ER stress mediated­autophagy contributes to neurological dysfunction in traumatic brain injury via the ATF6 UPR signaling pathway.

A major public health problem, traumatic brain injury (TBI) can cause severe neurological impairment. Although autophagy is closely associated with the pathogenesis of TBI, the role of autophagy in neurological deficits is unclear. The purpose of the present study was to investigate the molecular mechanisms of endoplasmic reticulum (ER) stress­induced autophagy and its detrimental effects on neurological outcomes following TBI. A rat model of TBI was established by controlled cortical impact. ER stress activation, autophagy induction and autophagic flux dysfunction were examined in the damaged hippocampus post­TBI. Pharmacological inhibition of ER stress significantly blocked post­traumatic autophagy activation, as evidenced by decreased conversion of microtubule­associated protein 1 light chain 3 (LC3)­I to LC3­II and Beclin­1 expression levels in the hippocampus region. Short hairpin RNA­mediated activating transcription factor 6 knockdown significantly prevented ER stress­mediated autophagy stimulation via targeting essential autophagic genes, including autophagy related (ATG)3, ATG9 and ATG12. Furthermore, neurological scores, foot fault test and Morris water maze were used to evaluate the neurological functions of TBI rats. The results revealed that the blockage of ER stress or autophagy attenuated TBI­induced traumatic damage and functional outcomes. In conclusion, these findings provided new insights into the molecular mechanisms of ER stress­induced autophagy and demonstrated its potential role in neurological deficiency following TBI.

Effect of Graft Composition on Acute Graft-Versus-Host Disease in Aplastic Anemia after Haploidentical Hematopoietic Stem Cell Transplantation / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between acute graft-versus-host disease and graft composition in patients with aplastic anemia(AA) after haploidentical hematopoietic stem cell transplantation.</p><p><b>METHODS</b>Fifty-seven cases of AA after haploidentical hematopoietic stem cell transplantation were retrospectively analyzed. All the patients were divided into 2 groups according to whether presence or absence grade Ⅱ-Ⅳ aGVHD, the relationship between aGVHD and graft composition was analyzed by comparing the differences of graft components between the 2 groups.</p><p><b>RESULTS</b>Fourteen out of 57 patients had grade Ⅱ-Ⅳ aGVHD and the other 43 did not have grade Ⅱ-Ⅳ aGVHD. The mononuclear cells, CD3, CD4, CD8, NK cells, NKT cells, B cells and Treg cells were not significantly different between the 2 groups (P>0.05), the CD34 cell count in the patients with grade Ⅱ-Ⅳ aGVHD was 3.85（1.73-10.61）×10/kg, which was significantly lower than that without grade Ⅱ-ⅣaGVHD: 6.31（2.98-19.35）×10/kg (P<0.05).</p><p><b>CONCLUSIONS</b>The incidence of grade Ⅱ-Ⅳ aGVHD may be related with CD34 cell count in AA after haploidentical hematopoietic stem cell transplantation..</p>

Clinical Observation of Haploidentical Hematopoietic Stem Cell Transplantation Combined with Bone-marrow Derived Mesenchymal Stem Cells Transfusion for Treatment of Children with Severe Aplastic Anemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the efficacy and safety of haploidentical hematopoietic stem cell transplantation(hi-HSCT) combined with bone-marrow derived mesenchymal stem cell (BM-MSC) tranfusion for treatment of children with severe apastic anemia(SAA).</p><p><b>METHODS</b>The clinical data of 25 children with SAA undergoing hi-HSCT and BM-MSC tranfusion were retrospectively analyzed from August 2014 to July 2016.</p><p><b>RESULTS</b>neutrophil engraftment was achieved in all 25(100%) children, with the median time 12(11-22) days. The median time of platelet engraftment was 21(11-130) days in 23(92%) children. Acute graft-versus-host disease(aGVHD) was observed in 16(64%) cases, including 11 case of grade I and 5 cases of aGVHD grade II-IV, and one of them died of grade IV of skin, gut and liver at day 115; 5 cases of chronic GVHD were found, all of them were limited cGVHD. Cytomegalovirus (CMV) viremia was observed in 23(92%) cases, but no CMV disease was developed after therapy. 3 cases of post-transplant lymphoroliferative disease with 23 EBV viremia positive occurred, all of them were cured after rituximab. Hemorrhagic cystitis appeared in 9 cases with only one case of grade III, 22 children suffered from infection, involving 10 cases in lung and 4 cases in liver, 1 patient was diagnosed as Guillain-Barre syndrome. Autoimmune hemolytic anemia was recorded in 1 patient, 22 children survived during a median following-up time of 14(3-27) months.</p><p><b>CONCLUSION</b>The hi-HSCT combined with BM-MSC transfusion for treatment of children with SAA has been confirmed to be safe and feasible.</p>

