LUNG DOSE DETERMINATION IN TOTAL BODY IRRADIATION PRIOR TO BONE MARROW TRANSPLANTATION

Total body irradiation(TBI)combined with chemotherapy prior to bone marrow transplantation(BMT)is used successfully for treatment leukemias.It need a high and homogeneous radiation dose to all target cells,dispersed In the whole body.The lung is the most sensitive vital organ at risk in TBI.The lung dose must be within it's tolerable level.So,the determination of the lung dose is most Important for TBI.The determination of the lung dose is dependent on at least 8 parameters.In order to determine the effect of 8 parameters on the lung dose,using a system of phantom of Essen University hospital in F.R.Germany,a lot of measurements and a systematical investigation was made by varying 8 parameters,under the Essen translation TBI conditions.A analysis and discussion of results was made.

A prophylactic approach for bone marrow transplantation from a hepatitis B surface antigen-positive donor

It has been accepted that bone marrow transplantation （BMT） is the only curative therapeutic option for certain hematologic malignancies. The southeast Asia region is an endemic area of hepatitis B virus （HBV） infection; thus, BMT using a hepatitis B surface antigen （HBsAg）- positive donor is occasionally unavoidable. Organ transplantation using a HBsAg-positive donor can lead to post-transplantation de novo HBV infection and severe HBV-related hepatitis if no effective prophylactic measures are taken prior to and after transplantation. In this report, a four-level approach was designed for a patient with chronic myeloid leukemia, beginning with a booster HBV vaccination before performing BMT with a HBsAg-positive donor. Prior to BMT, the HBV viral load of the donor was reduced to an undetectable level by anUviral therapy. After BMT, hepatitis B immunoglobulin was administered intramuscularly for 1 wk together with a long-term antiviral drug, lamivudine. One year after discontinuation of lamivudine, the patient is still free of HBV infection.

Alpha-GalCer Administration after Allogeneic Bone Marrow Transplantation Improves Immune Reconstitution in Mice

Objective To explore the effect of α-galactosyleramide （α-GalCer） on immune reconstitution under acute graft-versus-host disease （aGVHD）. Methods BALB/c mice were transplanted with allogeneic C57BL/6 bone marrow cells and splenocytes （both 1 × 10^7） after receiving lethal total-body irradiation, α-GalCer （100 ug/kg） or vehicle （dimethyl- sulfoxide） was administered intraperitoneally immediately after transplantation. The effects of α-GalCer on immune reconstitufion, proliferation of T cells and B cells, hematopoiesis, and thymic microenvironment were assessed. Results The α-GalCer group exhibited higher percentages of CD3^＋, CD4^＋, CD8^＋, B220^＋, CD40＋, and CD86＋cells compared with the vehicle group. The number of colony forming unit per 1000 CD34^＋ cells in the et-GalCer group was higher than in the vehicle group （P=0.0012）. In vitro proliferation assays showed that the α-GalCer group had higher percentages of CD3^＋, CD4^＋, CD8^＋, and B220^＋ cells compared with the vehicle group. As for the results of in vivo proliferation assays, the numbers of CD3^＋, CD4^＋, CD8^＋, and B220^＋ cells were higher in the α-GalCer group than in the normal group, especially the number of B220^＋ cells （P=0.007）. Significant difference was not found in thymocyte count between the α-GalCer group and the vehicle group, nor in the percentages of CD3^＋, CD4^＋, and CD8^＋ cells. Conclusion Administration of tl-GalCer after allogeneic bone marrow transplantation may promote immune reconstitution in the presence of aGVHD.

Effects of dendritic cell and subgroup changes on bone marrow transplantation treatment of multiple sclerosis

