白蛋白微球作为肝靶向给药载体的研究

用均匀设计方法和计算机技术筛选了乳化化学交联法制备白蛋白微球的六个因素,十二个水平。优化出最佳制备工艺,制备了平均粒径0.41～0.47μm的白蛋白微球。将此工艺制备的¹²⁵I-白蛋白微球做动物体内研究,结果表明微球iv后主要浓集在肝脏,可达注入总剂量的68%,此微球在靶组织肝脏的变化规律可用二室模型契合。     

Further study on the vascular basis for the reimplantation of the hand amputated through the palm

Summary Based on the anatomic data obtained from earlier studies on the vascular anatomy of the hand, the vascular architecture in the palm of the hand was studied on 60 sides of unembalmed adult upper extremities. Each palm was divided into 64 squares by 8 sagittal and 8 transverse sections. The vascular architecture in these squares and the arterial relations between them were observed and measured by angiography, operative microscopic dissection and computerised three-dimensional reconstruction. According to the pattern of the blood-vessels, the amputated palms can be classified into 4 types. The anatomic basis for the vascular anastomosis in each type is defined. There are three key-areas for the blood-supply of the palm and their significance is discussed. Apart from the 4 types of transversely amputated palms, the repair programe of the blood-vessles in 4 types of common obliquely amputated palms are also discussed.

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Etude complémentaire de l'anatomie vasculaire de la main pour la réimplantation des amputations transpalmaires

Résumé Sur la base de données anatomiques obtenues lors de précédents travaux sur l'anatomie vasculaire de la main, l'architecture vasculaire palmaire a été étudiée sur 60 extrémités supérieures de cadavres d'adultes, non embaumés. Chaque paume a été divisée en 64 carrés par 8 sections sagittales et 8 sections transversales. L'architecture vasculaire à l'intérieur des carrés et les relations artérielles entre eux ont été étudiées et mesurées par angiographie, dissection au microscope opérateur et reconstruction computérisée en 3D. Les paumes amputées ont été regroupées en 4 types d'après la distribution des vaisseaux sanguins. Les données anatomiques concernant les anastomoses vasculaires sont précisées. Il existe trois zones clés pour l'irrigation de la paume. Leur importance quant à l'irrigation de la main est exposée. Outre la division des paumes amputées transversalement en 4 types, le programme de réparation de vaisseaux dans les 4 types d'amputations obliques communes de la paume et aussi discuté.

     

Simultaneous Detection of 15 Human Cytokines in a Single Sample of Stimulated Peripheral Blood Mononuclear Cells

Cytokines secreted by cells of the immune system can alter the behavior and properties of immune or other cells. At a site of inflammation, sets of cytokines interact with immune cells, and their combined effect is often more important than the function of one isolated component. Conventional techniques, such as enzyme-linked immunosorbent assays, generally require large quantities of cells to characterize a complete cytokine profile of activated lymphocytes. The Bio-Plex system from Bio-Rad Laboratories combines the principle of a sandwich immunoassay with the Luminex fluorescent-bead-based technology. We developed a multiplex cytokine assay to detect different cytokines simultaneously in culture supernatant of human peripheral blood mononuclear cells stimulated with antigen and with mitogen. Fifteen human cytokines (interleukin 1α [IL-1α], IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-15, IL-17, IL-18, gamma interferon, and tumor necrosis factor alpha) were validated with a panel of healthy individuals, rheumatoid arthritis patients, and juvenile idiopathic arthritis patients. Comparing the multiplex assay with a regular enzyme-linked immunosorbent assay technique with this donor panel resulted in correlation coefficients for all cytokines ranging from 0.75 to 0.99. Intra-assay variance proved to be less then 10%, whereas interassay variability ranged between 10 and 22%. This multiplex system proved to be a powerful tool in the quantitation of cytokines. It will provide a more complete picture in differences between activated lymphocyte cytokine profiles from healthy individuals and those from patients with chronic inflammatory diseases.     

Quantitation of human IgG subclass antibodies to Haemophilus influenzae type b capsular polysaccharide. Results of an international collaborative study using enzyme immunoassay methodology.