Allogeneic hematopoietic stem cell transplantation for chronic myelomonocytic leukemia and juvenile myelomonocytic leukemia / 中国实验血液学杂志

This study was purposed to explore the therapeutic efficacy and influencing factors of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients with chronic myelomonocytic leukemia (CMML) and in patients with juvenile myelomonocytic leukemia (JMML). The clinical data of 3 cases of CMML and 2 cases of JMML underwent allo-HSCT were analysed in term of multiparameter. The results showed that the hematopoietic stem cells in 5 patients grafted successfully. One case of JMML died of pulmonary disease, other 4 cases survive without disease. The analysis found that the disease burden before transplant, chromosome karyotype, acute GVHD II-IV and poor risk cytogenetics all associated with the relapse rate and disease-free survival rate of CMML. The low intensity conditioning regimen was better than myeloablative conditioning regimen. Type of donor and source of stem cells did not statistically and significantly affect OS and RFS. The splenectomy before allo-HSCT as well as spleen size at time of the alloHSCT did not influence on posttransplantation outcome of JMML. However, cord blood HSCT for JMML patients delayed hematologic recovery as compared to that of bone marrow or peripheral blood HSCT. The age, GVHD, HbF level played an important role in leukemia replace. It is concluded that the allogeneic hematopoietic stem cell transplantation is a curative regimen for CMML and JMML, but there also is a serial problems to be resolved.

Analysis of T lymphocyte absolute number and function in the early phase after haploidentical hematopoietic stem cell transplantation / 中国实验血液学杂志

This study was purposed to investigate the immune reconstitution of T-cells in patients who received haploidentical hematopoietic stem cell transplantation (hiHSCT). The peripheral blood was harvested from 22 patients before transplantation and at month 1, 3, 6 after hiHSCT. The proportions of T lymphocyte subtypes including CD3(+), CD4(+), CD8(+), CD45RO(+), and CD45RA(+)CD62L(+) were analyzed by flow cytometry, followed by the calculation of T cell numbers according to the amounts of peripheral blood leukocytes. Adenosine triphosphate (ATP) value in CD4(+) T cells was measured by ImmuKnow method to evaluate the function of lymphocytes. The results showed that the CD3(+) cell absolute value before transplantation was 833.75 ± 359.84/µl, but those values at month 1, 3, 6 after transplantation were 318.87 ± 266.71/µl, 1006.76 ± 512.32/µl and 1296.38 ± 958.77/µl respectively. The CD4(+) cell absolute value before transplantation was 336.99 ± 211.11/µl, but such values at month 1, 3, 6 after transplantation were 45.89 ± 44.21/µl, 142.97 ± 114.85/µl, and 181.78 ± 120.61/µl respectively. The CD8(+) cell absolute value before transplantation was 430.21 ± 159.48/µl, but those values at month 1, 3, 6 after transplantation were 230.44 ± 195.89/µl, 621.64 ± 318.83/µl, and 823.07 ± 633.55/µl respectively. The CD4(+)CD45RO(+) memory T cell absolute value before transplantation was 227.44 ± 73.34/µl, but such values at month 1, 3, 6 after transplantation were 43.47 ± 43.40/µl, 138.69 ± 110.17/µl, 147.73 ± 82.94/µl respectively. The CD8(+)CD45RO(+) memory T cell absolute value before transplantation was 212.70 ± 98.48/µl, but such values at month 1, 3, 6 after transplantation were 184.76 ± 168.65/µl, 445.90 ± 252.50/µl, 519.80 ± 475.53/µl respectively. CD4(+)CD45RA(+)CD62L(+) naive T cell number before transplantation was 68.94 ± 59.74/µl, but such cell numbers at month 1, 3, 6 after transplantation decreased to 2.44 ± 2.93/µl, 3.14 ± 3.48/µl, 23.22 ± 38.38/µl respectively. The CD8(+)CD45RA(+)CD62L(+) naive T cell absolute value before transplantation was 124.82 ± 60.95/µl, but those values at month 1, 3, 6 decreased to 19.37 ± 17.71/µl, 76.63 ± 50.85/µl, and 114.49 ± 174.29/µl respectively. The ATP value in CD4(+) T cells decreased to 210.19 ± 119.37 ng/ml at month 1 after transplantation and increased to 280.62 ± 110.03 ng/ml at month 3, and 357.28 ± 76.18 ng/ml at month 6 after transplantation. It is concluded that CD8(+) memory T cell reconstruction contributes critically to T cell recovery early after hiHSCT, while the thymic output function remains low. However, T cell function recovers to normal range at month 3 after transplantation.

