万古霉素/磷酸钙骨水泥复合材料的细胞毒性研究

目的:研究不同浓度配比(0%、1%、5%、10%)万古霉素/磷酸钙复合材料对大鼠骨髓间充质干细胞的毒性作用。方法:原代培养大鼠间充质干细胞并鉴定;采用CCK-8法测定不同浓度配比万古霉素/磷酸钙复合材料对大鼠骨髓间充质干细胞增殖的影响、TUNEL法测定细胞凋亡率、扫描电镜观察细胞形态学改变。结果:CCK-8结果显示,5%及10%万古霉素/磷酸钙复合材料显著抑制大鼠骨髓间充质干细胞增殖(P0.05);TUNEL结果显示,5%及10%万古霉素/磷酸钙复合材料组细胞凋亡率显著增高(P0.05);扫描电镜结果显示,高浓度万古霉素毒性作用下细胞失活,形态学发生显著变化;1%万古霉素/磷酸钙复合材料组对细胞影响相对于空白对照组无显著差异。结论:低浓度(1%)万古霉素/磷酸钙复合材料基本无细胞毒性,细胞相容性好。     

Approach to the direct intramolecular localization of antigenic determinants in Androctonus australis hemocyanin with monoclonal antibodies by molecular immunoelectron microscopy

Monoclonal antibodies (mAb) directed vs. subunits from hemocyanin (Hc) of the scorpion Androctonus australis were used in molecular immunoelectron microscopy (MIEM) to directly localize the epitopes within the subunits. Four types of mAb were used. First, mAb 6302, an IgG clone highly specific for subunit Aa 2, produced with native hemocyanin long strings composed of hemocyanin molecules in the side view and in the 45 degrees view. At lower concentration, "parachute" and "butterfly" structures composed of two Hc molecules and one monoclonal immunoglobin G (IgG) molecule were obtained. Fab fragments prepared from mAb 6302 bound exactly on the top and bottom edges of the molecule. The second type of mAb (6003), directed vs. subunit Aa 2, produced nice immunocomplexes with the free subunit but nothing with the native oligomer. It is suggested that due to steric hindrance or to conformational changes the epitope is not accessible in the native molecule. The third mAb belonged to the IgM class and apparently bound Hc in the Aa 2 area. However, because of the difficulty of separating the immunocomplexes from the residual mAb and the polymorphism of the IgM molecules, monoclonal IgM are no longer used for MIEM. The last type of mAb (5701) had a high affinity and a high specificity for subunit Aa 6. It produced two types of immunocomplexes with native Hc. The two types differed by a 180 degrees rotation around one of the Fab arms. These complexes, which support recent results of Wrigley et al. [Wrigley, N. G., Brown, E. B., & Skehel, J. J. (1983) J. Mol. Biol. 169, 771-774] and of Roux [Roux, K. H. (1983) Eur. J. Immunol. 14, 459-464], indicate that monoclonal IgG have a high degree of rotational flexibility around the Fab arm. Monoclonal antibody 5701 bound exactly at the corner of the molecule in the area where subunit Aa 6 is known to be located. The MIEM approach of the location of the epitope requires the model of the architecture and of the quaternary structure to be very precise. Thus, recent findings of Gaykema et al. [Gaykema, W. P. J., Hol, J. M., Vereijken, J. M., Soeter, N. M., Bak, H. J., & Beintema, J. J. (1984) Nature (London) 309, 23-29] and of Van Heel et al. [Van Heel, M., Keegstra, W., Schutter, W., & Van Bruggen, E. F. J. (1983) Life Chem. Rep., Suppl. Ser. 1, 69-73] led to a reexamination of previous models.(ABSTRACT TRUNCATED AT 400 WORDS)     

Genetic Structure and Variation of Van Cats

To determine the genetic structure and variation of Van cats and some other cats, seven enzyme loci were examined using horizontal starch gel electrophoresis. ME bands were observed for the first time in cats. For the enzyme loci CA ( 1 ), SOD, GPI, and GOT, neither the individual Van cats nor the specimens of other cat species exhibited any variation. These enzymes presented identical bands, all of which were homozygous. With respect to the PGD, ME, and ESD loci, however, genetic variation was observed in all of the cats. Hence, three of the seven gene-enzyme systems (43%) were polymorphic with two alleles, contributing to an estimate of average heterozygosity of 0.33-0.49 for the Van cats. PGD was the most discriminatory among the three polymorphic loci. The phylogenetic tree indicated that the Van, Persian, Turkish Angora, and Turkish Tekir cats are distinct from Siamese and Bombay cats.     

