Rapid migration of host lymphocytes into the lymphoid tissue of the intestinal graft at early stage after transplantation

AIM:To determine the migration mode of host lymphocytes into the intestinal graft by analyzing different lymphoid compartments of the graft. METHODS:2, 4, 6 days after transplantation, murine intestinal grafts were harvested for histologic assessment. Lymphocytes were collected from mesenteric lymph nodes (MLN), Peyer’s patch (PP), lamina propria lymphocytes (LPL) and intraepithelial lymphocytes (IEL) in the graft for analyzing by cytometry. RESULTS:At the day 2 and day 4, no evidence of rejection in intestinal allograft was found, while the rejection signs were apparent at the 6th day. At day 2 and day 4, host lymphocytes had rapidly replaced donor cell in graft MLN and PP. The migration of host lymphocytes into the LPL and IEL was not as fast as MLN and PP, however, the near-total replacement took place at the 6th post transplant day. The host lymphocytes were activated at the early stage of recruitment into the graft. CONCLUSION:The rapid recruitment of recipient lymphocytes and early activation could be responsible for the formidable rejection of intestinal transplantation.     

Effect of granulocyte colony-stimulating-factor on acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation in a murine model

AIM: To explore the impact of granulocyte colony-stimulating factor (G-CSF) on acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in a murine model and its possible mechanisms. METHODS: Male C57BL/6 (H-2^b) and BALB/c (H-2^d) mice were used as the allogeneic and syngeneic donor mice, respectively. Moreover, female BALB/c mice were used as recipient mice. The recipient mice were conditioned by a single dose (8 Gy) of total body irradiation (TBI). The recipient mice were randomly divided into 7 groups: TBI group, Syn-BMST control group, post-Syn-BMST G-CSF administration (Syn-BMST+G-CSF)group, allo-BMT control group, post-allo-BMT G-CSF administration (allo-BMT+G-CSF)group, allo-BMST control group and post-allo-BMST G-CSF administration (allo-BMST+G-CSF) group. The mice in control groups and G-CSF administration groups were subcutaneous injected with 0.1 mL normal saline (NS) and 0.1 mL NS containing 2 μg G-CSF per day from 1st day, respectively. The effect of G-CSF on aGVHD was evaluated by clinical manifestations and pathological changes, as well as survival time of the mice in different groups. The serum levels of IL-2, IL-4, IFN-γ and TNF-α in allo-BMST and allo-BMST+G-CSF groups were detected by ELISA at 10th day. Flow cytometry was used to analyze the immunophenotypes of splenocytes at 10th day. RESULTS: The mice in TBI group were all died for hematologic failure on 9~15 d after TBI. No effect of G-CSF on the survival of the mice underwent Syn-BMST and transplantation of single allogeneic marrow cells was observed. The mean survival days in allo-BMST group and allo-BMST+G-CSF group were (34.8±4.5) d and (19.8±6.1) d'respectively (P<0.01). Moreover, post-transplant administration of G-CSF increased the spleen total nucleated cells count (SpTNC), NK cells subset, and DC1/DC2 ratio in the spleen with over 99% of donor chimerism rate at 10th day. No difference in the levels of serum IL-2, IL-4, IFN-γ and TNF-α between the 2 group at 10th day was found. CONCLUSION: The administration of G-CSF after allo-BMST significantly aggravates mouse aGVHD. The expansion of NK cells stimulated by G-CSF may be involved in the mechanism of generating alloreactivity against host cells. These results imply there may be potential risk of evoking or aggravating acute GVHD if G-CSF is administered in the early stage of clinical allo-HSCT.     

