A rare fatal case of a novel bunyavirus-associated hemophagocytic lymphohistiocytosis.

Herein we describe a rare fatal case of a novel bunyavirus-associated hemophagocytic lymphohistiocytosis (HLH) in a 62-year-old female patient. The novel bunyavirus infects patients with or without HLH who have similar clinical features such as fever, thrombocytopenia, and leukocytopenia. Therefore, the diagnosis of HLH can be easily missed. When HLH occurs, the disease worsens and the fatality rate rises. Our finding highlights the importance of bone marrow biopsy performed as soon as possible for patients suspected of having a novel bunyavirus infection and showing marked cytopenia in three cell lines.

[The induced differentiation and apoptosis of THP-1 cells by anti-CD44 antibody and its mechanism].

OBJECTIVE: To investigate the effects of anti-CD44 mAb A3D8 on the cell proliferation of human acute monocytic leukemia cell line THP-1 and its mechanism. METHODS: Cell proliferation was assayed with MTT method, the expression of CD33, CD15, CD11b, CD14, Annexin-V, caspase-3 and cell cycle with flow cytometry, and the expression of p-Akt, p-ERK, bcl-2 and p27kip1 with Western blot. RESULTS: A3D8 could remarkably inhibit the proliferation capacity of the THP-1 cells in a dosage- and time-dependent manner. THP-1 differentiation was observed when treated with A3D8 (2.0 µg/ml) for one to six days. Expression of CD33 (68.9 ± 2.0 vs 39.3 ± 1.5), CD15 (61.7 ± 5.5 vs 12.9 ± 2.6), CD11b (67.3 ± 3.8 vs 14.0 ± 2.0) and CD14 (83.0 ± 5.7 vs 8.0 ± 1.0) was significantly increased at day 4 compared with the control group (all P < 0.01). Cell cycle of the THP-1 cells was arrested in G(0)/G(1). Expression of the Annexin-V \[(32.5 ± 2.5)% vs (2.4 ± 0.3)%\] and caspase-3 \[(33.3 ± 2.5)% vs (3.6 ± 0.3)%\] was much higher than that in normal controls (all P < 0.01), and apoptosis was observed in THP-1 cells at day 5. Expression of p-Akt (0.24 ± 0.06 vs 1.20 ± 0.15), p-ERK (0.32 ± 0.05 vs 1.24 ± 0.09), and bcl-2 (0.11 ± 0.05 vs 0.65 ± 0.07) was much lower than that of the controls (all P < 0.01), while p27kip1 (1.08 ± 0.09 vs 0.10 ± 0.02) was significantly increased at day 4 (P < 0.05). CONCLUSION: Anti-CD44 antibody can induce the differentiation and apoptosis of THP-1 cell through inhibiting PI3K/AKt and ERK1/2 signaling pathway.

[Expression of CD123 in lymphocytic leukemia and its significance for monitoring minimal residual diseases.].

OBJECTIVE: To investigate the expression of CD123 and its significance in lymphocytic leukemia. METHODS: CD123 expression in 139 lymphocytic leukemia patients and in lymphocytes from 10 normal bone marrows (BM) was analyzed by multi-parameter flow cytometry. Cytogenetic and minimal residual disease (MRD) analysis were performed in acute B-lymphocytic leukemia (B-ALL) patients. RESULTS: CD123 expression was absent in B lymphoid lineage stem-progenitor cells, mature B and T lymphocytes from 10 normal BM. Among 139 lymphocytic leukemia patients, CD123 was negative in 5 T-ALL and 23 B-CLL patients. However, among 111 B-ALL patients, CD123 was expressed in 106 (12 pro B-ALL, 57 common B-ALL and 37 Pre B-ALL) (95.49%) but not in 5 mature B-ALL patients. There was a positive correlation between CD123 and p-Akt expression, and CD123 expression was much higher in hyperdiploid than in non-hyperdiploid B-ALL patients. A statistically significant difference in relapse rate within 12 months (MRD positive group: 63.04% vs MRD negative group 21.56%)and in disease free survival (DFS) time was found beween patients with MRD\[(36.06 +/- 2.62)%\] or not \[(48.23 +/- 1.82)%\] (P < 0.01). Moreover, stable CD123 expression could be observed in B-ALL patients in relapse. CONCLUSIONS: CD123 was predominantly expressed in B-ALL patients and remained in patients in relapsec, indicating that it may be an useful MRD marker in B-ALL patients.

[Effect of rapamycin on apoptosis in human myelodysplastic syndrome cell line MUTZ-1 and its possible mechanisms].