An international collaborative study was conducted at ten sites to examine the performance of enzyme immunoassays (EIAs) for the quantitation of IgG1, IgG2, IgG3, IgG4 and total IgG anti-Haemophilus influenzae type b (Hib) capsular polysaccharide in human serum. All groups used the same reagents: microtiter plates coated with polyribosylribitol phosphate (PRP) conjugated to poly-L-lysine (PLL), reference, control and test human sera, biotin-conjugated International Union of Immunological Societies (IUIS)-documented monoclonal anti-human IgG1-4 and IgG Pan detection antibodies, avidin-peroxidase and TMB substrate. Initial mixing of soluble PRP antigen or an equal volume of buffer with the 20 test sera prior to analysis confirmed PRP antigen specificity in all five EIAs with greater than 80% competitive inhibition at most sites. Positive correlation between the total IgG anti-Hib and sum of IgG1-4 anti-Hib was demonstrated (r2 = 0.99, Y = 1.13X -0.15). Good agreement was shown between the total IgG anti-Hib as measured by EIA and the total Hib-specific antibodies measured by the current radiolabeled antigen binding assay (r2 = 0.97, Y = 4.6X -5.8). Assay parallelism was demonstrated with an average interdilutional %CV of 22% and parallel dose-response curve slopes. The interdilutional %CVs were calculated as an average per sample of the variation of microgram/ml (corrected for dilution) at different dilutions per laboratory for all participating sites. The interlaboratory variation was the only performance parameter studied that exceeded the target level of 35% CV in all IgG1-4 and total IgG anti-Hib assays. IgG subclass distributions in the test sera demonstrated a predominance of IgG1 anti-Hib in the pediatric serum pools and IgG2 anti-Hib in the adult sera, with low but detectable levels of IgG3 and IgG4 anti-Hib in each group.     

Effect of carrier priming on immunogenicity of saccharide-protein conjugate vaccines

Previous studies with saccharide-protein conjugates have demonstrated that antibody responses to the saccharide can be improved by the preexistence of carrier immunity. Here we report that prior exposure to the carrier protein can either enhance or suppress antibody response to polysaccharides administered in saccharide-protein conjugates. A dose-dependent role for carrier priming in the antisaccharide antibody response to three saccharide-protein conjugate vaccines, i.e., a Streptococcus pneumoniae type 4 polysaccharide-tetanus toxoid (TT) conjugate (PS4TT), a Neisseria meningitidis group C polysaccharide-TT conjugate (MenCTT), and a N. meningitidis group C oligosaccharide-diphtheria mutant toxin conjugate (MenCCRM), was investigated. The results showed that an increase in the antipolysaccharide antibody response could be obtained for both PS4TT and MenCTT but not for MenCCRM with low-dose carrier priming (0.025 to 0.25 microgram). However, suppression of the antipolysaccharide antibody response was observed with the PS4TT and MenCTT vaccines with high-dose (25-micrograms) carrier priming. There was no suppression effect with MenCCRM. The increase in the antipolysaccharide antibody response was shown to be restricted to the immunoglobulin G1 (IgG1) subclass, whereas suppression with high-dose carrier priming affected all antipolysaccharide subclass antibodies induced by PS4TT (IgG1, IgG2b, and IgG3) and only two of the four subclass antibodies induced by MenCTT (IgG2a and IgG2b). The increase in the antipolysaccharide antibody response was also present at the antipolysaccharide IgM antibody level but was not observed at the anti-carrier IgG antibody level.     

Pneumococcal conjugate vaccines.

We have prepared conjugates of pneumococcal type 4 polysaccharides (PS4) or oligosaccharides to tetanus toxoid using the carbodiimide method. The use of a spacer, 6-aminohexanoic acid, resulted in higher incorporation of carrier protein. Conjugates contained up to 10% free polysaccharide, but no free protein. In general, polysaccharide conjugates induced higher anti-PS4 IgG antibody titers than oligosaccharide conjugates. Conjugates with the highest amount of incorporated protein were the most immunogenic. The response to conjugated PS4 does show characteristics of a T cell-dependent antibody response, in terms of both isotype distribution and induction of immunological memory. Repeated immunization with high doses of PS4TT conjugate resulted in a virtually negative anti-PS4 IgG response, suggestive of the induction of high dose tolerance.     

