Effects of mycophenolic acid on human bone marrow-derived mesenchymal stem cells in vitro / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effects of mycophenolic acid (MPA) on the proliferation and differentiation of human bone marrow-derived mesenchymal stem cells (MSCs).</p><p><b>METHODS</b>MSCs were treated with MPA at the concentration of 1 μ mol/L, 10 μ mol/L, 50 μ mol/L, and 100 μ mol/L, respectively. Cell proliferation was analyzed using CCK-8 method. Apoptosis was detected by PI/Annexin V assay kit. The mRNA expression of inosine-5'-monophosphate dehydrogenase (IMPDH) in MSCs was analyzed by RT-PCR. Osteogenic differentiation was analyzed by Von Kossa staining, Ca(2+) quantification and real-time PCR.</p><p><b>RESULTS</b>In the range of 1 μ mol/L to 100 μ mol/L, MPA caused a significant subdued proliferation rate of MSCs in a concentration-and time-dependent manner by guanosine depletion, and PI/Annexin staining showed no apoptosis induced by MPA. RT-PCR results showed that MSCs expressed both IMPDH I and IMPDH II. von Kossa staining and Ca(2+) quantification indicated that MPA inhibited osteogenic differentiation of MSCs, and real-time PCR detected a dose-dependent decrease in expression of Osteopontin and BMP-2. Further investigation showed that MPA down-regulated the expression of Runx2 and Osterix.</p><p><b>CONCLUSION</b>MPA can inhibit the proliferation of MSCs by guanosine depletion in a concentration-and time-dependent manner and inhibit the osteogenic differentiation of MSCs by down-regulation of the expression of Runx2 and Osterix.</p>

Clinical features and treatment of serious brainstem encephalitis caused by enterovirus 71 infection / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the clinical features and treatment of serious brainstem encephalitis caused by enterovirus 71 (EV71) infection.</p><p><b>METHODS</b>The clinical data of 32 hospitalized children with serious brainstem encephalitis caused by EV71 infection between May and December 2008 were retrospectively reviewed.</p><p><b>RESULTS</b>The children whose age was younger than 3 years old accounted for 88% (22 cases). Fever(>38.5 degrees centigrade)lasting at least 3 days, frequent vomiting and limb twitch were presented as the main manifestations in the 32 children. Cyanosis, tachypnea, tachycardia and cold extremities were observed, and pulmonary edema or even pulmonary hemorrhage occurred in 8 children 3 to 4 days after the onset. The 32 children received a medical treatment: reduction of intracranial pressure with mannitol or frusemide, inhibition of inflammation reactivity with gamma globulin and methylprednisolone, and improvement of cardiac function and pulmonary edema with innotropic agents, fluid restriction and positive mechanical ventilation.</p><p><b>CONCLUSIONS</b>Vegetative nerve functional disturbance is the main clinical feature of brainstem encephalitis caused by EV71 infection in children. An early identification and treatment of pulmonary edema or hemorrhage is of great importance.</p>

Response to therapy of 13 children with rhabdomyosarcoma / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the clinical response to comprehensive therapy in children with rhabdomyosarcoma.</p><p><b>METHODS</b>Clinical data of 13 children (8 males and 5 females) with rhabdomyosarcoma from January 1998 through October 2005 were retrospectively studied. Their ages ranged from 7 months to 12 years. The 13 cases of rhabdomyosarcoma consisted of 2 cases in stage I, 2 cases in stage II, 3 cases in stage III, and 6 cases in stage IV. Rhabdomyosarcoma was confirmed by biopsy, 12 cases (92.3%) presenting as embryonal type and 1 as alveolar type in histology. One patient underwent surgery treatment alone, one patient received surgery plus local radiation treatment, one patient received surgery plus chemotherapy and 10 patients were administered with a combination of surgery, local radiation treatment and chemotherapy. The chemotherapy protocol before 2002 was VDCA, VAC or VadrC. After 2002, the COG protocol was employed, with CDV+IE for stage III, and CT+VAC or CT+VAC+VCT for stage IV patients.</p><p><b>RESULTS</b>The 2-year overall survival was 60% in the 10 patients who received a combination of surgery, local radiation treatment and chemotherapy, but the three patients died without receiving combination therapy. The 2-year overall survival in the 13 patients was 46.2%. The 2-year overall survival of the patients after 2002 (60%, 3/5) was higher than that before 2002 (37.5%, 3/8).</p><p><b>CONCLUSIONS</b>Embryonal rhabdomyosarcoma dominates the histology type in children, which is highly malignant. A combination therapy of surgery, local radiation and chemotherapy can result in a satisfactory therapeutic effect in children with rhabdomyosarcoma.</p>