BACKGROUND： Bone marrow transplantation is an effective treatment for severe forms of various autoimmune disorders. Dendritic cell reconstitution is thought to be one factor contributing to host immune recovery and therapeutic efficacy. OBJECTIVE： To assess the effects of bone marrow transplantation on an animal model of experimental autoimmune encephalomyelitis （EAE）, and to investigate changes in dendritic cells and subgroups following bone marrow transplantation. DESIGN, TIME AND SETTING： This experimental, neuroimmunological study was performed in Sun Yat-sen University between August 2006 and May 2007. MATERIALS： A total of 30 female C57BL/6 mice, aged 6-8 weeks, served as recipients, and 20 female adult C57BL/6 served as donors. Myelin oligodendrocyte glycoprotein 35-55 amino acid peptide （MOG35-55） of mudne origin was synthesized by Bio-Scientific （Xi＇an, China, purity 〉 95%）. Complete Freund＇s adjuvant was purchased from Difco Laboratories, Detroit, MI; pertussis vaccine was purchased from Alexis, San Diego, CA; radiation device and flow cytometry for FACS analysis were purchased from Theratron 780-C, Canada and Coulter, Fullerton, CA, respectively. METHODS： The C57BL/6 mice, aged 6-8 weeks, were immunized by subcutaneous injection of MOG35-55 peptide emulsified in complete Freund＇s adjuvant, which contained 500 μg Mycobacterium tuberculosis H37RA. The mice were subsequently intravenously injected with pertussis vaccine to induce EAE. The mice were randomly assigned to transplantation and EAE model groups （n = 12 for each）. Bone marrow cells [（5-10） × 10^6] were transplanted into EAE mice via the tail vein 4-6 hours following total body irradiation, and the model group was not treated. MAIN OUTCOME MEASURES： Mouse behavioral changes following EAE were evaluated daily. Injured spinal cord tissue sections were stained with hematoxylin and eosin 20 days after the initial immunization to observe inflammatory infiltration using light microscopy. Flow cytometry was used to detect the ratio and absolute number of DC1 （CD6aCD11c＋） and DC2 （CD8a＋CD11c＋） in peripheral blood 36 days after bone marrow transplantation. RESULTS： Significant improvement in clinical signs was observed in EAE mice following bone marrow transplantation compared with the model group （P 〈 0.01 ）, as well as attenuated lymphocyte and macrophage infiltration. Compared with the model group, the absolute number of dendritic cells and DC1, as well as the DC1/DC2 ratio, was significantly greater following bone marrow transplantation （P 〈 0.05 or P 〈 0.01）. CONCLUSION： The increased proportion of dendritic cells and DC1 is proposed to contribute to EAE remission following bone marrow transplantation.

Enhancing Effects of Ligustrazine on Expression of CD31 and Hematopoietic Reconstitution in Syngenic Bone Marrow Transplantation of Mice

Summary： The effect of ligustrazine on the expression of CD31 in syngenic bone marrow transplantation （BMT） mice was studied. Fifty-six Balb/c mice were divided into 3 groups： normal control group. BMT control group, and ligustrazine treated group. Syngenic BMT mouse models were established according to the literatures. In BMT control group and the ligustrazine treated group, the mice were given respecxively orally 0.2 mL saline and 2 mg ligustrazine twice a day. On the 7th, 14th, and 21st day after BMT, the mice were killed. The expression of CD31 on the surface of bone marrow nuclear cells （BMNC） was detected by flow cytometry. Peripheral blood leukocytes, platelets and BMNC were counted. Histological observation of bone marrow was made. The results showed thai in ligustrazine treated group the peripheral blood leukocylcs, platelets and BMNC counts, and the expression of CD31 on the day 7, 14, 21 after BMT were higher than in BMTcontrol group （P〈0.01 or P〈0.05）. In conclusion, ligustrazine could obviously enhance the CD31 expression on the surface of BMNC after syngcnic BMT in mice, which may be one of the mecha- nisms underlying the ligustrazine accelerating hematopoietic reconstitution in syngenic BMT.

Effects of Ligustrazine on Expression of Bone Marrow Heparan Sulfates in Syngeneic Bone Marrow Transplantation Mice

To explore the effects of ligustrazine on bone marrow heparan sulfates (HS) expression in bone marrow transplantation (BMT) mice, the syngeneic BMT mice were orally given 2 mg ligustrazine twice a day. On the 7th, 10th, 14th, 18th day after BMT, peripheral blood cells and bone marrow nuclear cells (BMNC) were counted, and the expression levels of HS in bone marrow and on the stromal cell surfaces were detected by immunohistochemistry and flow cytometry assay respectively. In ligustrazine-treated group, the white blood cells (WBC) and BMNC on the 7th, 10th, 14th, 18th day and platelets (PLT) on the 7th, 10th day were all significantly more than those in control group (P<0.05). The bone marrow HS expression levels in ligustrazine-treated group were higher than those in control group (P<0.05) on the 7th, 10th, 14th, 18th day. However, the HS expression levels on the stromal cell surfaces showed no significant difference between the two groups on the 18th day (P>0.05). It was concluded that ligustrazine could up-regulate HS expression in bone marrow, which might be one of the mechanisms contributing to ligustrazine promoting hematopoietic reconstitution after BMT.

