壳聚糖/聚乙二醇琥珀酸酯薄膜的制备及其与肌成纤维细胞的相容性

目的：制备防粘连壳聚糖/聚乙二醇琥珀酸酯薄膜并观察其与肌成纤维细胞的相容性。方法：实验于2006-05/11在南方医科大学附属珠江医院中心实验室完成。实验材料：在透析后的壳聚糖与聚乙二醇琥珀酸酯或聚乙二醇共混后置入冻干机冻干制得壳聚糖/聚乙二醇琥珀酸酯薄膜或壳聚糖/聚乙二醇膜，并将新生2～5d的SD大鼠骨骼肌成纤维细胞种植于膜片上。实验评估：①MTT法测定肌成纤维细胞接种在不同膜片上的吸光度值，计算相对贴附率。相对贴附率=不同膜的A490nm/培养板的A490nm×100%。②MTT法测定肌成纤维细胞在不同膜片上的生长1，5d后的吸光度值。③相差显微镜下观察肌成纤维细胞的生长形貌。结果：①肌成纤维细胞在不同膜片上的贴附率：肌成纤维细胞在壳聚糖/聚乙二醇琥珀酸酯薄膜上能良好黏附、增殖，而在壳聚糖/聚乙二醇膜、壳聚糖膜上黏附性差。联合培养12h，5d后MTT法结果显示，壳聚糖/聚乙二醇琥珀酸酯组的A值分别为0.074±0.009，0.141±0.031，分别为壳聚糖组的6.17倍和6.13倍（P〈0.05）。②肌成纤维细胞的生长特性：肌成纤维细胞在壳聚糖/聚乙二醇琥珀酸酯膜上的活性最高，增殖能力最强，增长速度最快，其次为壳聚糖/聚乙二醇膜，但两者差异无显著性意义（P〉0.05），而细胞在壳聚糖膜上的增殖能力较低，膜上细胞数目较少，与其他组比较差异有显著性意义（P〈0.05）。③肌成纤维细胞与不同膜片联合培养1，5d时的生长形貌：壳聚糖膜上细胞未贴壁生长，为透明的圆球形，呈游离状态，未能很好舒展，且有些皱缩，生长活力也不旺盛;细胞与壳聚糖/聚乙二醇膜、壳聚糖/聚乙二醇琥珀酸酯膜片联合培养的生长情况要明显好于壳聚糖膜，细胞相互融合成片，多呈长梭形，细胞间隙狭窄，紧密排列成束，成指纹状结构且聚集生长的趋势也更明显。结论：将接支琥珀酰基的聚乙二醇与壳聚糖共混组成的网状系统改进了膜片的力学性能，提高了膜片的柔韧性，使其成膜性更好;壳聚糖/聚乙二醇琥珀酸酯薄膜具有良好的生物相容性，肌成纤维细胞在壳聚糖/聚乙二醇琥珀酸酯薄膜上的黏附及生长情况明显好于壳聚糖薄膜。     

Potential increased risk of virus transmission due to exclusion of older donors because of concern over Creutzfeldt-Jakob disease. The National Heart, Lung, and Blood Institute Retrovirus Epidemiology Donor Study