Squatting facet: a case study Dilkaya and Van-Kalesi populations in eastern Turkey

Anomalies of the skeleton provide information on living conditions, cultural structure and health problems in ancient societies. Squatting facet is a kind of anomaly that forms on the surfaces where the tibia and talus articulate is the squatting facet states the daily activities and living style of the society. The aim of this study is to learn the daily activities of the medieval societies in the Van region through studying of squatting facets. In this study, adult skeletons from Dilkaya and Van Kalesi-Eski Van Sehri societies dating to the Medieval Age were investigated (65 tibia and 82 tali from Dilkaya, 61 tibia and 52 tali from Van Kalesi-Eski Van Sehri). The lateral squatting facet had high ratios in both societies. The tibia lateral squatting facet found on females and males of Dilkaya was 97.2% and 96.9%, respectively, and on females and males of Van kalesi Eski Van Sehri was 87.5% and 89.2%, respectively. The talus lateral squatting facet found on females and males of Dilkaya was 72.1% and 51.3%, respectively, and on females and males of Van kalesi Eski Van Sehri was 91.2% and 83.7%, respectively. The results provide an opportunity to study the relationship between past and modern population, and also describe the daily activity of life and cultural structure.     

Van Gogh: a new Drosophila tissue polarity gene.

Mutations in the Van Gogh gene result in the altered polarity of adult Drosophila cuticular structures. On the wing, Van Gogh mutations cause an altered polarity pattern that is typical of mutations that inactivate the frizzled signaling/signal transduction pathway. The phenotype however, differs from those seen previously, as the number of wing cells forming more than one hair is intermediate between that seen previously for typical frizzled-like or inturned-like mutations. Consistent with Van Gogh being involved in the function of the frizzled signaling/signal transduction pathway, Van Gogh mutations show strong interactions with mutations in frizzled and prickle. Mitotic clones of Van Gogh display domineering cell nonautonomy. In contrast to frizzled clones, Van Gogh clones alter the polarity of cells proximal (and in part anterior and posterior) but not distal to the clone. In further contrast to frizzled clones, Van Gogh clones cause neighboring wild-type hairs to point away from rather than toward the clone. This anti-frizzled type of domineering nonautonomy and the strong genetic interactions seen between frizzled and Van Gogh suggested the possibility that Van Gogh was required for the noncell autonomous function of frizzled. As a test of this possibility we induced frizzled clones in a Van Gogh mutant background and Van Gogh clones in a frizzled mutant background. In both cases the domineering nonautonomy was suppressed consistent with Van Gogh being essential for frizzled signaling.     

Functional activity of different IgG subclass antibodies against type b capsular polysaccharide of Haemophilus influenzae

The biologic activity of different human IgG subclass antibodies directed against the Haemophilus influenzae type b (Hib) capsular polysaccharide (PRP) was compared by using an in vitro complement-mediated bactericidal assay and an in vivo passive protection assay in infant rats. An IgG pool was made by Sephacryl S-300 chromatography of sera from adults immunized with PRP vaccine. An IgG2 subclass fraction was prepared by column immunoabsorption of the IgG pool with anti-IgG1 monoclonal antibody. An IgG1 subclass fraction was eluted from the affinity matrix. IgG1, IgG2, IgG3, and IgG4 concentrations in the fractions were measured by solid-phase competitive radioimmunoassays, and anti-PRP antibody was measured by a modified Farr assay. Each fraction was greater than 90% pure IgG2 or IgG1, respectively. There were no significant differences in the minimal anti-PRP antibody concentrations required to kill 50% of Hib cells in vitro (IgG, 0.22; IgG1, 0.21; and IgG2, 0.42 microgram/ml). Similarly, equivalent amounts of anti-PRP antibody of the IgG1 or IgG2 fractions protected against bacteremia (IgG1, 0.12; IgG2, 0.24 microgram per rat). IgG absorbed to remove anti-PRP antibody was neither bactericidal nor protective. Thus IgG1 and IgG2 anti-PRP antibody have equivalent functional activities against Hib as determined by these biologic assays.     