Characteristics of chronic active Epstein-Barr virus infection-associated hematological disorders in children / 中国实验血液学杂志

The aim of this study was to analyze characteristics of chronic active Epstein-Barr virus (CAEBV) infection associated hematological disorders in children. Clinical characteristics were summarized; the morphology of hematopoietic cells in bone marrow was observed by microscopy; the lymphocyte subpopulations were analyzed by flow cytometry; the immunophenotype of liver biopsies was assayed by immunohistochemistry; EBV-related antibodies were measured by ELISA; serum EBV-DNA loads were detected by real-time quantitative PCR; EBV-encoded small RNA 1-positive cells in peripheral blood mononuclear cells were identified by in situ hybridization. The results indicated that the clinical manifestations in patients included persistent or recurrent fever, hepatosplenomegaly, liver dysfunction, anemia, thrombocytopenia, systemic inflammatory reaction. Bone marrow presented as hypocellularity, dysmaturation, myelodysplasia and hemophagocytosis. CD8(+) cell high counts were demonstrated in all 4 patients, one of them developed into a T cell lymphoma. Serum EBV-DNA load was 3.26 x 10(3) copies/ml in one patient, EBER1(+) cells were detected at a frequency of 1.7% in PBMNCs from another patient; the titers of IgG to EBV-VCA were >or= 1:5120 in the rest 2 patients. All 4 patients described above were diagnosed as CAEBV infection. In conclusion, the immune-related cytopenia, macrophage activation syndrome and lymphoproliferative disorders are characteristics of CAEBV infection associated hematological disorders in these 4 children patients.

Skin lesions and myelodysplastic syndrome as initial manifestations of biphenotypic acute leukemia / 中国实验血液学杂志

The aim of this study was to investigate the clinical, pathological and biological features of biphenotypic acute leukemia. The morphology of tumor cells was observed by bone marrow examination; the immunophenotype was assayed by flow cytometry and immunohistochemistry; the chromosomal aberrations were detected by conventional chromosomal analysis and RT-multiplex nested PCR. The results showed that extramedullary skin lesions and myelodysplasia occurred before the onset of overt disease. At the time of diagnosis, this case had more than 30% blasts in bone marrow with meningeal involvement. Large-sized tumor cells predominated morphologically over other cells. Flow cytometry revealed the co-expression of myeloid antigens (cMPO, CD33 and CD117) and T-lymphoid antigens (cCD3, CD5, CD7, dual expression of CD4 and CD8). Immunohistochemical staining showed that CD43 and CD99 were strong positive which define the earliest hematopoietic progenitors. Partial tandem duplication of the MLL gene could be detected with normal cytogenetic method. All above-mentioned results led to the diagnosis of biphenotypic acute leukemia. It is concluded that the biphenotypic acute leukemia is an uncommon type of leukemia which may be preceded by myelodysplastic syndrome and has aggressive clinical and biological behavior. Immunophenotype, cytogenetics and molecular analysis can contribute to early diagnosis of BAL and evaluation of prognosis.