Role of NK cells in mouse allogeneic bone marrow transplantation

AIM: To study the effect of natural killer(NK)-cells on graft-versus-host disease(GVHD), graft rejection, engraftment and reconstituting of hematopoiesis in mouse allogeneic bone marrow transplantation. METHODS: Lethally irradiated BALB/c(H-2^d) mice were transplanted with C57/6j(H-2^b) bone marrow containing donor peripheral T cells and/or NK cells. Recipients CD₃₄⁺ cell counts and the expression of H-2K^b＋ cell were detected by flow cytometry, peripheral white blood cell(WBC) was detected by auto-cytometry, and the recipients survival rates, GVHD, engraftment and hematopoiesis recovery were then observed. RESULTS: In the group of transplanted with NK cells infusion, the incidence of GVHD was evidently lessened, the survival rates, WBC and CD₃₄⁺ cell counts and the expression of H-2K^b＋ cell were significantly high than that without NK cells infusion. CONCLUSION: In mouse allogeneic bone marrow transplantation, alloreactivity NK cells prevents GVHD, reduces graft rejection, and promotes engraftment and reconstituting of hematopoiesis.     

Role of B cells in CD45RB antibody-induced transplantation immune tolerance

AIM: To investigate the role of B cells in CD45RB antibody-induced transplantation immune tolerance. METHODS: Single cell suspension was made from the spleen of BALB/c nude mice disposed by CD45RB antibody, then mixed cultured with T cells of BALB/c mice and spleen cells of C57BL/6 mice. The Th1, Th2, Treg and Tm cells were monitored by flow cytometry during the culture process. The skin graft model was set up with B6.μMT^-/- mice as receptors and BALB/c mice as donors. CD45RB antibody was intraperitoneally injected into the receptors after transplantation and then CD3⁺CD45RB^hi cells were detected by flow cytometry. In another mixed lymphocyte culture, CD45RB antibody was added, and then B cells were isolated and injected into B6.μMT^-/- mice through the tail vein. The heart transplantation model was established with B6.μMT^-/- mice as receptors and BALB/c mice as donors, and then the survival and the migration of B cells to the thymus were observed. RESULTS: When T lymphocytes were co-cultured with B lymphocytes treated with anti-CD45RB monoclonal antibody(mAb) in vivo, the percentages of Th2 and Treg cells were up-regulated and Th1 cells were down-regulated, but Tm cells were not altered as compared with the control. In vivo without B lymphocytes, anti-CD45RB mAb also down-regulated the expression of CD45RB in T lymphocytes. The reduction was faster and the percentage of CD3⁺CD45RB^hi T cells was not altered as compared with the control. The B lymphocytes treated with anti-CD45RB mAb in vitro prolonged the lifetime of receptor in heart transplantation model but failed to induce complete tolerance. After recieving B cells treated with anti-CD45RB mAb and allogeneic heart transplantation, B cells migrated to the thymus in B6.μMT^-/- mice. CONCLUSION: B lymphocytes play a definite role in the transplantation immune tolerance induced by anti-CD45RB mAb through their affection on T-cell subgroups and also in the central tolerance. However, the induction of immune tolerance can not only rely on B cells.     

Chinese herbal preparation Qing Yi Tang Ⅱ granule protects against experimental acute pancreatitis in mice

AIM: To investigate the protect effect of Chinese herbal preparation, Qing Yi TangⅡgranule (QYT), on acute pancreatitis (AP) mice and its mechanism. METHODS: Adult male and female C57BL/6 mice (n=24) were randomly divided into control group, AP group and AP+QYT group. Severe AP was induced by combined intra-peritoneal injection of caerulein (50 μg/kg) and lipopolysaccharide (LPS; 10 mg/kg). Drinking water or 24% QYT solution was given to the mice in AP group or AP+QYT group by oral gavage. The mice in control group were intraperitoneally injected with equivalent volume of normal saline and gavaged with water. The mice were sacrificed 3 h after the last injection. Severity of AP was assessed by biochemical markers and histology. The plasma level of IL-6 and MCP-1, and lung myeloperoxidase (MPO) levels were determined for assessing the extent of systemic inflammatory response. The intestinal microflora, T lymphocytes and T-lymphocyte subgroups were examined for assessing the function of the intestinal barrier. RESULTS: Compared with control group, the mice in AP group presented significant increases in pathological histological scores, plasma amylase activity and IL-6 and MCP-1 levels, as well as the MPO activity in the lung and pancreatic tissues. QYT attenuated these changes to some extent. Furthermore, the increased intestinal microflora was significantly reversed by QYT. No difference of the numbers of Peyer's patches in small intestine in the 3 groups was observed, but the percentage of CD3⁺ T lymphocytes decreased significantly in AP group, and increased percentage of CD4⁺ and CD4⁺/CD8⁺ ratio were found in AP group and AP+QYT group. CONCLUSION: QYT protects against cearulein and LPS-induced acute pancreatitis in mice. The mechanisms may be related to the suppression of the inflammatory response, promoting intestinal bacteria removal, and regulating the functions of T lymphocytes in the intestinal barrier.     