Temperature dependence of mutant mevalonate kinase activity as a pathogenic factor in hyper-IgD and periodic fever syndrome

Hyper-IgD and periodic fever syndrome (HIDS) and mevalonic aciduria are autosomal recessive disorders characterized by recurrent episodes of fever and generalized inflammation. Both syndromes are caused by specific mutations in the gene encoding mevalonate kinase (MK), resulting in a depressed enzymatic activity mainly due to reduced protein levels. We studied the effect of temperature on the activity of wild-type and several mutant MKs in fibroblasts. All fibroblast cell lines from HIDS patients and harbouring the common V377I MVK allele displayed substantially higher MK activities at 30 degrees C as compared to 37 degrees C. As shown by temperature inactivation experiments this resulted in a protein nearly as stable as in control cell lines, indicating that primarily the maturation of the protein is affected. Accordingly, when HIDS cell lines were cultured at 39 degrees C, MK activity decreased further. This triggered a compensatory increase in 3-hydroxy-3-methylglutaryl-CoA reductase activity, indicating that MK becomes progressively rate-limiting. A similar phenomenon occurs in vivo. MK activity in peripheral blood mononuclear cells drops 2-8-fold when HIDS patients experience febrile attacks. Our results suggest that minor elevations in temperature can set off a chain of events with MK becoming progressively rate-limiting, leading to a temporary deficiency of isoprenoid end-products, which induces inflammation and fever.     

Expression and modulation of cell surface determinants on human adult and neonatal monocytes

The difference between newborn and adult mononuclear cells in the antigen dose required for optimal antibody production in vitro can be ascribed to differences in the antigen-presenting capacities of the respective monocytes (Van Tol et al., 1984b). We have therefore studied the expression of cell surface determinants on human neonatal and adult monocytes by the use of monoclonal antibodies to membrane proteins including MHC antigens. No difference was observed in the expression of LeuM3 with regard to both the percentage of positive cells and the density of the respective determinant. In contrast, neonatal cells express the antigens OKM5, LFA1, OKM1 and LeuM5 at a lower density than adult cells do. The same holds for beta 2-microglobulin, but neonatal and adult monocytes express MHC class I alpha-chains at a similar density, whereas among the class II MHC antigens, HLA-DR is significantly more highly expressed on neonatal cells. This difference remains after treatment in vitro with gamma-interferon (gamma-IFN). Treatment with gamma-IFN also resulted in a less dense expression of the LeuM3 antigen. Preincubation of monocytes with LeuM3 monoclonal antibody partially abrogates subsequent upregulation of class II MHC antigens by gamma-IFN, a phenomenon observed with both neonatal and adult monocytes. These data indicate a functional involvement of LeuM3 with the cellular action of gamma-IFN. Taken together, the cell surface phenotype of neonatal monocytes is that of a highly efficient antigen presenting cell.     

After chemotherapy,functional humoral response capacity is restored before complete restoration of lymphoid compartments

Chemotherapy has, besides the beneficial effects, several adverse effects. Suppression of the immune system is one of the most important problems. Infections caused by encapsulated bacteria like Streptococcus pneumoniae are responsible for a major part of infectious problems during and after treatment. The splenic marginal zone is essential in the initiation of an immune response to encapsulated bacteria. In this study, we analysed the effects of three different cytostatic agents on humoral immune responses. We found a reduced, but detectable immune response capacity at two days after treatment although the marginal zone B cell population is severely reduced at this time point. Twenty-four days after cessation of treatment, the immune response capacity was largely restored although lymphoid compartments were still not completely restored at that time point. Apparently, the presence of only few marginal zone B cells is sufficient to evoke a rise in antibody titres and although antibody titre increases are low, even small rises are most likely clinically relevant.