blockade and CTL therapy compared with either treatment alone.

Recombinant human IgG1 based Fc multimers, with limited FcR binding capacity, can effectively inhibit complement-mediated disease.

There is a lack of effective targeted therapies for the treatment of complement dependent diseases. We developed two recombinant Fc multimers, G207 and G211, with limited ability to interact with low/moderate affinity FcγRs, but with high avidity for C1q. These drugs effectively inhibited complement dependent cytotoxicity (CDC) in vitro, and prevented the deposition of C1q, C3b and MAC, on the surface of Ab-opsonized cells. Importantly, these inhibitory effects were both C1q dependent and independent. In order to determine the biologic relevance of our findings, we evaluated the clinical efficacy of these drugs in three different animal models, acute RBC hemolysis, anti-Thy-1 nephritis and passive Heymann's nephropathy (PHN), in which disease pathophysiology relies preferentially on complement activation. While G207 was protective in the anti-Thy-1 nephritis and PHN models, G211 was protective in all of the models tested and could effectively treat PHN. In the anti-Thy-1 nephritis model, G211 prevented the characteristic histologic changes associated with the disease and limited glomerular deposition of C3. Collectively, these data suggest that "complement preferential" Fc multimers offer a novel approach to the treatment of complement mediated diseases.

The inhibitory effect of MEG3/miR-214/AIFM2 axis on the growth of T-cell lymphoblastic lymphoma.

T-cell lymphoblastic lymphoma (T-LBL) is an aggressive malignancy with poor prognosis and high recurrence rate. Long non-coding RNA (lncRNA)-MEG3 is an important tumor suppressor in various cancers. The present study investigated the potential role of maternally expressed gene 3 (MEG3) in the progression of T-LBL. Suppressed expression of MEG3 was detected in T-LBL tissues compared with adjacent histologically normal tissues. Down-regulated level of MEG3 was also found in three T-LBL cell lines (CCRF-CEM, Jurkat and SUP-T1) compared with human T-cell line H9. The proliferation of T-LBL cells was inhibited and cell apoptosis rate was largely promoted when MEG3 was upregulated by a lentiviral vector. Further research revealed that microRNA (miRNA)-214 is a direct target of MEG3. The expression of miR-214 was increased in T-LBL tissues and cell lines compared with control groups. Besides, decreased level of miR-214 was elevated adding miR-214 mimic in SUP-T1 cells transfected with LncRNA-MEG3. Similarly, upregulated level of miR-214 was downregulated adding miR-214 inhibitor in SUP-T1 cells transfected with MEG3 siRNA. Luciferase activity assay further confirmed the targeting relationship between MEG3 and miR-214. Moreover, AIFM2 protein was predicted as a target of miR-214. The expression of AIFM2 was increased by MEG3 and was downregulated by miR-214 mimic. miRNA-214 reversed the effect of MEG3 on inhibiting cell proliferation and inducing cell apoptosis and cell cycle arrest in SUP-T1 cells. Moreover, relative expression of AIFM2 had a positive correlation with the expression of MEG3 and was negatively affected by miR-214. In vivo, MEG3 effectively suppressed tumor growth and the expression of proliferation markers Ki-67 and proliferating cell nuclear antigen (PCNA). Taken together, our research revealed that MEG3 worked as an anti-oncogene in T-LBL, and the MEG3-miR-214-AIFM2 pathway regulated the growth of T-LBL, providing potential prognosis markers as well as new potential targets for T-LBL treatment.

Enhanced antitumor effect of combining chemotherapy with Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes in mice with EBV-related non-Hodgkin's lymphoma.

Epstein-Barr virus (EBV)-related non-Hodgkin's lymphoma (NHL) represents a major problem in hematological clinical studies due to its drug tolerance and refractoriness. EBV infection is a key factor driving the process of tumor growth. Immune therapy is an important biotherapeutic method of treating cancer, which is attracting increasing attention. We hypothesized that combining conventional chemotherapy with immune therapy in the treatment of EBV-related NHL may achieve better outcomes. First, we successfully cloned large numbers of EBV-specific T cells by immune stimulation ex vivo. Subsequently, the combined therapy was applied in a murine model of human EBV-related NHL. As expected, combined therapy inhibited tumor growth more effectively compared with monotherapy. In addition, we continuously tested the tumor-associated immune microenvironment and observed that the numbers of tumor-infiltrating cytotoxic T lymphocytes (CTLs) and macrophages were elevated following combined therapy. These effects suggest that EBV-specific CTLs may indirectly promote an innate immune reaction in lymphoma by activating tumor-infiltrating macrophage proliferation. Our findings may provide a guide for the prospective treatment of EBV-related NHL.

Umbilical cord blood-derived stromal cells regulate megakaryocytic proliferation and migration through SDF-1/PECAM-1 pathway.

We have previously reported that human umbilical cord blood-derived stromal cells (hUCBDSCs) are able to enhance the expansion of CFU-Meg in vitro, particularly promote the megakaryocytic lineage recovery, and effectively protect the survival of irradiated mice. In this study, we demonstrated that hUCBDSCs secreted SDF-1 to stimulate PECAM-1 expression in HEL cells (MK cell line), and consequently promoted the proliferation and migration of HEL cells. On the other hand, SDF-1 knock down in hUCBDSCs or PECAM-1 knock down in HEL cells diminished or abrogated the above effect. In addition, SDF-1/PECAM-1 probably activated PI3K/Akt and MAPK/ERK1/2 pathways. This report for the first time defines a SDF-1/PECAM-1 signaling pathway in the proliferation and migration of MKs, which provides supportive evidence for the clinical applications of hUCBDSCs in the treatment of megakaryocytic injury.