Restoration of visual function of mice with MNU-induced retinal degene-ration by expression of melanopsin in ON-bipolar cells

AIM：To investigate the changes of light response and visual behavior after transfecting intrinsic photosensitive protein melanopsin (opsin 4, Opn4) into ON-bipolar cells in a mouse retinal degeneration model. METHODS：N-methyl-N-nitrosourea (MNU)-induced CD1 mice served as the retinal degeneration model. CD1 mice on postnatal day 0 (P0) and postnatal day 1 (P1) were subretinally injected with Grm6-Opn4-GFP plasmid, and those injected with Grm6-GFP plasmid served as negative control group. Electroporation was applied to transfect the plasmids into cells. Five weeks later, the transfected mice received intraperitoneal injection of MNU to induce the apoptosis of photosensory cells in 7 days. The mice were divided into 5 groups：normal control group, normal saline (NS) intraperitoneal injection with Grm6-Opn4-GFP transfection (NS+melanopsin) group, MNU intraperitoneal injection with Grm6-Opn4-GFP transfection (MNU+melanopsin) group, MNU intraperitoneal injection with Grm6-GFP transfection (MNU+GFP) group and MNU intraperitoneal injection (MNU) group. Visual behavior was tested by Dark/Light Box test for 7 days after MNU injection, in which the time spending in each area of the box was measured every day for each animal. Electroretinagram was applied to measure the peak amplitude and the latency time of b-wave, and photopic negative response (PhNR), which stand for the functions of ON-bipolar cells and retinal ganglion cells, respectively. Immunohistochemistry was performed to check the expression of melanopsin in the ON-bipolar cells in the transfected retina. RESULTS：Melanopsin was specifically transfected into ON-bipolar cells and was expressed in the transfected cells. The dark/light box test showed that the mice in MNU+melanopsin group stayed significantly longer in the dark zone than those in negative control group (P<0.05). Their b-waves tended to recover as well (P<0.05 vs negative control group). CONCLUSION：Specific transfection of melanopsin into ON-bipolar cells can partially restore the visual function of mice.     

Distribution and identification of immunocytes in humanized SCID mouse model

AIM: Humanized-NOD/SCID(hu-NOD/SCID) mouse model was established and the level of immune reconstitution was assessed in this model. METHODS: Mononuclear cells (MNC) and CD34⁺ cells were isolated or sorted from cord blood(CB). Human CD45, CD19, CD3 markers on cells from NOD/SCID murine peripheral blood(PB), bone marrow(BM), thymus were detected by FCM from 4 to 10 weeks after hematopoietic stem cell transplantation. After 10 weeks, the gene expressions of the human β₂M and RAG2 were detected by RT-PCR in PB or bone marrow of mice model. RESULTS: Human CD45, CD19, CD3 cells populations in PB and BM were found by flow cytometry in mice model transplanted with CD34+ cells or CB MNC from 4 to 10 weeks. The highest positivity of human lymphocytes was at 8 week after transplantation. The levels of human cell engraftment in mice transplanted with CD34⁺ cells were higher than those in mice transplanted with CB MNC. The mRNA of human β₂M and RAG2 were found by RT-PCR in BM.CONCLUSION: The higher level of human lymphocyte engraftment is established in NOD/SCID mouse model transplanted with CD34⁺ compared with CB MNC. The maturation of T lymphocytes could be happened in bone marrow of mice model.     