Ex vivo chemical modification of graft cells by methoxy polyethylene glycol alleviates graft versus host disease after haploidentical stem cell transplantation in mice / 中国实验血液学杂志

This study was aimed to explore the effect of ex vivo chemical modification of graft cells with methoxy polyethylene glycol (mPEG) on graft versus host disease (GVHD) after haploidentical stem cell transplantation in neonatal mice and its influence on activity of the stem cells. The modified and non-modified spleen cells of adult CB6F1 mice were injected into the abdominal cavity of neonatal BALB/c mice with 5x10(6) spleen cells per mouse, and GVHD were measured by spleen index (SI). Furthermore, the modified and non-modified mixture of bone marrow and spleen cells (BMS) were transplanted to haploidentical lethally irradiated adult BALB/c mice via tail vein with 2x10(5) BMS per mouse, and the colony forming units of spleens (CFU-S) were counted on the eighth day after irradiation. The results indicated that SI1 in modification group were lower than that in non-modification group, and SI2 in modification group was <1.3, showing that GVHD in modification group were less severe. The numbers of CFU-S formed in both modification group and non-modification group were not significantly different (p>0.05), indicating that the activity of the stem cells were not affected by mPEG modification. In conclusion, the modification of graft cells with mPEG alleviates GVHD after haploidentical stem cell transplantation in neonatal mice, and do not influence the activity of the stem cells.

Detection of MYCN mRNA in neuroblastoma cell lines by quantitative RT-PCR / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To examine the feasibility and practicability of quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) with SYBR GREEN I fluorescence for detecting the MYCN mRNA expression in neuroblastoma cell line LA-N-5.</p><p><b>METHODS</b>MYCN mRNA expression in LA-N-5 cells was measured using real time RT-PCR with SYBR GREEN I. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as internal control. The level of the MYCN mRNA was calculated as MYCN copies/GAPDH copies.</p><p><b>RESULTS</b>Standard curves were linear and showed high correlations (R2>0.99). The ratio of MYCN mRNA copies to GAPDH mRNA copies was calculated based on specific PCR products. The MYCN mRNA level in LA-N-5 cells was obtained (17.4 +/- 1.2).</p><p><b>CONCLUSIONS</b>Quantitative RT-PCR with SYBR GREEN I fluorescence may be a sensitive and reliable method for detecting the MYCN mRNA expression. It may also be potential applicable for detecting the MYCN mRNA expression in the small amount neuroblastoma tissues.</p>

Treatment of high-risk neuroblastoma with intensive chemotherapy, autologous peripheral blood stem cell transplantation, and 13-cis-retinoic acid / 中华儿科杂志

<p><b>OBJECTIVE</b>The prognosis for neuroblastoma in advanced stage is still poor, even under conventional chemotherapy. This study aimed to investigate if very high dose chemotherapy in conjunction with autologous peripheral blood stem cell transplantation and 13-cis-retinoic acid could get excellent results in children with high risk neuroblastoma.</p><p><b>METHODS</b>Six children, aged from 4 to 8 years, with stage IV neuroblastoma were included in the study. The duration of the illness before admission was 1 to 12 months. Primary sites of the diseases were in the abdominal cavity (n = 5) and thoracic cavity (n = 1). All of patients had bone marrow metastasis, and one had multiple bone metastasis and orbital metastasis. All of the patients received very high dose chemotherapy, surgery, local radiation (20-30 Gy), and autologous peripheral blood stem cell transplantation as well as 13-cis retinoic acid. Induction chemotherapy included vincristine 0.67 mg/(m2 x 24 h, x 3), cyclophosphamide 2.1 g/(m2 x 24 h, x 2) and doxorubicin 25 mg/(m2 x 24 h, x 3) for 4 courses. Drugs were given as 24 hour-continuous intravenous infusion. Etopside 200 mg/(m2 x 24 h, x 3) and cisplatin 50 mg/(m2 x 24 h, x 3) were given for 2 courses. Conditioning regimen included carboplatin 400 mg/(m2.d) for 4 days, etoposide 300 mg/(m2.d) for 4 days and melphalan 70 mg/(m2.d) for 3 days. 13-cis retinoic acid 160 mg/(m2.d) started on +59 days for 6 courses, each course including 14 days therapy and 14 days rest.</p><p><b>RESULTS</b>Six patients got a complete response before stem cell transplantation. Their bone marrow metastasis disappeared and so did bone and orbital metastasis. However, marrow suppression due to very high dose chemotherapy occurred in all of the patients, which lasted for 3-4 weeks for peripheral leukocyte recovery. Fever occurred after they finished 1/3 course of chemotherapy. Infection, however, was cured with the use of Fortum and Imipenem, ect. Autologous peripheral blood stem cell transplantation was initiated and successful in all cases. Follow-up studies revealed that all the patients were in CR status 4-18 months after transplant, and the cardiac and liver and renal functions were normal. Meanwhile, bone marrow was recovered or in the process of recovery.</p><p><b>CONCLUSION</b>The new strategies focused on very high dose chemotherapy, autologous peripheral blood stem cell transplantation and biological therapy might be a good option for patients with advance neuroblastoma.</p>

