DIFFICULTIES AND DILEMMAS IN THE MANAGEMENT OF CONGENITAL ANOMALIES IN TWIN PREGNANCY

Both the incidence of twin pregnancy and the demand for prenatal diagnosis are increasing. Unfortunately, biochemical screening and ultrasound scanning are less reliable for prenatal diagnosis in twin pregnancies than in singletons. Amniocentesis and chorionic villous biopsy are usually diagnostic in singleton pregnancies but may be marred by sampling errors in twin gestations. Where a congenital anomaly has been diagnosed in a twin pregnancy, difficult decisions may have to be made, especially if one twin is unaffected. In these cases, special skills are required to ensure that adequate information, psychological support and optimal medical care are provided.     

Plasma P-selectin is increased in thrombotic consumptive platelet disorders

P-selectin is a 140-kD protein found in the alpha-granules of platelets and the Weibel-Palade bodies of endothelial cells that on cell activation is expressed on the cell surface and also secreted into the plasma. The secreted form of P-selectin, like plasma P-selectin, differed from platelet membrane P-selectin in that its molecular mass was approximately 3 kD lower under reducing conditions. Both the secreted and plasma forms of P-selectin contained cytoplasmic sequence as determined by Western blot analysis with an affinity-purified rabbit anti-P-selectin cytoplasmic peptide antibody. We have measured plasma P- selectin and beta-thromboglobulin (beta TG) concurrently in (1) patients with consumptive thrombotic disorders, including disseminated intravascular coagulation (DIC), heparin-induced thrombocytopenia (HIT), and thrombotic thrombocytopenic purpura (TTP)/haemolytic uremic syndrome (HUS); (2) patients with idiopathic thrombocytopenic purpura (ITP); and (3) healthy controls. Patients with DIC, HIT, and TTP/HUS, but not ITP, had significantly elevated plasma P-selectin and beta TG levels when compared with their age-matched healthy controls. The increased plasma P-selectin and beta TG in patients with thrombotic disorders were likely to be the result of in vivo platelet and endothelial cell damage or activation. We also found that avoidance of veno-occlusion and other tedious measures customarily taken during blood collection and sample preparation to prevent in vitro platelet activation did not affect plasma P-selectin assay results. In addition, plasma P-selectin levels were not influenced by the presence of renal failure or heparin administration. These results indicate that plasma P- selectin may be a useful new marker for thrombotic diseases.     

Three unrelated Rh D gene polymorphisms identified among blood donors with Rhesus CCee (r'r') phenotypes

Human red blood cells are traditionally typed as Rhesus (Rh)-positive or -negative depending on the presence or absence of the Rh D antigen. A recent report demonstrated that the Rh D gene is completely absent in Rh D-negative individuals. In this study, Rh D-negative blood donors with ccee (n = 25) and CCee (n = 3) phenotypes were examined for the presence of absence of the D gene. Polymerase chain reaction (PCR) probes that hybridize to the 5' and 3' regions of the Rh CcEe gene and the closely related D gene were used in a Southern analysis. The D gene was absent in all ccee phenotypes examined. The CCee phenotypes showed three Rh D polymorphisms: one donor lacked the D gene, one donor had a partial deletion on one D gene at the 3' region, and the remaining donor appeared to have one normal D gene within the intron/exon regions examined. We conclude that, while the D gene may be absent in the majority of Rh D-negative phenotypes, rarer polymorphisms also occur that prevent expression of the D antigen resulting in the Rh D-negative phenotype.     