BACKGROUND: Concern over the theoretical possibility of disease transmission via blood from donors who develop Creutzfeldt-Jakob disease has led to proposals to exclude older individuals from donating plasma for further manufacture into pooled plasma donations. The impact of extending this age-deferral policy to blood donors was examined with respect to the risk for known transmissible viruses. STUDY DESIGN AND METHODS: Demographic characteristics and confirmed prevalence rates (/10(5) first-time donations) and incidence rates (/10(5) person-years for repeat donors) for viral markers were compared for donors < 50 years old (n = 1,259,805 [85%]) and > or = 50 years old (n = 219,856 [15%]) and for donors < 60 years old (n = 1,409,176 [95%]) and > or = 60 years old (n = 70,485 [5%]). Incidence rates were combined with infectious window-period estimates for each virus, to calculate the risk of virus transmission per 10(6) donations. RESULTS: Unadjusted prevalence rates were significantly greater for younger than for older donor groups for human immunodeficiency virus (HIV), hepatitis B surface antigen (HBsAg), and hepatitis C virus (HCV) (p < or = 0.05). Incidence rates (and transmission risk estimates) for HBsAg were significantly higher in the < 50 donor group than in the > or = 50 group (p < or = 0.05), and those for HIV, human T-lymphotropic virus, and HCV were not significantly higher (p > 0.05). Blanket removal of donors over the age of 50 would potentially lead to the following significant increases in the risk of infected units: HIV, 12 percent; HCV, 21 percent; and hepatitis B virus (HBsAg), 22 percent. CONCLUSION: Removal of donors over the age of 60 would not significantly affect the risk of infected units. Deferral of donors > or = 50 years of age from whole-blood donations for unfounded concerns about Creutzfeldt-Jakob disease could have adverse effects on both blood availability and safety.     

Blood group A immunodeterminants on human red cells differ in biologic activity and sensitivity to alpha-N-acetylgalactosaminidase

BACKGROUND: Epitopes of blood group A antigen can be enzymatically cleaved from red cells (RBCs), but the extent of cleavage required for normal survival in allogeneic blood transfusion recipients is unknown. Therefore, the cleavage rates were studied for A antigen epitope binding of 1) complement-activating anti-A, 2) Dolichos biflorus anti- A, lectin, and 3) hemagglutinating anti-A during incubation with a purified alpha-N-acetylgalactosaminidase, E.C. 3.2.1.49 (alpha- GalNAc'ase). STUDY DESIGN AND METHODS: Suspensions of group A RBCs were incubated with alpha-GalNAc'ase. Cells were removed at intervals, washed, and tested for loss of binding by monoclonal, polyclonal, and complement-activating anti-A, D. biflorus anti-A1 lectin, and Ulex europaeus anti-H lectin. RESULTS: A epitopes binding D. biflorus lectin were highly susceptible to alpha-GalNAc'ase; simultaneously with their loss, binding with U. europaeus lectin emerged. Loss of complement- mediated hemolysis was slower. A epitopes binding hemagglutinating anti- A were most resistant. Cleavage of A epitopes from membrane glycosphingolipids with short oligosaccharide chains was similarly resistant. Rates of cleavage from A1 and A2 RBCs were similar. CONCLUSION: RBC epitopes of blood group A differ in susceptibility to cleavage and biologic reactivity, which suggests that subsets mediating important biologic functions exist on functionally and topographically distinct membrane glycoconjugates.     

In vitro interactions of PGE and cAMP with murine and human erythroid precursors

Addition of prostaglandins of the E series (PGE1, PGE2) in methylcellulose cultures of murine marrow results in a dose-dependent inhibition of the cloning efficiency of both BFU-E and CFU-C. However, CFU-E growth is unaffected. The inhibitory action of PGE is progressively overcome by increasing amounts of colony-stimulating factor (CSF), and with some limitations, also of erythropoietin (Ep). Addition of PGF2 alpha' associated or not with indomethacin, does not exert any significant effect on these hemopoietic precursors. In an attempt to unvail the mechanism(s) underlying these phenomena, dibutyryl-cyclic AMP (db-cAMP), theophylline (an inhibitor of phosphodiesterase), or theophylline + PGE were plated at various concentrations. Both db-cAMP and theophylline induce an inhibitory influence on both BFU-E and CFU-C growth, which mimicks that by PGEs; additionally, theophylline potentiates the inhibitory action of PGE1. In all these studies, the CFU-E number was not significantly modified. PGE action on BFU-E proliferation is clearly species-dependent, since PGE1 addition to human marrow methylcellulose cultures induces a significant enhancement of the number of both BFU-E and CFU-E derived colonies. This action was abolished upon removal of adherent cells, thus suggesting that PGE1 evokes a release of factor(s) enhancing human erythroid colony growth by adherent cells.     