Effect of Ligustrazine on the Expression of LFA-1, ICAM-1 Following Bone Marrow Transplantation in Mice

Summary: The effects of ligustrazine on the expression of LFA-1, ICAM-1 in bone marrow tissue and the mechanism promoting hematopoietic reconstitution following bone marrow transplantation (BMT) were investigated. The 150 mice were randomly divided into 3 groups: normal group, saline group and ligustrazine group. The normal group received no treatment, while in the saline group and ligustrazine group, the mice were subjected to normal saline (0.2 ml, twice a day) and ligustrazine (0.2 ml, twice a day) respectively through a gastric tube. At the 7th, 14th, 21st and 28th day after BMT, survival rate, colony forming unit of spleen (CFU-S), peripheral blood cells and bone marrow mononuclear cells (BMMNC) were measured, histological changes in bone marrow tissue were observed and the expression level of LFA-1, ICAM-1 was detected. In ligustrazine group CFU-S counts on the 10th day and the peripheral blood WBC, PLT, BMMNC counts, hematopoietic tissue volume as well as the expression level of LFA-1 on the 7th, 14th, 21st, 28th day after BMT were higher than in saline group (P<0.01 or P<0.05). Mature RBC volume and the expression level of ICAM-1 were significantly lower in the ligustrazine group than in the saline group (P<0.01 or P<0.05). In the ligustrazine group, fat tissue volume was higher on the 7th, 14th day after BMT (P<0.01) and was lower on the 21st, 28th day (P<0.01) after BMT than in the saline group. It was concluded that Ligustrazine could improve bone marrow microenvironment and promote hematopoietic reconstitution.

Attenuation of GVHD for Allo-Bone Marrow Transplantation Recipient by FasL-Fas Pathway in an H-2 Haplotype Disparate Mouse Combination

In order to explore a new special and effective way to prevent graft versus host disease (GVHD) after allogenic bone marrow transplantation (allo-BMT), the stem cell antigen-1 (Sca-1) + early hematopoietic cells (EHC) from BALB/c mouse (H-2 d) were introduced with exogenous mouse Fas ligand (mFasL) cDNA gene by the retrovirus-mediated gene transfer and expanded for one week, and then they were co-cultured with the spleen mononuclear cells (SMNC) from BAC mouse (H-2 d×b) as one way mixed lymphocyte reaction (OWMLR). The cytotoxicity of treated BAC mouse SMNC against Na 2 51CrO 4 labeling SMNC from BALB/c mouse was observed. The bone marrow mononuclear cells (BMMNC) from BAC mouse treated by the above methods were transplanted into lethally-irradiated congenic BALB/c mice to observe the occurrence of GVHD. The results showed that the SMNC from BAC mouse after OWMLR with exogenous mFasL cDNA gene-transduced hematopoietic cells (HC) from BALB/c mouse in a ratio of 1 to 5 exhibited an obvious inhibition of the cytotoxicity against the BALB/c mouse spleen cells at different effector/target ratios as compared to the control group (P<0.01). The gradeⅠ GVHD or no GVHD and the 80 % survival rate at day 60 post-BMT were observed in the BALB/c mouse receiving BAC mouse BMMNC treated with similar way, while the grade Ⅱ-Ⅲ GVHD and the 20 % survival rate were noted in the control group (P<0.01). It is suggested that the attenuation of GVHD in allo-BMT recipient could be successfully achieved through FasL-Fas pathway in an H-2 haplotype disparate mouse combination.