Direct demonstration that autologous bone marrow transplantation for solid tumors can return a multiplicity of tumorigenic cells

Patients with solid tumors are increasingly being treated by autologous bone marrow transplantation (BMT). Although response rates appear to be increased, disease recurrence is the commonest cause of treatment failure. Whether relapse is entirely due to residual disease in the patient or arises also from infiltrating malignant cells contained in the autologous marrow transplant has not been resolved. If the latter explanation is correct, then purging would be required as part of the transplantation procedure. We used retrovirally mediated transfer of the neomycin-resistance gene to mark BM harvested from eight patients with neuroblastoma in clinical remission. The marked marrow cells were subsequently reinfused as part of an autologous BMT. At relapse, we sought the marker gene in malignant cell populations. Three patients have relapsed, and in each the marker gene was detected by phenotypic and genetic analyses of resurgent malignant cells at medullary and extramedullary sites. Analysis of neuroblast DNA for discrete marker gene integration sites suggested that at least 200 malignant cells, each capable of tumor formation, were introduced with the autologous marrow transplant and contributed to relapse. Thus, autologous BMTs administered to patients with this solid tumor may contain a multiplicity of malignant cells that subsequently contribute to relapse. The marker-gene technique we describe should permit evaluation of the mechanisms of relapse and the efficacy of purging in patients receiving autologous marrow transplantation for other solid tumors that infiltrate the marrow.     

Oxidative metabolism of the human eosinophil

We have compared the oxidative metabolism of human eosinophils (80%-90% purity) to that of neutrophils. Hexose monophosphate (HMP) shunt activity of eosinophils was higher than that of neutrophils under either resting or phagocytizing conditions. Eosinophil HMP shunt activity also was stimulated by phorbol myristate acetate, a membrane- active agent. Eosinophils showed a marked incorporation of 125I into trichloroacetic acid-insoluble material under resting conditions, which increased markedly during phagocytosis. Eosinophils likewise showed a greater reduction of nitroblue tetrazolium dye during phagocytosis than did neutrophils. Measurement of other parameters of oxidative metabolism indicated that eosinophils generated superoxide anion following phagocytosis and also elicited a burst of chemiluminescence similar to that observed during phagocytosis by neutrophils. Measurement of NADPH oxidase activity demonstrated that this enzyme was 3-6 times more active in fractions isolated from eosinophils than in corresponding fractions isolated from neutrophils; this was observed over a range of substrate concentrations. The eosinophil enzyme sedimented differently than the neutrophil enzyme with differential centrifugation; neither showed sedimentation characteristics of peroxidase. These data indicate that eosinophils possess a similar, although in some ways more potent, oxidative burst than neutrophils and are consistent with a role for NADPH oxidase in the initiation of that burst.     

Inositol 1,4,5-trisphosphate turnover enzymes--activities and subcellular distribution in hepatocarcinogenesis

The metabolism of inositol 1,4,5-trisphosphate and inositol 1,3,4,5- tetrakisphosphate in homogenates and sub-fractions from normal rat liver and premalignant liver nodules was investigated. The activities of 5-phosphatase, expressed as pmol converted substrate per minute and mg protein, were equal when using the two substrates, and did not differ between normal and nodular homogenates. Subcellular fractions were purified by sequential steps of differential centrifugation and density gradient fractionation procedures. The total phosphatase activity was found to be distributed between cytosol (15%) and membraneous fractions (75%), with most of the enzyme activity residing in the plasma membranes. A doubling of phosphatase specific activity was seen in the nodular low density membrane fraction, containing Golgi apparatus and endosomes, as compared with normal liver. Inositol 1,4,5- trisphosphate 3-kinase activity was found to be exclusively cytosolic. No difference in this enzyme was seen between the two tissue types studied. Vasopressin (0.2 or 2 microM) had no effect either on phosphatase or kinase activity. The compartmentalization of inositol polyphosphate 5-phosphatase activity presents a possible explanation of earlier findings that premalignant liver tissue was able to respond with inositol 1,4,5-trisphosphate, but not inositol 1,3,4,5- tetrakisphosphate formation after agonist stimulation.      