Interaction among the subunits of Golgi membrane mannosyltransferase complexes of the yeast Saccharomyces cerevisiae

Saccharomyces cerevisiae Mnn9 protein is a type II Golgi membrane protein which concerns in protein mannosylation. When solubilized by Triton X-100, it was recovered in two distinct complexes both having mannosyltransferase activity; one with Van1 protein (V-complex) and the other with Anp1, Hoc1, Mnn10, and Mnn11 proteins (A-complex). Characterization of the null mutants suggested that A-complex is also concerned in protein O-glycosylation. A-complex was more resistant than V-complex to dissociating conditions. Interaction between the lumenal domains of Van1 and Mnn9 was detected by a two-hybrid experiment. The anchor domain of Mnn9 protein could be replaced with other membrane anchors without losing the ability to form complexes similar to V- and A-complexes. Thus the lumenal domains are important to assemble these distinct complexes.     

Os trigonum syndrome in ancient Anatolian settlements

An os trigonum is a small, round bone situated just behind the ankle joint. Clinical research shows that the os trigonum is present in about 5-15 % of normal feet. It occurs when one part of the bone does not fuse with the rest of the talus during growth. The present study is made on the skeletons, which were found in the Karagündiiz, Dilkaya and Van Kalesi-Eski Van Sehri settlements, Van province, Anatolia, dated in the Middle Ages. In this study 228 skeletons were examined, 120 males and 108 females. 376 tall were studied: 227 from Karagiindtiz, 93 from Dilkaya, and 56 from Van Kalesi-Eski Van Sehri. The percentage of the os trigonum syndrome was recorded as 6.6 %.     

Demonstration of neutralizing autoantibodies against IL-1 alpha in sera from patients with rheumatoid arthritis

We have recently reported the presence of IgG which has a potent inhibitory activity against IL-1 alpha in some sera from patients with rheumatoid arthritis. The mechanism of this inhibition by IgG against IL-1 alpha is now elucidated. IgG with IL-1 alpha-inhibitory activity inhibited the binding of 125I-IL-1 alpha to receptors on rheumatoid synovial cells. In addition, preincubation of synovial cells with the inhibitory IgG did not block the binding of 125I-IL-1 alpha to receptors, suggesting a direct interaction between IgG and IL-1 alpha. To examine which region of the IgG, namely Fab or Fc region, has the inhibitory activity, the IgG was digested with papain, and Fab and Fc fragments were purified. Fab fragments, but not Fc fragments, inhibited both IL-1 alpha-induced thymocyte-proliferation and the binding of 125I-IL-1 alpha to receptors. We further demonstrated that the inhibitory IgG which was bound to protein A Sepharose could bind a significant amount of 125I-IL-1 alpha, whereas only a negligible binding of the radiolabeled ligand was detected when IgG without the inhibitory activity was used as control. Moreover, the binding of 125I-IL-1 alpha to IgG with the inhibitory activity was clearly blocked by Fab fragments of IgG having the inhibitory activity. Finally, affinity-purified IgG over an IL-alpha affinity column showed approximately 100-fold more potent inhibitory activity on IL-1 alpha-induced thymocyte proliferation compared with untreated IgG. From these results, we conclude that IgG molecules with IL-1-alpha-inhibitory activity are neutralizing autoantibodies against IL-1 alpha.     

Neutralization of sendai virus by the IgG subclass antibodies of the guinea pig

A comparative study was made of the neutralizing activities of IgG subclasses IgG1 and IgG2, fractionated from guinea pig antisera against Sendai virus. The yields of IgG2 from the antisera were about 16 times as much as those of IgG1. The neutralizing activity of IgG2 per unit weight was four times as high as that of IgG1. This neutralizing activity of both IgG subclasses was enhanced about 10 times by addition of antibodies to the L-chain of guinea pig immunoglobulin. It is suggested that, in the complement-dependent neutralization of the virus, IgG1 and IgG2 activate the complement through the alternative and the classical pathway, respectively.     

The kinetic behaviour of radiolabelled IgG1 derived from different sources in adult sheep and neonatal lambs.