HIM_(1) AND HIM4, TWO MONOCLONAL ANTIBODIES POTENTIALLY USEFUL FOR AUTOLOGOUS BONE MARROW TRANSPLANTATION IN CHRONIC MYELOGENOUS LEUKEMIA

We developed two complement-fixing MoAbsHIMand HIM(murine)that were specifically reac-tive with chronic myelogenous leukemia (CML) cells.They were capable of fixing human or rabbit com-plement and suitable for CML cells purging of re-mission marrow from CML patients.HIMreactedwith majority leukemic cells form 7 out of 10 CMLpatients by complement-mediated cytotoxicity(C’MC)assay(positive cells 80%—90%),HIMreacted withmajority CML cells from 4 out of 5 CML by C’MCassay(positive cells 80%—90%).Treatment withHIMor HIMand human C’was capable of lysing97% of K562,U937,HL-60 and CML cells in a 20fold excess of unrelated cells by indirect FITC+EBstain.Using limited dilution culture,incubation withHIMand C’produced 1.5 logs inhibition of growthin K562 cells,and 1.9 logs in U937 cells,and withHIMand C’produced 2.9 logs inhibition in HL-60cells and 3.0 logs in U937 cells.Both MoAbs cocktailwas shown 1.8 logs in K562 cells and 3.2 logs in U937cells.They were no suppression on the growth o

Effects of Platelet Factor 4 on Expression of Bone Marrow Heparan Sulfate in Syngenic Bone Marrow Transplantation Mice

To explore the effects of platelet factor 4(PF4) on hematopoietic reconstitution and its mechanism in syngenic bone marrow transplantation (BMT). The syngenic B MT mice models were established. 20 and 26 h before irradiation, the mice were injected 20 μg/kg PF4 or PBS twice into abdominal cavity, then the donor bone marrow nuclear cells (BMNC) were transplanted. On the 7th day, spleen clone forming units (CFU S) were counted. On the 7th, 14th and 21st day after BMT, the BMNC and megakaryoryocytes in bone marrow tissue were counted and the percentage of hematopoietic tissue and expression level of heparan sulfate in bone marrow tissue were assessed. In PF4 treated groups, the CFU S counts on the 7th day were higher than those in BMT groups after BMT. The BMNC and megakaryoryocyte counts and the percentage of hematopoietic tissue and heparan sulfate expression level were higher than those in BMT group on the 7th, 14th and 21st day after BMT ( P <0.01 or P <0.05). PF4 could accelerate hematopoietic reconstitution of syngenic bone marrow transplantation. The promotion of the heparan sulfate expression in bone marrow may be one of mechanisms of PF4.

Study on the Effect of Ligustrazine on Hematopoietic Reconstitution in Bone Marrow Transplantation Mice

To explore tile effects of ligustrazine on hematopoietic reconstitution and its mechanism after bone marrow transplantation (BMT), the allogenic BMT mice were given intra-abdominal injection of 2,mg ligustrazine twice a day. On the 1st, 7th, 14th, and 28th day after BMT, peripheral blood cells and bone marrow nuclear cells (BMNC) were counted, and the histological features were evaluated. On the 7th, 14th, 21st day after BMT, CXCR4 expression on the BMNC was assayed. The results showed that peripheral blood cell counts and BMNC counts in ligustrazine-treated group on the 7th, 14th, 28th day were higher than those in BMT group (P<0. 01 or P<O. 05). The percentage of hematopoietic tissue volume, fat tissue hyperplasia and congestion and dilation degree of microvessel in ligustrazine-treated group on the 7th, 14th, 21st, 28th day was higher than those in BMT group. The CXCR4 expression levels in ligustrazine-treated group were higher than in BMT group (P<0.01 or P<0. 05) on the 7th and 14th day, and were lower than in BMT group on the 21st day (P<0. 01 ). It is concluded that the ligustrazine can accelerate hematopoietic reconstruction, enhance growth of hematopoietic tissues and promote the repair of microvessels. The CXCR4 expres- sion levels on BMNC may be responsible for the effect of ligustrazine.