A humanised tissue‐engineered bone model allows species‐specific breast cancer‐related bone metastasis in vivo

Bone metastases frequently occur in the advanced stages of breast cancer. At this stage, the disease is deemed incurable. To date, the mechanisms of breast cancer‐related metastasis to bone are poorly understood. This may be attributed to the lack of appropriate animal models to investigate the complex cancer cell–bone interactions. In this study, two established tissue‐engineered bone constructs (TEBCs) were applied to a breast cancer‐related metastasis model. A cylindrical medical‐grade polycaprolactone‐tricalcium phosphate scaffold produced by fused deposition modelling (scaffold 1) was compared with a tubular calcium phosphate‐coated polycaprolactone scaffold fabricated by solution electrospinning (scaffold 2) for their potential to generate ectopic humanised bone in NOD/SCID mice. While scaffold 1 was found not suitable to generate a sufficient amount of ectopic bone tissue due to poor ectopic integration, scaffold 2 showed excellent integration into the host tissue, leading to bone formation. To mimic breast cancer cell colonisation to the bone, MDA‐MB‐231, SUM1315, and MDA‐MB‐231BO breast cancer cells were cultured in polyethylene glycol‐based hydrogels and implanted adjacent to the TEBCs. Histological analysis indicated that the breast cancer cells induced an osteoclastic reaction in the TEBCs, demonstrating analogies to breast cancer‐related bone metastasis seen in patients.     

自体骨髓干细胞移植治疗失代偿期肝硬化

选择36例失代偿期肝硬化患者,年龄37～59岁,患者在无菌条件下,从髂后上棘抽取骨髓100～200mL,在体外分离纯化骨髓干细胞后,局部麻醉下经股动脉插管经肝动脉将分离的骨髓干细胞移植于肝脏。自移植后12周,25例(69.4%)患者谷丙转氨酶逐渐降低,由平均(2788.56±357.90)nkat/L降至(1077.05±440.25)nkat/L;22例(61.1%)患者总胆红素逐渐下降,由平均(151.47±25.77)μmol/L降至(69.93±18.86)μmol/L;27例(75%)患者白蛋白逐渐升高,由平均(25.17±11.79)g/L升至(30.87±12.17)g/L。在干细胞移植后凝血酶原活动度逐渐上升,由术前平均(25.89±12.67)%上升至术后12周的(50.39±19.38)%,患者凝血机制明显改善。移植后大多数患者身体状况有明显的改善;移植后12周腹水减轻的19例(52.7%),食欲改善的28例(77.7%),体力好转20例(58.3%),腹胀减轻17例(47.2%),36例干细胞移植患者未出现严重并发症。     

犬内皮与平滑肌细胞的短期静态联合培养

目的:为获得组织工程化自体血管,观察体外静态培养条件下犬内皮细胞与平滑肌细胞联合培养组织学及形态学的特征。方法:实验于2004-07/2005-06在首都医科大学宣武医院外科院级实验室完成。①实验材料:雄性杂种犬,3个月龄,体质量8～12kg。②实验方法:贴块法及酶解法对犬内皮细胞及平滑肌细胞进行原代分离培养及扩增,将第Ⅱ代平滑肌细胞以1×109L-1的密度种植于胶原膜上培养13d,再将第Ⅱ代内皮细胞接种于生长平滑肌细胞的胶原膜上2d。③实验评估:行苏木精-伊红染色同时扫描电镜和透射电镜观察平滑肌细胞和内皮细胞在胶原载体上联合培养后的形态。结果:苏木精-伊红染色见平滑肌细胞较均匀的分布于支架材料表面及内部;扫描电镜下,平滑肌细胞可以在胶原载体材料上生长,增殖明显并在短期内形成多层细胞。内皮细胞与平滑肌细胞联合培养2d就可在平滑肌细胞层表面获得连续的单层内皮细胞层。结论:在短期静态培养条件下犬的血管平滑肌细胞和内皮细胞可以在胶原载体材料上形成具有两层细胞结构的组织工程化动脉血管组织片。