Effect of c-Jun NH2-terminal kinase-mediated p53 expression on neuron autophagy following traumatic brain injury in rats.

BACKGROUND: Activation of c-Jun NH(2)-terminal kinase (JNK) has been implicated in neuron apoptosis as well as autophagy in response to various stressors after traumatic brain injury (TBI). However, the underlying molecular pathway remains unclear. Our study assessed whether JNK-mediated p53 phosphorylation might be an important mechanism for enhancing neuron autophagy in response to TBI. METHODS: A total of 186 male Sprague-Dawley (SD) rats (300 - 350 g) were used in this study. By randomized block method rats were randomly divided into four groups: sham-operated (n = 46), TBI (n = 60), TBI + dimethyl sulfoxide (DMSO) (n = 40), and TBI + SP600125 (n = 40). JNK was treated with SP600125, a specific JNK inhibitor. JNK, p-P53, Beclin-1, damage-regulated autophagy modulator (DRAM) and p-bcl-2 were evaluated by Western blotting analysis. The cellular localization and expression of Beclin-1 and DRAM was observed by immunofluorescence and immunohistochemistry, and the expression of Beclin-1-Bcl-2/Bcl-xL complexes was evaluated by immunoprecipitation. Multiple-group comparisons were conducted using analysis of variance (ANOVA). P values of less than 0.05 were considered statistically significant. RESULTS: It was observed that the expression of JNK, p-P53, Beclin-1, DRAM and p-bcl-2 was increasing after TBI, and the expression of Beclin-1 and DRAM was mainly located in the cytoplasm of neurons. But these were significantly inhibited in SP600125 group compared with sham group and TBI + SP600125 group (P < 0.05). The expression of Beclin-1-Bcl-2/Bcl-xL complexes was reduced after TBI. CONCLUSION: JNK-mediated p53 phosphorylation might be an important mechanism for enhancing neuron autophagy in response to TBI.

[Effect of expression of c-jun N-terminal kinase on neuron autophagy following diffuse brain injury in rats].

OBJECTIVE: To study the effect and potential mechanism of expression of c-jun N-terminal kinase (JNK) signal pathway on neuron autophagy after diffuse brain injury (DBI). METHODS: Male Sprague Dawley rats (n = 216) were randomly divided into four groups: DBI group (n = 54), SP600125 intervene group (n = 54), DMSO group (n = 54) and sham operation group (n = 54). DBI rat model was established according to the description of Marmarou DBI. At different time points (1, 6, 12, 24, 48 and 72 h) after operation, the histopathologic changes of neurons in cortex were observed by HE staining method; The expression of p-JNK, p-P53, DRAM and Beclin-1 were detected by Western blot and immunohistochemistry. RESULTS: The results showed that under light microscope degenerated and necrotic neurons were observed to be scattered in cortex at 6 h after operation in DBI group, but these changes were low in SP600125 intervene group. Compared with SP600125 intervene group, the expression of p-JNK in DBI group were enhanced obviously at 6, 12 and 24 h (F = 17.902, P < 0.05); the expression of p-P53 in DBI group were enhanced obviously at 12, 24, 48 and 72 h (F = 7.107, P < 0.05); the expression of DRAM in DBI group were enhanced obviously at 6, 12, 24, 48 and 72 h (F = 15.455, P < 0.05); the expression of Beclin-1 in DBI group were enhanced obviously at 6, 12, 24, 48 and 72 h (F = 11.517, P < 0.05). Compared with DBI group, the expression of p-JNK, p-P53, DRAM and Beclin-1 in DMSO group were similar at 1, 6, 12, 24, 48 and 72 h (F = 1.509, P > 0.05). CONCLUSIONS: The present results indicate that SP600125 can dramatically improve trauma brain injury from autophagy after DBI and the molecular mechanism is related to the modulation of JNK signal pathway following DBI, while it measures the neuron autophagy by means of intervening JNK signal pathway.