The turnover rate and pool volume of radiolabelled autologous IgG1 was similar to homologous IgG1 in adult sheep. In neonatal lambs there was no significant difference for either of these parameters between homologous serum IgG1 and maternal colostrum IgG1. However, the pool volume for IgG1 expanded by approx. 30% over the experimental period in the lambs. When the turnover rates were corrected for this increase they were found to be similar to those observed for IgG1 in adult animals.     

IgG subclass expression by human B lymphocytes and plasma cells: B lymphocytes precommitted to IgG subclass can be preferentially induced by polyclonal mitogens with T cell help

The human IgG subclasses expressed by circulating B lymphocytes, tissue plasma cells, and plasma cells generated from B cell precursors in response to the polyclonal mitogens LPS and PWM were examined by immunofluorescence using subclass-specific monoclonal antibodies. The subclass distribution observed for circulating B lymphocytes was IgG2 (48%) greater than IgG1 (40%) greater than IgG3 (8%) greater than IgG4 (1%), while the distribution among IgG plasma cells in bone marrow, blood, spleen, and tonsils was IgG1 (64%) greater than IgG2 (26%) greater than IgG3 (8%) greater than IgG4 (1%). Multiple IgG isotypes were not observed on B cells or in plasma cells. Although IgG plasma cell responses to both LPS and PWM were T cell dependent, the distributions of IgG subclasses elicited were strikingly different. In control and LPS-stimulated cultures of blood mononuclear cells, the induced plasma cells expressed the IgG subclass distribution: IgG2 greater than 80%, IgG1 less than 20%, IgG3 less than 1%, IgG4 less than 1%. In PWM-stimulated cultures, the subclass distribution, IgG1 approximately 65%, IgG2 approximately 25%, IgG3 approximately 7%, IgG4 approximately 1%, was in perfect concordance with the in vivo subclass distribution of IgG plasma cells. Selective inhibition of suppressor T cell activity by x-irradiation and mitomycin C treatment did not alter the IgG subclass distribution pattern induced by LPS and PWM. Monoclonal antibodies were used to deplete selectively the B cell precursors bearing IgG1, IgG2, or IgG3 before PWM stimulation of blood mononuclear cells. In each instance, a reduction was observed only in the subpopulation of plasma cells producing the homologous IgG subclass. The results indicate that T cells can preferentially influence the terminal differentiation of B cells that are precommitted to different IgG subclasses.     

Interaction Among the Subunits of Golgi Membrane Mannosyltransferase Complexes of the Yeast Saccharomyces cerevisiae

Saccharomyces cerevisiae Mnn9 protein is a type II Golgi membrane protein which concerns in protein mannosylation. When solubilized by Triton X-100, it was recovered in two distinct complexes both having mannosyltransferase activity; one with Van1 protein (V-complex) and the other with Anp1, Hoc1, Mnn10, and Mnn11 proteins (A-complex). Characterization of the null mutants suggested that A-complex is also concerned in protein O-glycosylation. A-complex was more resistant than V-complex to dissociating conditions. Interaction between the lumenal domains of Van1 and Mnn9 was detected by a two-hybrid experiment. The anchor domain of Mnn9 protein could be replaced with other membrane anchors without losing the ability to form complexes similar to V- and A-complexes. Thus the lumenal domains are important to assemble these distinct complexes.     

Antibody isotype responses in Balb/c mice immunized with the cytoplasmic repetitive antigen and flagellar repetitive antigen of Trypanosoma cruzi

In the present report we analyzed the levels of IgG1, IgG2a, IgG2b and IgG3 isotypes from Balb/c mice immunized with cytoplasmic repetitive antigen (CRA), and flagellar repetitive antigen (FRA) of Trypanosoma cruzi. The immunization was done by subcutaneous route three times (20 days apart) and the analysis was performed 14 days after each treatment. CRA-immunized mice produced high levels of all IgG isotypes, mainly IgG3 and IgG1. FRA-immunization elicited only high levels of IgG1.     

Studies on the Fc gamma receptor of the murine macrophage-like cell line P388D1. II. Binding of human IgG subclass proteins and their proteolytic fragments.