T CELL REPERTOIRE COMPLEXITY IN SEVER COMBINED IMMUNODEFICIENCY PATIENTS AFTER BONE MARROW TRANSPLANTATION

Objective. To study thymus-dependent T cell development and T cell repertoire in human s ever com-bined immunodeficiencypatients after HLA-identical or haploid entical T cell-depleted allogeneic bone marrow transplantation.Methods .Blood samples were obtained from15SCID patients before transplantation and a t varying intervals thereafter.Quantitative competitive PCR assay and immunosco pe analysis of the T cell receptorVarepertoire were performed.Results. Before and within the first100days after transplantation,patients’ periphera l blood mononuclear cellpresented an oligoclonal or polyclonal skewed T cell repertoire,low T cell re-ceptor excision circlesvalues and pred ominance of CD45RO + T cell.In contrast,the presence of high numbers of CD45RA + T cells in bone marrowcirculation reconstituted SCID patients(>10 0days post-transplantation)correlated with active T cell production by the th ymus as revealed by high TREC values,and a polyclonal T cell repertoire demonst rated by a Gaussian distribution of Va-specific peaks.Conclusions.Within one year after BMT ,a normal T cell repertoire develops in SCID patients as a resu lt of thymic output.The T cell receptor diversity is highly and positively corr elated in these patients with TREC levels.

EFFECTS OF INTERLEUKIN-4 ON GRANULOCYTE-MACROPHAGE-COLONY FORMATION FROM MURINE BONE MARROW CELLS AND HEMATOPOIETIC RECONSTITUTION FOLLOWING MURINE ALLOGENEIC BONE MARROW TRANSPLANTATION

We investigated the effects of mouse recombinant IL-4 on hematopoiesis in vitro and in vivo. IL-4 alone was found to be incapable of stimulating colony formation, but it inhibited both IL-3-and GM-CSF-induced colony formation by murine hematopoietic progenitor cells. In contrast, colony formation induced by G-CSF was enhanced in the presence of IL-4. We also studied the influence of IL-4 on hematopoietic reconstiution after allogeneic bone marrow transplantation in a murine medel, and found that IL-4 had significant inhibitory effects on neutrophil recovery and that neutrophil recovery accelerated by IL-3 and G-CSF was significantly suppressed by IL-4. The combination of IL-4 and GM-SF caused a significant decrease in the absolute number of neutrophils.

Experimental study of G-CSF alleviating graft-versus-host disease after mixed bone marrow transplantation in mice

Objective： How to reduce the incidence and severity of acute graft-versus-host disease （aGVHD） is a crucial step to improve the overall survival of allogeneic bone marrow transplantation （allo-BMT）. The low incidence of severe aGVHD observed in allogeneic peripheral blood stem cell transplantation （allo-PBSCT）, which may be related to modulating immune function of T lymphocytes by granulocyte colony-stimulating factor （G-CSF） primed donors. The study aimed to explore whether aGVHD could be alleviated by syngeneic bone marrow mixed with G-CSF-mobilized H-2 haploidentical marrow grafting. Methods： Female BALB/c mice and neonatal BALB/c mice were recipients and male （BALB/c × C57BL/6）F1（BCF1） mice were donor mice respectively. Donor mice were injected subcutaneously with G-CSF daily at 0.01 μg/g body weight or saline for 6 days, and splenocytes were harvested on day 6. Spleen index （SI） represented GVHD in neonatal mice after the intraperitoneal injection of mixed spleen cells. Lethally irradiated （^60Co, 8.5 Gy） adult mice were transplanted with a mixture of syngeneic plus G-CSF-mobilized （control diluents） H-2 haploidentical marrow cells. Survival time and survival rate of the recipients were observed after mixed marrow transplantation （MBMT）. GVHD was assessed by observing signs of weight loss, ruffled fur, diarrhea and histological change of skin, liver and small intestines. Enzyme-linked immunosorbent assay （ELISA） method was used to detect cytokines （IL-2, IL-4 and INF-γ）. Fluorescence-activated cell sorting （FACS） analysis was used to detect T cells phenotype. Results： （1） The neonatal mice subject to injection of 2：1 and 1：1 mixed spleen cells and H-2 haploidentical spleen cells all suffered from aGVHD. The severity of aGVHD in recipient mice receiving G-CSF-mobilized splenocytes was dramatically reduced. （2） The aGVHD signs and histological change were observed in most mice of 2：1 and 1：1 MBMT groups. However, the survival time of G-CSF-mobilized MBMT was longer than in control groups and these mice had signs of moderate GVHD. （3） L3T4^＋ cells and relative ratio in both subsets was significantly reduced in G-CSF-treated donor mice. The total number of Thyl.2 and lyt2^＋ cells was increased after G-CSF pretreatment of donors, but no statistical difference. （4） The supernatants from a primary MLR were collected at 48 h for cytokine measurement. The results showed an increased production of IL-4 and a decreased production of IL-2 and INF-γ after stimulating with concanavalin A for 48 h. Conclusion： The GVHD could be reduced using syngeneic bone marrow mixed with H-2 haploidentical marrow cells. The severity of aGVHD in recipient mice receiving G-CSF-mobilized splenocytes or marrow cells could be further moderated, which is associated with increased IL-4 production and decreased IL-2 and INF-y production.