Clinical investigation on treatment of children leukemia with non-T-cell depleted haploidentical bone marrow transplantation / 中国实验血液学杂志

To investigate the efficacy and feasibility of parent non-T cell depleted haploidentical bone marrow transplants (haploidentical BMT) for children with leukemia, the efficacy of haploidentical BMT was evaluated in 8 leukemia children (1.9-9 years) received hematopoietic stem cell transplantation, donors were their parents with HLA-mismatched for two or three loci. Five children were pre-conditioned with a myeloablative regimen consisting of high-dose cytarabine (Ara-C), cyclophosphamide (CY) and total body irradiation. Busulfan (BU), Ara-C and CY were used for preconditioning regimen in other three children. The donors were given G-CSF prior to marrow harvest and the non-T-cell depleted grafts were used. A combination of CsA, MTX, ATG, mycophenolate mofetil (MMF) and CD25 monoclonal antibody were used for GVHD prophylaxis. The results showed that rapid engraftment was observed in all cases after transplantation by cytogenetic evidence. The mean time of neutrophil count exceeded 0.5 x 10(9)/L and the mean time of platelet count exceeded 20 x 10(9)/L were 16 and 17 days after transplantation respectively. Incidence of lethal aGVHD was lower, II-III acute aGVHD was found only in one out of eight patients. Chronic GVHD was observed in five patients, 4 from which showed local cGVHD, one developed extensive cGVHD. During the follow-up of 33 months (range 7-56 months), two patients died from relapsed leukemia, including one relapsed as donor-origin leukemia. Disease-free survival was achieved in the remaining six patients. No death occurred during the follow-up of 6 months. It is concluded that above-mentioned preconditioning regimen and GVHD prophylaxis procedure in non-T-cell depleted bone marrow transplantation from HLA-mismatched parents are effective approaches and safe strategy for the treatment of children leukemia.

Allogeneic bone marrow transplantation for chronic myeloid leukemia using HLA identical sibling donors primed with G-CSF / 中国实验血液学杂志

Many studies have shown that G-CSF mobilized peripheral blood progenitor cell transplants (PBPCT) manifests faster recovery kinetics than conventional bone marrow transplants. This potential advantage of PBPCT still needs to be balanced against the risk of acute and chronic GVHD associating with the infusion of 10 - 15 fold higher donor lymphocyte number in unmanipulated allogeneic PBPCT than the marrow graft. To evaluate the effect of G-CSF primed bone marrow as a source of stem cells in the HLA-matched sibling transplantation, G-CSF primed with non-primed donor marrow in engraftment and incidence of GVHD for a homogenous group of patients with chronic myeloid leukemia (CML) were compared. Fifty patients with CML underwent bone marrow transplant, thirty-two donors (study group) were given G-CSF 3 - 4 micro g/kg per day for seven days prior to marrow harvest and eighteen donors (control group) had marrow harvest without G-CSF stimulation. Conditioning regimen consisted of total body irradiation and cyclophosphamide (CY), busulfan and CY, or busulfan, total body irradiation and CY. Both groups received same post-grafting GVHD prophylaxis and postgrafting G-CSF treatment. It was found that G-CSF primed donor marrow yielded with significantly higher number of total nucleated cells as well as CD34(+) cells and CFU-GM compared to non-G-CSF primed marrow (P = 0.001). The median engraftment time for absolute neutrophil (ANC > 0.5 x 10(9)/L) was day 15 (range 10 - 22) in the group of G-CSF primed vs day 21 in the non-primed donor group (P = 0.001). The median time for platelets (> 20 x 10(9)/L) was day 17.5 (range 13 - 28) in the group of G-CSF primed vs day 24 in non-primed group (P = 0.001). The incidence of acute GVHD grade II - IV in G-CSF primed donor group was surprisingly as low,as only two cases of thirty-two transplants (6.3%) with acute GVHD grade II limited to the skin. Whereas, five of eighteen patients (27.8%) in the control group developed acute GVHD grade II - IV (P = 0.032). G-CSF primed donors showed reduced CD4(+) and increased CD8(+) cells, resulting in a significant reduction of CD4(+)/CD8(+) ratio as compared with non-primed marrow. The total CD3(+) cell count kept unchanged in G-CSF primed donors. There were not significant differences in the incidence of the chronic GVHD (24% vs 33.3%), relapse rate (12.5% vs 11.1%) and overall survival rate (78.1% vs 66.7%, P = 0.32) during 6 - 50 months of follow-up. In conclusion, G-CSF primed donor marrow accelerates engraftment. Although G-CSF did not change the total CD3(+) cells in bone marrow, it altered the ratio of CD4(+) and CD8(+) cells and significantly reduced the incidence of acute GVHD.