Binding studies of human IgG proteins to murine P388D1 cells indicated that they bind to an apparently homogeneous Fc receptor population. The association constant was 0.89 x 10(6)M-1 at 22 degrees C and was comparable to the binding affinities of homologous murine IgG2a and IgG2b. The number of receptor sites was found to be approximately 6 x 10(5)/cell. Fc gamma 1 and Fc gamma 3 fragments bound with an affinity comparable to that of the parent proteins. The P388D1 receptors could discriminate between the human IgG subclasses; the relative cytophilic activity was IgG3 greater than IgG1 greater than IgG4 and IgG2 was devoid of binding activity. Fragments corresponding to the C gamma 2 and C gamma 3 domains of human IgG1 were both unable to bind to the P388D1 receptors either alone or in equimolar combination. This suggests that the cytophilic site may be formed cooperatively by interaction between the two domains. The integrity of the hinge region appeared to be essential for full expression of cytophilic activity since reduction of the hinge-region disulfides in both human IgG1 and its Fc fragment markedly decreased their binding affinity. In addition, a mutant IgG1 molecule lacking the hinge region was significantly less cytophilic than its normal counterpart.     

Hyperacute rejection by anti-Gal IgG1, IgG2a, and IgG2b is dependent on complement and Fc-gamma receptors

We have previously reported that anti-Gal-alpha1,3Gal (Gal) IgG3 mAbs mediate a classical complement-dependent hyperacute rejection (HAR), while anti-Gal IgG1 mAbs mediate HAR that is dependent on complement, the Fc-gamma receptors FcgammaRII/III (CD32/CD16), and NK cells. IgG2a and IgG2b subclasses can activate complement and have FcgammaR binding properties in vitro. Whether these IgG subclasses can mediate HAR in vivo and the mechanisms by which they would do so are not known. In this study, we isolated spontaneous IgG switch mutants from an anti-Gal IgG1 hybridoma. In vitro complement-mediated hemolytic assays with mouse complement indicate that both anti-Gal IgG2a and IgG2b mAbs were more potent compared with the parent anti-Gal IgG1. In vivo administration of anti-Gal IgG2a and IgG2b mAbs into Gal-/- mice induced HAR of rat cardiac xenografts. HAR induced by anti-Gal IgG2a and IgG2b was dependent on complement activation and the presence of NK cells. Using FcgammaRIII-deficient (Gal-/-CD16-/-) recipients, we observed that HAR mediated by different anti-Gal IgG subclasses was variably dependent on FcgammaRIII, with IgG1>IgG2b>IgG2a=IgG3. Using FcgammaRI-deficient (Gal-/-CD64-/-) recipients, we observed that HAR mediated by anti-Gal IgG1, IgG2a, and IgG2b, but not by anti-Gal IgG3, was dependent on FcgammaRI. Collectively, these studies demonstrate the necessity and sufficiency of complement in IgG3-mediated HAR and the necessity of both complement and FcgammaR, especially FcgammaRI, in IgG1-, IgG2a-, and IgG2b-mediated HAR.     

In vivo allogeneic effects: shift in the isotype profile of primary TI-2 responses in mice undergoing graft-vs-host reaction

We showed previously that primary responses to T-dependent (TD) and T-independent type 2 (TI-2) antigens were differentially affected by allogeneic effects induced in vivo during a graft-vs-host reaction (GVH). TD responses were greater than or equal to 80% suppressed, whereas the TI-2 responses were greatly enhanced, particularly the IgG component, which normally is very low. We have analyzed the IgG subclass distribution in primary responses of normal and GVH F1 mice in order to determine whether the strong T cell signals that occur during GVH reactions also induce shifts in the isotype profile. The effect of GVH on responses to TI-2 antigens was of particular interest because they are usually dominated by IgM and IgG3 classes in normal mice. We found a threefold to 10-fold increase in the PFC numbers of all four IgG subclasses in the response to TI-2 antigens, with an apparent shift from the usual IgG3 dominance to IgG1 in GVH mice. This IgG1 dominance was not found in serum antibodies where IgG3, IgG1, and IgG2b were equally expressed, although total IgG was increased greater than 20-fold. No isotype shift was found in either the TNP-KLH response, which was greater than or equal to 75% suppressed (IgG1 dominance was retained), or in the TI-1 response to TNP-Ba. The latter response was reduced (25 to 50%) in GVH mice and continued to be dominated by IgG2b/2a and IgG3. Unlike the unique isotype patterns found in primary responses, TNP-KLH primed mice challenged with TD, TI-1, or TI-2 antigens gave memory responses with identical isotype profiles that were dominated by IgG1 PFC. The role of T cells in B cell differentiation and isotype expression is discussed.     