The aim of this study was to investigate the effect of rapamycin on cell growth and apoptosis in the myelodysplastic syndrome (MDS) cell line MUTZ-1 and possible mechanism. MUTZ-1 cells were treated with rapamycin, cell proliferation capability was determined with MTT, protein expression including Annexin V/PI, caspase 3, PTEN, p-Akt, p-mTOR and the cell cycle were analyzed with flow cytometry. The results indicated that the proliferation of MUTZ-1 cells was inhibited by rapamycin in concentration-and time-dependent manners (r=0.67, 0.61, 0.72). After treatment with rapamycin for 24-72 hours, cell count in G0/G1 were significantly higher than that of the control (p<0.01), and this effect showed a time-and concentration-dependency (r=0.94, 0.93, 0.92), the cell cycle was blocked in G0/G1 phase. As compared with control group, the proportion of Annexin V+PI-MUTZ-1 cells and the cellular PTEN levels increased in the treated group dramatically and in time-and dose-dependent manners (p<0.01). To the contrary, level of p-mTOR expression markedly decreased as compared with control group (p<0.05). It is concluded that the rapamycin inhibits the proliferation of MUTZ-1 cells, down-regulates the PTEN/PI3K-Akt/mTOR signaling pathway by interaction with mTOR, which induces the apoptosis of mUTZ-1 cells.

[The correlation study of PTEN gene expression and Akt phosphorylation in myelodysplastic syndrome].

OBJECTIVE: To investigate the relationship between PTEN gene expression and Akt phosphorylation (p-Akt) in myelodysplastic syndrome (MDS) and to explore the progression of MDS and the mechanism of high risk transformation to acute myeloid leukemia. METHODS: RT-PCR was used to detect the PTEN mRNA expression in leukemia cell lines K562 (as negative control) and Jurkat (as positive control) and 65 MDS and MDS/AML patients. Flow cytometry was used to detect p-Akt in HL-60 and Jurkat cells and 30 MDS patients. RESULTS: (1) K562 cells present PTEN gene expression while Jurkat cells did not. Of 65 MDS and MDS/AML patients, 27 (41.5%) expressed PTEN mRNA, being significantly lower than that in normal group (85.7%) (P < 0.01). (2) Jurkat cell showed high expression (86.9%) of p-Akt, while HL-60 cell as negative control did not express. P-Akt levels of 30 MDS patients were increased (1.35% - 58.23%), being much higher as compared with that of the normal contrast group (0.54% - 2.34%) (P < 0.01). Moreover, with the rate of blast cells increasing, the p-Akt level was rising up. There is a positive correlation (r = 0.93, P < 0.01) between the low expression rate of PTEN and the positive rate of p-Akt. CONCLUSION: The loss of PTEN gene expression is one of the important factors of p-Akt high expression in MDS patients, moreover, it may speed up the progress of the MDS or transformation to acute myeloid leukemia.

Possible roles of KIR2DL4 expression on uNK cells in human pregnancy.

PROBLEM: To investigate possible roles of the natural killer (NK) cell receptor killer immunoglobulin-like receptor (KIR)2DL4 expressed on uterine NK (uNK) cells during pregnancy, we investigated KIR2DL4 expression on uNK cells isolated from patients with early recurrent spontaneous abortion (RSA) and normal early pregnancy women, and functions of KIR2DL4 was analyzed in vitro. METHODS OF THE STUDY: Semi-quantitative RT-PCR analysis was introduced to detect KIR2DL4 messenger RNA (mRNA) expression on uNK cells. Cytotoxicity and cytokine production as the result of interaction of KIR2DL4 and its ligand human leukocyte antigen (HLA)-G were analyzed in vitro with lactic dehydrogenase releasing method and enzyme-linked immunosorbent assay, respectively. RESULTS: No significant difference in KIR2DL4 mRNA expression was observed, while the KIR2DL4 protein level in isolated uNK cells is much higher in normal controls than that in RSA patients. Data showed that HLA-G transfection could not reverse the lysis of uNK against HLA-G transfected K562 cells but induced cytokine production. Furthermore, we demonstrated that, via KIR2DL4, membrane-bound HLA-G could induce high cytotoxicity and cytokine production in a high cytotoxic, IL-2 dependent human NK cell line NK-92 cells. CONCLUSION: Our data suggest that KIR2DL4 might play a crucial implication for human pregnancy.

[Expression of CD66c (CEACM6) in adult acute leukemia and its significance].

OBJECTIVE: To explore the expression of CD66c (CEACM6) in adult acute leukemia and its significance. METHODS: Acute leukemia cell lines HL-60, K562, LCL721.221 and Jurkat were cultured in vitro. RT-PCR and multi-parameter flow cytometry were applied to analysis of CD66c mRNA and protein expression respectively in the cell lines and patient' s bone marrow leukemic cells. Cytogenetic analysis for 199 bone marrow samples from leukemia patients and Minimal Residual Disease (MRD) detection for 25 CD66c positive B lineage ALL were performed. RESULTS: (1) CD66c expression both on cell surface and in plasma were negative in all the cell lines. (2) Four of 127 AML (3.15%) (mainly of M2 and M4), and 28 of 79 ALL (35.44%) (all of B linage ALL) were CD66c positive the subtypes of the ALL being common B-ALL (20/54) and pre B-ALL (8/11) including 8 Ph + B-linage ALL. (3) Six-month relapse rate was significantly different between the MRD positive and negative patients. (4) CD66c mRNA was strongly expressed in B-linage ALL. For the cell lines, only the HL60 cells weakly expressed CD66c mRNA. CONCLUSION: CD66c expression could be a useful bio-marker for the MRD analysis in ALL, and is closely associated with its transcription level.