Effect of lupus recipe on immune system and lymphocyte subsets proliferation in splenic cells of lupus-prone BXSB mice

AIM: To investigate the effects of lupus recipe on immune system and lymphocyte subsets proliferation in splenic cells in BXSB mice. METHODS: Eighteen male BXSB mice model was used in the experiment. The model mice were divided into three groups: un-treated model group, lupus recipe (LR) treated group, and prednisone treated group. All model mice were killed in 10 weeks. The control group consisted of 6 syngeneic normal C57BL/6 male mice. The levels of total IgG and anti-dsDNA antibody in serum were detected by ELISA. The percentages of lymphocyte subsets (CD3+, CD4+, CD8+ T lymphocytes and CD19+, CD23+ B lymphocytes) were detected by using flow cytometry analysis. RESULTS: (1) The serum levels of total IgG and anti-dsDNA antibody in un-treated model group were higher than that in other groups. There was no differences among LR treated group, prednisone treated group and control group. (2) The percentages of CD3+, CD4+, CD8+ T lymphocytes and CD19+, CD23+ B lymphocytes in model group were obviously higher than that in normal control. (3) Compared to un-treated model group, the percentages of CD4+, CD8+ T lymphocytes and CD19+, CD23+ B lymphocytes in LR or prednisone treated group were significantly reduced, which closely reached the levels in normal group. CONCLUSIONS: The immune functions of T and B lymphocytes in BXSB mice are up-regulated. LR inhibits the activation of T and B lymphocytes, reduces the serum levels of IgG and auto-antibody production.     

Immunization with mixed peptides derived from glucose-6-phosphate isomerase induces rheumatoid arthritis in DBA/1 mice

AIM: To establish an animal model of rheumatoid arthritis(RA) in DBA/1 mice induced by immunodominant mixed peptides derived from glucose-6-phosphate isomerase(GPI). METHODS: The DBA/1 mice were immunized with emulsified mixed peptide fragments of hGPI325-339+hGPI469-483 or single peptide hGPI325-339 in complete Freund's adjuvant by subcutaneous injection to induce the model of RA. Body weight, ankle joint symptom scores, the pathological change of the ankle joint, the levels of CD4⁺ T cells in the spleen and peripheral blood, the proportion of iNKT cells in the peripheral blood, and the levels of TNF-α and IL-6 in serum were detected to evaluate and analyze the model. RESULTS: The hind paw of the model mice appeared red swelling on the 8th day, and then aggravated gradually to the limbs. The red swelling reached peak on the 14th day, and then relieved gradually. Inflammation response dominated by lymphocytes and monocytes was observed in the ankle joint. The inflammatory effect of mixed peptides was more obvious than that of the single one(P<0.05). Compared with control group and the mice treated with single peptide, the weight gain was slow, the amount of CD4⁺ T cells in the peripheral blood and spleen were increased, the proportion of peripheral iNKT cells in the inflammatory peak was decreased(P<0.05), and the serum level of TNF-α was increased significantly(P<0.05) in the mice treated with mixed peptide fragments. CONCLUSION: The immunological characteristics of RA model induced by mixed GPI peptides in DBA/1 mice is closer to that in RA patients, especially in the immunopathology of iNKT cells. Therefore, this model can be used as a new tool for studying the mechanism and immunological intervention of RA.     

The prevention of acute graft versus host disease by Rhodamine 123-mediated photodynamic therapy

AIM: To evaluate the effect and safety of Rhodamine 123 (Rh123)-mediated photodynamic treatment (PDT) on acute graft versus host disease. METHODS: An acute graft versus host disease (aGVHD) mice model was established using C57B/6 mice as donors and BALB/c mice as recipients. Mixed lymphocytic cells were cultured with Rh123 (50 nmol/L) and irradiated by argon laser 30 mW/cm2 for 3 min, then transplanted to BALB/c recipient mice mixed with donor bone marrow. Hepatopoietic recovery, aGVHD occurrence, survival time after transplantation and pathological changes were observed. In addition, CD3+CD69+ positive rates of MLC were examined by flowcytometry. RESULTS: Occurrence of aGVHD decreased, degree of pathological manifestation became milder, survival rates were higher than non PDT groups. CD3+CD69+ rates of MLC cells treated with photodynamic therapy (PDT) and cultured for 24 h significantly decreased. CONCLUSION: Rh123-mediated PDT can effectively prevent aGVHD of allogeneic bone marrow transplantation in mice.     