Chronic graft-versus-host disease in 52 patients: adverse natural course and successful treatment with combination immunosuppression

Fifty-two of 175 (30%) survivors of allogeneic marrow transplantation developed chronic graft-versus-hose diseases (GVHD). Five with limited chronic GVHD had an indolent clinical course with involvement of only the skin and liver. Forty-seven with extensive chronic GVHD had an unfavorable multiorgan disorder that resembled several autoimmune diseases. Thirteen patients with extensive disease (group I) were not treated and only 2 survive with Karnofsky scores >- 70%. Mortality resulted from infections and morbidity from sica syndrome, pulmonary and hepatic insufficiency, scleroderma-like skin disease, and contractures. Another 13 (group II) received a median of 8 mo prednisone and/or a brief course of antithymocyte globulin, and 3 survive without disability. The other 21 (group III) were treated with a combination of prednisone (1.0 mg/kg/q.o.d.) and either cyclophosphamide, procarbazine, or azathioprine (all 1.5 mg/kg/day) for a median of 13 mo. Combination therapy was well tolerated with only modest myelotoxicity. Fifteen in group III had a good and 4 a fair response to treatment while 2 with no response died. Azathioprine and prednisone was the most effective regimen. All therapy has been discontinued in 12 group III patients: GVHD returned in 5 (including 2 who died in spite of retreatment) while 7 remain free of GVHD for a median of 11 (range 6-30) mo observation. Only I group III survivor is disabled and 16 of the original 21 are alive 2-4 yr after transplant with Karnofsky scores of 70%-100%. Thus, combination immmunosuppression appears to favorably affect and, in some cases, premanently arrest the adverse natural course of extensive chronic GVHD.     

The effects of vitamin D binding protein-macrophage activating factor and colony-stimulating factor-1 on hematopoietic cells in normal and osteopetrotic rats

Osteopetrosis is a heterogeneous group of bone disorders characterized by the failure of osteoclasts to resorb bone and by several immunological defects including macrophage dysfunction. Two compounds, colony-stimulating factor-1 (CSF-1) and vitamin D-binding protein- macrophage activating factor (DBP-MAF) were used in the present study to evaluate their effects on the peritoneal population of cells and on cells within the bone marrow microenvironment in normal and incisors absent (ia) osteopetrotic rats. Previous studies in this laboratory have demonstrated that administration of DBP-MAF to newborn ia animals results in a substantial increase in bone marrow cavity size due to upregulated osteoclast function. To study the effects of these compounds on the macrophage/osteoclast precursors, DBP-MAF, CSF-1, and the combination of these compounds were given to newborn ia and normal littermate animals. Both the normal and mutant phenotypes responded similarly when treated with these compounds. Rats exhibited a profound shift toward the macrophage lineage from the neutrophil lineage when compared with vehicle-treated control animals after treatment with these compounds. In the in vivo peritoneal lavage study, animals received injections of CSF-1, DBP-MAF or DBP-MAF/CSF-1 over a 4-week period. The various types of cells in the peritoneal cavity were then enumerated. The in vitro study consisted of cells isolated from the bone marrow microenvironment and cultured on feeder layers of CSF-1, DBP-MAF, or DBP-MAF/CSF-1 for colony enumeration. The increase in macrophage numbers at the expense of neutrophil numbers could be seen in both the in vivo and in vitro experiments. The macrophage/osteoclast and neutrophil lineages have a common precursor, the granulocyte/macrophage colony-forming cell (GM-CFC). With the addition of CSF-1, the GM-CFC precursor may be induced into the macrophage/osteoclast lineage rather than the granulocyte lineage. This increased pool of cells in the macrophage/osteoclast lineage can be functionally upregulated with the subsequent addition of DBP-MAF to perform the activities of phagocytosis and bone resorption. The in vitro data also showed that DBP-MAF did not support colony development as in CSF-1 or the combination treatment. The recruitment and activation of cells into the macrophage/ osteoclast lineage may help to correct the bone and immune defects found in diseases demonstrating a significant lack of myeloid cells, as well as neutrophilia disorders and the disease, osteopetrosis.     