Effect of chemical modification of grafts on the survival improvement post haploidentical bone marrow transplantation in mice / 中华儿科杂志

<p><b>OBJECTIVE</b>Human leukocyte antigen (HLA) haploidentical bone marrow is a potential source of donor to children for its availability. The drawback is deleterious graft versus host disease (GVHD) reaction post transplantation because of the incompatibility of HLA antigen expression between donors and recipients, in which donor T lymphocyte is stimulated to proliferate and differentiate. The methoxy polyethylene glycol (mPEG) is a kind of amphoteric compound without immunogenicity, which was used to modify various proteins covalently and to prepare the versatile blood type. If mPEG modification blocks the activation of T cells in grafts, GVHD reaction probably would become less serious and transplantation might become successful. The aim of this study was to verify the improvement of haploidentical bone marrow transplantation (BMT) in a murine model by using mPEG of certain concentration to modify the grafts.</p><p><b>METHODS</b>Male BALB/c mice were chosen as the donor, and female CB(6)F(1) mice as the recipient. There were three groups of mPEG modification, non-modification and irradiation control, and 20 mice in each group. The modified and non-modified mixture of bone marrow and spleen cells (as T lymphocytes) were transplanted to haploidentical lethally irradiated CB(6)F(1) mice via the tail vein. After the transplant, the hematopoietic recovery, survival rate, acute graft versus host disease (aGVHD) and chromosomal karyotype were analyzed and compared with controls.</p><p><b>RESULTS</b>Seventy-five percent (15/20) of mice survived in the group of mPEG modification, while only 40% (8/20) survived in the group without the modification (chi(2) = 5.01, P = 0.025). And 100% mice died in the group of the irradiation control within 2 weeks. The hematopoietic recovery in the group of mPEG modification was show n to be faster than that in the group without modification (P < 0.05). Histopathological examination of the skin, liver and intestine showed typical signs of aGVHD, but the GVHD grading in the group of modification was less severe. The recipient mice in both groups of transplantation surviving for more than 75 days showed complete donor-type implantation by the chimerism examination.</p><p><b>CONCLUSION</b>The modification of grafts by mPEG could alleviate aGVHD and improve the survival rate of mice after the haploidentical bone marrow transplantation.</p>

Clinical Characteristics of Basal Ganglia Calcification Combined with Cerebral Infarction / 实用儿科临床杂志

Objective To explore the clinical characteristics and pathologic mechanism of basal ganglia calcification combined with cerebral infarction.Methods The clinical data,radiation demonstration and prognosis in 53 cases of cerebral infarction were reviewed retrospectively,31 cases with basal ganglia calcification and 22 cases without basal ganglia calcification.Results Cerebral infarction with basal ganglia calcification mostly occurded in infants and after slight traumar,CT scan all showed lacunar infarction in basal ganglia and MRA demonstrated no severe changes on cerebral vessal.Compared with the infants without basal ganglia calcification,the infants with basal ganglia calcification recovered quickly and had better prognosis.Conclusions The infants of cerebral infarction with basal ganglia calcification had unique clinical characteristics.The occurrence of cerebral infarction is closely associated with basal ganglia calcification which perhaps resulted from cerebral hypoxia and ischemia previously.