Effect of Liang Xue Huo Xue capsules on mouse psoriasis-like lesions induced by imiquimod

AIM: To observe the effects of Liang Xue Huo Xue (LXHX) capsules on mouse psoriasis-like lesions induced by imiquimod (IMQ). METHODS: BALB/c female mice (n=48) were randomly divided into 6 groups: normal group, model group, LXHX capsules groups with high, medium or low doses, and glycosides of Tripterygium wilfordii (GTW) group. On day 8, skin lesions were determined by pathological examination. The lesions were evaluated according to the psoriasis area and severity index (PASI). The histology and epidermal thicknesses were observed under light microscope. The expression of proliferating cell nuclear antigen (PCNA) was detected by immunohistochemical staining. Meanwhile, the positive expression of CD3, CD11c, F4/80, CD31 and Gr-1 was counted by immunohistochemical staining. RESULTS: Compared with model group, the cutaneous symptoms in LXHX capsules groups were alleviated, with PASI scores decreased, epidermal parakeratosis and epidermal over-proliferation relived, the numbers of dermal T lymphocytes, dendritic cells, macrophages, neutrophils and monocytes reduced significantly. CONCLUSION: LXHX capsules improve imiquimod-induced mouse psoriasis-like lesions by inhibiting over-proliferation of keratinocytes, parakeratosis, inflammatory infiltration and angiogenisis.     

Expression of CD158 in refractory chronic graft-versus-host diseases treated with tacrolimus，mycophenolate mofetil combined with methylprednisone

AIM: To investigate the sequence expression of CD158 molecule after tacrolimus (FK506), mycophenolate mofetil (MMF) combined with methylprednisone (MP) treatment for refractory chronic graft-versus-host diseases (cGVHD). METHODS: The efficacy and the side effect were observed in 6 child patients with extensive cGVHD after allogeneic hematopoietic stem cell transplantation treated with the combination of FK506, MMF and MP, meanwhile the changes of the CD158 expressions on T lymphocytes and NK cells in peripheral blood before and after treatment were observed. RESULTS: The expression of CD4+CD158a+ and CD4+CD158b+ were very low before and after transplantation and treatment, there was no statistical significance. The expression of CD3+CD158b+ and CD3+CD8+CD158b+ were 4.97%±2.36% and 4.58%±2.90% respectively in five patients with acute GVHD, and there was statistical significance compared with that of before-transplantation (P＜0.05). The expression of CD3-CD16+CD158b+ was also higher than that of before transplantation (P＜0.05). In the six patients with chronic GVHD, the expressions of CD3+CD158b+ and CD3+CD8+CD158b+ were higher than those of before transplantation (P＜0.05), and decreased gradually after the effective combined treatment, there was statistical significance (P＜0.05) compared with those during the time of cGVHD, but were still higher than that before transplantation. The expression of CD3-CD16+CD158b+ decreased at the late stage after transplantation and was closed to the level of before-transplantation. CONCLUSIONS: The increase in expression of CD158b on T cells might be related to cGVHD. The combined immunosuppression with FK506, MMF and MP is feasible for the treatment of cGVHD. The possible mechanism of the combined immunosuppression with FK506, MMF and MP may be the down-regulation of CD158 expression on T lymphocytes and NK cells.     