Changes in corticotrophin releasing factor of the stalk median eminence in rats with various cuts around the medial basal hypothalamus.

The activity of corticotrophin releasing factor (CRF) in extracts of the stalk median eminence (SME) complex proper (average protein content, 30.6 micrograms) of male rats was assayed by monolayer cultures of anterior pituitary cells using the release of immunoreactive ACTH. Extracts which were equivalent to 0.025 SME of control rats usually had detectable CRF activity, while there was no detectable activity in extracts of 0.4 SME equiv. taken 8 days after complete surgical isolation of the medial basal hypothalamus (MBH). The activity of CRF in extracts from rats with an anterolateral cut around the MBH was at least ten times less than that in the control rats. One day after placing an anterolateral cut around the MBH the ACTH releasing activity of the SME was not significantly different from that of the control animals but activity decreased significantly 3 days after the operation and was at least ten times less than in the control animals on day 7 after the operation. It is suggested that most of the CRF activity of the SME is contained in nerve fibres entering the neurohaemal region from outside the MBH and that transection of these fibres produced the fall in CRF content of the SME in rats with partial or total surgical isolation of the MBH.     

Nuclear RNA sequences coding for alpha and beta globins in erythroid cells: evidence for multiple intermediate molecules

The poly (A)-containing nuclear RNA from dimethylsulfoxide-induced Friend leukemia cells was fractionated by acrylamide gel electrophoresis in denaturing conditions and analyzed for alpha and beta globin RNA sequences. The results indicate that nuclear RNA contains one species of large-size RNA (0.6 X 10(6) daltons), which is the putative precursor for beta globin mRNA only. In addition, it was shown by electrophoretic analysis that the complex of RNA molecules not resolved by sucrose gradient centrifugation (11S) comprises sequences of decreasing size (0.34, 0.28, and 0.26 X 10(6) daltons), which might be the precursors of alpha and beta globin mRNA.     

Immunophenotypic diagnosis of clinical and preclinical chronic lymphatic leukemia by using monoclonal antibodies against the cCLLa, a CLL-associated antigen

The clinical usefulness of monoclonal antibodies (MoAbs) against the cCLLa, an antigen restricted to B-chronic lymphatic leukemia (CLL) and its variants, was ascertained in 65 patients with overt CLL and 25 individuals with unexplained mild lymphocytosis. Healthy volunteers (n = 25) and patients with malignant and nonmalignant disorders (n = 58) served as controls. The following observations were made in CLL. (a) Anti-cCLLa MoAbs identified neoplastic CLL cells as judged by the high correlation (r = .985) between monoclonal surface immunoglobulins (Slgs) and cCLLa expression in all patients, and dual-label flow cytometry studies showing cCLLa expression by monoclonal Slg-bearing B- CLL cells but not by normal B lymphocytes. (b) The size of the circulating cCLLa-positive clone paralleled the degree of lymphocytosis (r = .987) and was associated with reciprocal (r = .893) relative T lymphopenia. Ten patients with borderline lymphocytosis exhibited a subset of monoclonal Slg/cCLLa-positive cells ranging from 16% to 45% of the total. These patients were indistinguishable from those with CLL in terms of age, clone lineage, and reciprocal relative T lymphopenia. Two patients have progressed to overt CLL within 19 months, but eight have not (observation time, 18 to 82 months). These data suggest that anti-cCLLa MoAbs are sensitive probes useful to identify and monitor cCLLa clones during their clinical and preclinical phases.