Human umbilical cord blood-derived stromal cells are superior to human umbilical cord blood-derived mesenchymal stem cells in inducing myeloid lineage differentiation in vitro.

Stromal cells and mesenchymal stem cells (MSCs), 2 important cell populations within the hematopoietic microenvironment, may play an important role in the development of hematopoietic stem/progenitor cells. We have successfully cultured human umbilical cord blood-derived stromal cells (hUCBDSCs). It has been demonstrated that MSCs also exist in hUCB. However, we have not found any reports on the distinct characteristics of hUCBDSCs and human umbilical cord blood-derived mesenchymal stem cells (hUCBDMSCs). In this study, hUCBDSCs and hUCBDMSCs were isolated from the cord blood of full-term infants using the same density gradient centrifugation and cultured in the appropriate medium. Some biological characteristics and hematopoietic supportive functions were compared in vitro. hUCBDSCs were distinct from hUCBDMSCs in morphology, proliferation, cell cycle, passage, immunophenotype, and the capacity for classical tri-lineage differentiation. Finally, quantitative real-time polymerase chain reaction analysis revealed that granulocyte colony-stimulating factor (G-CSF) gene expression was higher in hUCBDSCs than that in hUCBDMSCs. Enzyme-linked immunosorbent assay revealed that the secretion of G-CSF, thrombopoietin (TPO), and granulocyte macrophage colony-stimulating factor (GM-CSF) by hUCBDSCs was higher than that by hUCBDMSCs. After coculture, the granulocyte/macrophage colony-forming units (CFU-GM) of hematopoietic cells from the hUCBDSC feeder layer was more than that from the hUCBDMSC feeder layer. Flow cytometry was used to detect CD34(+) hematopoietic stem/progenitor cell committed differentiation during 14 days of coculture; the results demonstrated that CD14 and CD33 expression in hUCBDSCs was significantly higher than their expression in hUCBDMSCs. This observation was also true for the granulocyte lineage marker, CD15. This marker was expressed beginning at day 7 in hUCBDSCs. It was expressed earlier and at a higher level in hUCBDSCs compared with hUCBDMSCs. In conclusion, hUCBDSCs are different from hUCBDMSCs. hUCBDSCs are superior to hUCBDMSCs in supporting hematopoiesis stem/progenitor cells differentiation into myeloid lineage cells at an early stage in vitro.

[Clinical efficacy of immunotherapy of dendritic cell and cytokine-induced killer cell combined with chemotherapy for treatment of multiple myeloma].

OBJECTIVE: This research was aimed to evaluate the immune mechanism and clinical effect of immunotherapy of dendritic cells (DC) and cytokine-induced killer cell (CIK) combined with chemotherapy on multiple myeloma (MM). METHODS: 60 patients with MM were randomly divided into two groups. 30 patients in chemotherapy group were treated by chemotherapy only, 30 patients in joint group were treated by adoptive immunotherapy (DC-CIK) combined with chemotherapy. A variety of immunological indexes (Hsp70, Th1/Th2, TGF-ß) of all patients before and after chemotherapy were recorded; Also the clinical outcomes between two groups were compared. RESULTS: After chemotherapy, the immunological indexes of all patients were better than those of before chemotherapy (P < 0.05); After treatment, quality of life, clinical index and survival in joint group were better than in chemotherapy group (P < 0.05). CONCLUSION: Chemotherapy could break the immunosuppression of MM and improve the anti-tumor response of DC-CIK; Chemotherapy and DC-CIK may have synergistic effect for MM.

An experimental study of extracellular signal-regulated kinase and its interventional treatments in hepatic fibrosis.

BACKGROUND: The pathogenesis of hepatic fibrosis and cirrhosis is still not fully understood. The extracellular signal-regulated kinase (ERK) pathway is involved in the regulation of cell proliferation and differentiation. The aim of this study was to investigate the effects of PD98059, a specific inhibitor of ERK, on the cell cycle, cell proliferation, secretion of type I collagen and expression of cyclin D1 mRNA, CDK4 mRNA and transforming growth factor-beta1 (TGF-beta1) mRNA in rat hepatic stellate cells (HSCs) stimulated by acetaldehyde. METHODS: Rat HSCs stimulated by acetaldehyde were incubated with PD98059 at different concentrations. The cell cycle was analysed by flow cytometry. Cell proliferation was assessed by the methyl thiazolyl tetrazolium colorimetric assay. The mRNA expression of cyclin D1, CDK4 and TGF-beta1 was examined using the reverse transcriptase-polymerase chain reaction. Type I collagen in the culture medium was detected by enzyme-linked immunosorbent assay. RESULTS: 20, 50 and 100 micromol/L PD98059 significantly inhibited the proliferation and provoked a G0/G1-phase arrest of acetaldehyde-induced HSCs in a dose-dependent manner. The secretion of type I collagen and the expression of cyclin D1, CDK4 and TGF-beta1 mRNA in acetaldehyde-induced HSCs were markedly inhibited by 50 and 100 micromol/L PD98059, respectively. CONCLUSIONS: The ERK pathway regulates the cell proliferation, secretion of type I collagen and the expression of TGF-beta1 mRNA in rat HSCs stimulated by acetaldehyde, which is likely related to its regulative effect on the cell cycle.