Establishment of an acute GVHD animal model in EL9611 erythroleukemia mice

AIM: To establish an acute graft-versus-host disease (GVHD) model in EL9611 erythroleukemia mice. METHODS: Using C57BL/6 (H-2^b) mice as the donor and BALB/c (H-2^d) mice as the recipient in allogeneic bone marrow transplantation (allo-BMT), the acute GVHD model was established. The mice were divided into leukemia group (n=10), radiation control group (leukemic mice given radiation without allo-BMT, n=4), GVHD group (leukemic mice given radiation+allo-BMT, n=10) and normal control group (n=4). In leukemia group, 2×10⁶/mouse EL9611 erythroleukemic cells were transfused via tail vein into BALB/c mice to build the erythroleukemia model. In GVHD group, 7 days after leukemic cell transfusion, the mice received total dose of 8.0 Gy γ of total body irradiation(TBI), and within 5 h, 2×10⁶ C57BL/6 bone marrow cells and 1×10⁷ C57BL/6 spleen cells per mouse were transfused via tail vein to build the acute GVHD model in EL9611 erythroleukemia mice. The clinical manifestations of posture, fur, stool and so on were observed. Pathological examination was conducted to examine the changes of liver, spleen, skin, small intestine and peripheral blood. The survival rate was also calculated. RESULTS: (1) In leukemia group, the mean survival time (MST) was (14.5±2.1) days,or (7.5±0.7) days when irradiation day was as day 0(P<0.01 compared with GVHD group). The death rate was 100% with no spontaneous remission. The dead mice showed splenohepatomegalia and high WBC count . Pathological examination showed disorganization of normal tissues and leukemic cell infiltration. (2) In radiation control group, MST was (9.0±0.7) d, with significant difference as compared with GVHD group and normal group (P<0.01). The death rate was 100%. Pathological examination showed hematopoiesis exhaustion. (3) In GVHD group, MST was (32.0±3.2) d (P<0.01 compared with other groups). The death rate was 100%, the symptoms were observed on day 10-13 after allo-BMT. Clinical manifestations and pathological examination corresponded to those of I degree to II degree of GVHD. CONCLUSION: Intravenous infusion of 2×10⁶/mouse EL9611 leukemic cell successfully establishes the EL9611 erythroleukemia animal model. Seven days after EL9611 leukemic cell transfusion, lethal dose of TBI and allo-BMT can successfully build the acute GVHD model of EL9611 leukemic mice.     

Transplantation of mouse fetus-derived MSCs alleviates physical fatigue in mouse

AIM:To observe the effect of mouse fetus-derived mesenchymal stem cell(MSC) transplantation on alleviating fatigue in mice. METHODS:MSCs were derived from the mouse fetus at 13.5 d. The 6-month-old BALB/c mice were randomly divided into non-swimming group, swimming control group and swimming+MSCs group. The mice in swimming+MSCs group were injected with fetus-derived MSCs through the tail vein, while the mice in non-swimming group and swimming control group were injected with equal volume of normal saline. The anti-fatigue activity was evaluated using a weight-loaded swimming test, along with the determination of blood urea nitrogen(BUN), blood lactic acid(BLA),and glycogen in the hepatic and muscular tissues 24 h later. The antioxidant activity was evaluated by determining the activity of superoxide dismutase(SOD) and the content of malondialdehyde(MDA). The cardiac functions were measured by echocardiography. RESULTS:Primary cultured cells were spindle-shaped in scattered colony. The cells in fifth passage grew parallelly or swirlingly. The cells expressed CD44 and CD105, but not CD34 or CD45. Inducing experiments showed that the cells differentiated into osteoblasts or adipocytes. Transplantation of MSCs alleviated fatigue in mice. In swimming+MSCs group, weight-loaded swimming time was longer, the levels of BUN, BLA, hepatic and muscular MDA were lower, and the levels of glycogen and SOD in the hepatic and muscular tissues were higher than those in control group. Stroke volume, cardiac output and left ventricular diastolic volume in swimming+MSCs group increased, indicating that the cardiac functions were enhanced. CONCLUSION: Transplantation of mouse fetus-derived MSCs alleviates physical fatigue.     

Elevated levels of serum IL-6, ICAM-1 and P-selectin in stable survivors with liver transplantation