Effect of NK cells on GVHD in H-2 haploidentical bone marrow transplantation in mice

To study the effect of natural killer （NK） cells on graft-versus-host disease （GVHD） after H-2 haploidentical bone marrow transplantation （BMT） in mice. Methods ：Murine model of H-2 haploidentical BMT was established by using Balb/c （H- 2d） mouse as recipient, and Balb/c （H-2d）×C57BL/6 （H-2b） （H-2db） mouse as donor. Lethally irradiated Balb/c （H-2d） mice were transplanted with the bone marrow cells from Balb/c（H-2d）×C57BL/6（H-2b） （H-2db） mice containing donor spleen cells and/or NK cells. GVHD and survival rates were studied by observation of clinical manifestations and pathological changes. Results：In the group of bone marrow ＋spleen cells, GVHD was induced in 90% mice; but in the group plus with low amount of NK cells, GVHD was induced in 20% mice; and in the group transplanted with high amount of NK cells, GVHD was induced only in 10% mice. Compared to the group transplanted only with BM plus spleen cells, the incidences of GVHD in the latter two groups decreased significantly （P 〈 0.01） and the survival rates at different periods of 15, 30, 45 and 60 days increased obviously （P 〈 0.01）. Conclusion： In mouse H-2 haploidentical BMT, alloreactive NK cells can reduce the incidence of GVHD and increase the survival rate.

Cost Effectiveness Analysis of Filgrastim versus Placebo in Post AIIogentic Bone Marrow Transplantation

Filgrastim is used to accelerate hematopoietic recovery after ABMT （allogeneic bone marrow transplantation）. Its impact on the total cost of patient care remains to be explored. We therefore undertook a cost effectiveness analysis in the context of a randomized single blinded clinical trial of Filgrastim versus placebo in post ABMT. A primary endpoint, duration of myelosuppression, and three secondary end points （number of days of fever, length of hospital stay, survival at one hundred days） were used to assess efficacy. Direct costs were evaluated and allowed the calculation of the ICER （incremental cost-effectiveness ratios） for the major endpoint of the trial. Sixteen patients were included in the study. The duration of myelosuppression was significantly decreased in the Filgrastim arm with medians of 15 days vs. 19 days in the placebo arm （p = 0.023）. Cost analysis showed no statistically significant difference between the two arms. According to the calculation of ICER, Filgrastim was more costly and more effective than placebo for the number of days of aplasia avoided and the number of days with fever avoided. Placebo strictly dominated filgrastim for days of hospitalization avoided. Filgrastim has proven effective in reducing the duration of aplasia without increasing costs.

Cytomegalovirus infection in the bone marrow transplant patient

Cytomegalovirus(CMV) infection is an important contributor to the morbidity and mortality associated with bone marrow transplantation(BMT). Infection may lead to CMV disease involving multiple organs such as pneumonia, gastroenteritis, retinitis, central nervus system involvement and others. CMV seropositivity is an important risk factor and approximately half of BMT recipients will develop clinically significant infection most commonly in the first 100 d post-transplant. The commonly used tests to diagnose CMV infection in these patients include the pp65 antigenemia test and the CMV DNA polymerase chain reaction(PCR) assay. Because of its greater sensitivity and lesser turnaround time, the CMV PCR is nowadays the preferred test and serves as a main guide for pre-emptive therapy. Methods of CMV prevention include use of blood products from seronegative donors or leukodepleted products. Prophylaxis or pre-emptive therapy strategies for CMV prevention may be used post-transplant with the latter becoming more common. The commonly used antivirals for pre-emptive therapy and CMV disease management include intravenous gancyclovir and foscarnet. The role of intravenous immunoglobulin, although used commonly in CMV pneumonia is not clear.