[Study on platelet activated state and platelet activated function in adults with acute leukemia].

To investigate the changes of platelet activated state and platelet activated function by trace whole blood flow cytometry (FCM), and to explore the mechanism of hemorrhage and infiltration in adults with acute leukemia, the expression percentage and changes of these expressions of CD62p and PAC-1 on platelet surface were determined by FCM of trace whole blood after platelet activated by ADP in patients with new diagnosed AL (group I), complete remission (CR, group II) and continuously complete remission (CCR, group III). Healthy adults were used as control group. The result showed that the expression of CD62p in group I and II was higher than that in control group, before and after platelet activated by ADP (P < 0.01). The expression of PAC-1 in group I was higher than that in control group (P < 0.01), the expression of PAC-1 in group II was lower than that in control group (P > 0.01), There was no significant difference in expression of CD62p and PAC-1 between group III and control group (P > 0.01), and no significant difference was found between AL group with megakaryocyte malignant pathological changes and AL group without megakaryocyte malignant pathological changes before platelet activated by ADP (P > 0.01). After platelet activated by ADP, the expression of PAC-1 in the former was lower than that in the latter (P < 0.01). It is concluded that (1) high level activated platelet in peripheral blood of AL patients show that interaction between activated platelet and leukemia cells can be one of reason resulting in widespread hemorrhage and infiltration AL patiens; (2) the decrease of number and activted function of platelet at the first stage of AL patients may be caused by malignant hyperplasia of leukemia cells and damage of megakaryopoiesis in bone marrow.

[Study on NB4 cell apoptosis induced by trichosanthin].

In order to study the influence of trichosanthin (TCS) on apoptosis and growth inhibition of human NB4 cells in vitro, the expression of annexin V and the change of DeltaPsim of NB4 cells induced by TCS was analyzed by FACS, and MTT assay was adopted to measure the growth inhibition ratio of NB4 cells treated with TCS. Apoptosis was assayed by agarose gel electrophoresis. The results showed the higher concentration of TCS and the longer the acting time, the stronger growth inhibition of NB4 cells. The expression of annexin V was positive, and the positive ratio was greatly enhanced with prolongation of acting time. DeltaPsim reduced gradually while the apoptosis cells increasing. DNA agarose gel electrophoresis showed a gradient, which confirmed that TCS could induce NB4 cells apoptosis. In conclusion, taken together, data show that TCS can inhibit NB4 growth in vitro, and induce apoptosis. Experiment provides an important evidence for application of TCS in clinical treatment of acute promyelocytic leukemia.

[Sensitivity and specificity analysis of the lineage related antibodies in acute leukemia immunophenotyping by flow cytometry].

To evaluate the sensitivity and specificity analysis of the lineage related antibodies in acute leukemia immunophenotyping by flow cytometry (FCM), immunophenotyping in 184 patients with acute leukemia was performed by FCM analysis. The results showed that in the lineage-related antibodies of acute myelocytic leukemia (AML), the sensitivity of CD13 and CD33 was higher (95.5% and 91.2%, respectively), the specificity of them was deficient (72.5% and 62.2%, respectively); the sensitivity of MPO was low (69.1%), but the specificity was high (100%); the sensitivity and specificity of CD117 were high (88.2% and 100%, respectively); the sensitivity of CD14 and CD15 was low (18.4% and 27.2%, respectively); the specificity of CD14 with monocytes was high. As the lineage-related antibodies of B-lineage ALL were concerned, CD19 showed high sensitivity and low specificity (100% vs 83.4%); the sensitivity and specificity of CD79a (96.4% vs 100%) and CD22 (100% vs 100%) were high; the sensitivity and specificity of CD10 (53.6% vs 82.5%) and CD20 (70.4% vs 87.5%) were low. In T-lineage ALL, the specificity of CD3 was high (97.5%), but the sensitivity was below the mark (80.0%); the sensitivity of CD7 was high (100%), but the specificity was low (77.9%); while the sensitivity and specificity of CD5, CD2 and CD1a were all deficient. In conclusion, the sensitivity and specificity analysis of the lineage-related antibodies in acute leukemia immunophenotyping are coincident with St Jude immunophenotyping project. It seems only that CD117 is superior to MPO in defining AML, but the sensitivity and specificity analysis of CD22 and CD79 are similar in defining B-lineage ALL, therefore, anyone of them may be selected as your need.