Serologic aspects of IgG4 antibodies. II. IgG4 antibodies form small, nonprecipitating immune complexes due to functional monovalency

Human IgG4 antibodies directed against phospholipase A, the P1 antigen from Dermatophago?des pteronyssinus extracts, and cat albumin were found unable to cross-link antigen. Previously, it was demonstrated that IgG4 antibodies, in contrast to IgG1 antibodies, did not cross-link Sepharose-bound antigen and antigen added in solution. To eliminate the possibility that this phenomenon was caused by preferential binding of both IgG4 Fab fragments to the solid-phase-bound antigen, cross-linking of antigen was studied in a fluid-phase system. In this test, incapability of IgG4 antibodies to bridge two antigens was also found. As a result of such a phenomenon, it is expected that immune complexes formed by IgG4 antibodies will be considerably smaller than complexes formed by IgG1. This was confirmed by analysis of the molecular size profiles of IgG1- and IgG4-containing immune complexes in sucrose-density gradients. Moreover, IgG1 was able to precipitate antigen in a radioimmunoprecipitation test, whereas precipitation was not demonstrable by the same amount of IgG4 antibodies. Even 3% polyethylene glycol 8,000 did not precipitate the small IgG4-containing immune complexes efficiently. The antibodies studied were of a high-affinity type, and there was no significant difference in association constants between IgG1 and IgG4 antibodies. Therefore, we were not able to confirm observations reported in the literature that the IgG4 subclass is associated with a low-affinity antibody response; probably, the affinity of the IgG4 antibodies was underestimated by other investigators because of the polyethylene glycol precipitation technique used to separate antibody-bound and free antigen. Our findings stress the point that IgG4 antibodies take a special place in the immune response upon chronic exposure to antigen.     

The immune response in the hamster. VII. Studies on cytophilic immunoglobulin.

Hamster 7S IgG1 and IgG2 antibodies to hen-egg albumin (HEA) were tested for their capacity to bind to macrophage cytophilic Ig receptors. Both IgG1 and IgG2 were cytophilic for hamster macrophages though the membrane receptor had a predominant specificity for IgG1. Hamster IgG1 bound primarily to homologous macrophages whereas IgG2 bound to macrophages from other rodent species as well. The binding of hamster Ig to hamster macrophages was inhibited by a wide range of heterologous rodent sera. The only exception was guinea pig serum since guinea pig IgG2 was found to bind only to homologous macrophages. Sheep red blood cells (SRBC) coated with hamster IgG2 were ingested by macrophages more readily than those coated with hamster IgG1. Thus, there appeared to be a paradoxical relationship between the apparently strong affinity of IgG1 for the hamster macrophage Ig receptor and its reactivity weak ingestion promoting activity. Implications of this phenomenon are discussed.     

Human serum immunoglobulin G3 subclass bound preferentially to asialo-, agalactoimmunoglobulin A1/Sepharose.

As we reported before, exoglycosidase treatment of human serum IgA1 changed it to a sticky molecule. In order to examine the presence of the specific binding protein to the sticky IgA1 in human serum, IgA1, asialo-IgA1 (IgA1-S) and asialo-, agalacto-IgA1 (IgA1-SG)/Sepharose column chromatography of normal human serum was carried out. Purified hinge glycopeptide (HGP33) prepared from IgA1 was used for the preparation of HGP/Sepharose. A portion of the serum protein was bound to those columns and eluted with the buffer containing 1.0 M NaCl. About four times the amount of protein was eluted from the IgA1-SG/Sepharose column than that from IgA1/Sepharose. Most of the eluted protein was IgG, and the IgG1 and IgG3 subclasses but neither IgG2 nor IgG4 was dominant. Under the lower salt concentration, a portion of IgG was also bound to the HGP-SG/Sepharose column. The obtained results coincide well with the previous report of the co-present of IgG1 and IgG3 with deposited IgA1 in IgA nephropathy patients (Aucouturier, P., et al. (1989) Clin. Immunol. Immunopathol. 51, 338-347). Thus, the results solved the question of why the IgG3 was co-present with deposited IgA1 in IgA nephropathy patients.