AIM: To study the profile of serum IL-6， ICAM-1 and P-selectin in stable survivors with clinical liver transplantation (LTx). METHODS: Flow cytometric analysis was used to determine the phenotype of T cell subsets in peripheral blood mononuclear cells （PBMCs） from stable survivors with liver transplantation (n=22), and healthy volunteers (n=12). Serum levels of the pro-inflammatory cytokines, tumor necrosis factor (TNF-α) and interleukin (IL-6), intercellular adhesion molecules (ICAM-1) and P-selectin in stable survivors with liver transplantation and healthy volunteers were assessed by enzyme-linked immunoabsordent assay (ELISA). Recently performed 6 cases of liver transplantation were also dynamically observed in this study. RESULTS: Percentage of CD4+ T cells， CD8+ T cells and CD3+ T cells, as well as ratio of CD4 to CD8 were no difference between two groups (P>0.05). However, a significant higher percentage of CD3+CD25+ T cells was found in stable liver transplantation group as compared to healthy group (P<0.05). Significantly increased concentrations of IL-6, ICAM-1 and P-selectin were found in stable liver transplantation group as compared to healthy group (P<0.05). A high TNF-α level was detected in stable liver transplantation group while no significant difference was found as compared to healthy volunteers group (P>0.05). There was not found no regular change of serum cytokines (IL-6, TNF-α) and adhesion molecules (ICAM-1, P-selectin) in 6 liver transplanted patients during post-operation from day 1 to day 30, indicating that was associated with the different status of patients before or after transplantation. CONCLUSIONS: Our data suggesting that increased levels of ICAM-1 and P-selectin, appears to participate in the processing of immunoregulation to transplanted livers, whereas elevated concentrations of IL-6 appear to be involved in the repair of the injury induced by TNF-α in allo-transplanted livers.     

Generation of hematopoietic stem/progenitor cells with property of strengthened cell mediated immunity from an embryonic stem cell line

AIM:To induce lymphoid stem cells and/or T-cell precursors to differentiate into functional mature T lymphocyte, and to increase the surface marker of T lymphocytes such as CD₃⁺, while embryonic stem(ES) cells differentiated into hematopoietic stem/progenitor cells(HSPCs) in vitro. When they were injected into lethally irradiated mice, these differentiated cells had the advantage in immune reconstitution. METHODS:Embryonic stem cells formed embryoid bodies(EBs) in the medium containing methycellulose, hematopoietic growth factors(HGFs) was added to the culture system on the 6th day, thymopeptide was added at the same time. Flow cytometry were performed to detect the surface marker CD₃₄⁺ and CD₃⁺ of the differentiated cells. Finally the differentiated cells were injected into lethally irradiated mice, 60 days later, the incidence rate of graft versus host disease(GVHD) was taken as the mark of cell mediated immunity, PCR was performed to detect the sex determining region of the Y-chromosome(Sry) in bone marrow cells and spleen cells of the survival host female mice. RESULTS:The percentage of CD₃⁺ T lymphocytes was 10.52% and the incidence rate of GVHD was 0% on the 13th day, but they respectively rose up to 22.93% and 100% if thymopeptide was added in the procedure of inducing ES cells to differentiate into HSPC in vitro. CONCLUSION:The quantity of CD₃⁺ T lymphocytes increased in medium containing thymopeptide when ES cells differentiated into CD₃₄⁺ HSPC.     

Change of antithrombin Ⅲ in patients with atherosclerotic cerebral infarction

AIM：To explore the change of antithrombin Ⅲ (AT-Ⅲ) in the patients with atherosclerotic cerebral infarction. METHODS：Chromogenic substrate assay was used to measure the activity of AT-Ⅲ in 55 patients with atherosclerotic cerebral infarction and 55 healthy controls, and the correlation analysis was applied to determine the AT-Ⅲ activity with the severity of damage in central nervous system and general biochemical parameters. The levels of TNF-α and IL-6 in the plasma were detected by ELISA. Immunocomplex in the plasma was measured by enzyme immunoassay （EIA）. The number and phenotype of the monocytes in peripheral blood were analyzed by flow cytometry. ELISA was also applied to determine the secretion of TNF-α and IL-6 from the monocytes after the stimulation of immunocomplex. The expression of AT-Ⅲ in human brain vascular endothelial cells after the stimulation of TNF-α and IL-6 was observed by Western blotting. RESULTS：The activity of AT-Ⅲ significantly decreased in the patients with atherosclerotic cerebral infarction, and negatively correlated with the damage degree of nervous system function, systolic pressure, diastolic pressure, glucose, cholesterol, triglyceride, low-density lipoprotein cholesterol and homocysteine, while positively correlated with high-density lipoprotein. In addition, the plasma levels of TNF-α and IL-6 increased significantly, accompanied with the enhancement of immunocomplex level. The numbers of CD14+ CD16+ and CD14+ CD32+ monocytes in peripheral blood were not changed, while CD14+ CD64+ monocytes increased obviously. The secretion of TNF-α and IL-6 by monocytes were significantly enhanced after stimulated with immunocomplex, while the protein expression of AT-Ⅲ in the human brain vascular endothelial cells was down-regulated after co-incubated with TNF-α or IL-6. CONCLUSION：Decreased AT-Ⅲ activity in the patients with atherosclerotic cerebral infarction is one of the risk factors of cerebral infarction, and related with the disease severity. The production of pro-inflammatory cytokines through immunocomplex from CD14+ CD64+ monocytes may be involved in the mechanism. Improvement of AT-Ⅲ activity may protect against cerebral ischemia.     

Changes of cellular immunological function after surgical operation in patients with locally advanced lung cancer

AIM: To study the change of cellular immunological function in patients with locally advanced lung cancer before and after operations. METHODS: A lung cancer group of 20 cases with locally advanced lung cancer (group A), a benign disease group of 20 cases with lung benign disease (group B) and a normal group of 20 cases from healthy volunteers (group C) were set up. The levels of the peripheral blood T lymphocyte subsets (CD+3, CD+4, CD+8, CD+4/ CD+8 ratio) were detected in the group A before operation and on the 10th day and 17th day after operation by indirect immuno-fluorescence assay and contrasted with the group B and group C. RESULTS: The levels of T lymphocyte subsets in group A were abnormal before operation, CD+3, CD+4/ CD+8 ratio were significantly lower than those in group B and group C (P<0.05), and CD+8 was significantly higher (P<0.05). CD+3 significantly increased (P<0.05) and CD+8 decreased (P<0.05) on 10th day and 17th day after operation. CD+4/ CD+8 ratio significantly increased on 17th day after operation (P<0.05). There was no significant difference of the levels of T lymphocyte subsets between the 10th day and 17th day after operation. CONCLUSIONS: The patients with locally advanced lung cancer have a remarkable impairment of immunological function, which mainly show stronger immunosuppression and have some recovery after operation. In the view of immunology, the surgical resection for locally advanced lung cancer shows active significance.     

Effect of ulinastatin plus thymosin-α1 therapy on improving immune function in septic patients

AIM: To investigate the effect of ulinastatin plus thymosin-α1 therapy on improving immune function in septic patients. METHODS: 70 patients were divided into two groups. One group was classical treatment group (CT) with regular therapy and another group was classical treatment plus immunotherapy group (CIT) with ulinastatin plus thymosin-α1 for a week.The immune index before and after treatment on day 0, 1, 3 and 7 was observed, including the clinical and survival data. RESULTS: The most common pathogen of sepsis was bacteria, and infection by fungi was in rare. The common locations of bacteria observed were sputum and abdominal drainage. The level of TNF-α was significant lower in CIT group than that in CT group (P<0.05). IL-10 level was significantly higher in CIT group than that in CT group (P<0.05). IgG level was significant lower in CIT group than that in CT group (P<0.05). No significant difference in the levels of IgA, IgM, C3 and C4 between two groups was observed (P>0.05). CD4+T lymphocytes were significant higher in CIT group than those in CT group (P<0.05). From day 7 to day 28, the lymphocytes and level of HLA-DR in CD14+ monocytes were significant higher in CIT group than those in CT group (P<0.05). The time of mechanical ventilation and vasopressors used in CIT group was shorter than those in CT group (P<0.05). But the length of stay and the cost in ICU showed no significant increase between these two groups (P>0.05). During hospitalization, 20 patients died in the CT group and 13 patients died in CIT group (P<0.05). The long-term survival time in CIT group was longer than that in CT group (P<0.05). CONCLUSION: Immunotherapy in septic patients can decrease TNF-α level and increase IL-10 level. Immunotherapy in septic patients can increase IgG level slightly, CD4+T lymphocyte, and HLA-DR in CD14+ monocytes, which improve the immune paralysis in septic patients. Immunotherapy can shorten the time of mechanical ventilation and vasopressors used, but it doesn’t increase the length of stay and the cost.