Immunopathological correlation between mesangial C3d-deposition and C3d-fixing circulating immune complexes in lupus nephritis

By a direct immunofluorescent technique, glomerular C3d deposition was examined in a total of 50 renal biopsy specimens from patients with lupus nephritis. C3d deposition was then compared with disease activity, glomerular IgG and C3c deposition, and the levels of circulating immune complexes (CIC) measured by a solid-phase anti-C3d assay. There was a good correlation between disease activity and the positivity of glomerular C3d deposits (P less than 0.001), as well as C3c deposits (P less than 0.001). Even in clinically inactive patients, a relatively high percentage (59%) of C3d deposits were positive compared with C3c deposits (17%). Mesangial C3d deposition correlated with clinical disease activity more significantly (P less than 0.005) than capillary wall C3d deposition (P less than 0.025). C3d deposits were detected in all of the 30 cases with positive C3c deposits, and moreover, in 15 of the 20 (75%) cases with negative C3c deposits. Glomerular IgG deposits were almost always associated with C3d deposits, both in mesangial areas and along capillary walls, with statistical significance (P less than 0.005, P less than 0.001, respectively). The serum levels of C3d-fixing immune complexes (IC) were significantly correlated with the positivity and intensity of mesangial C3d deposits. This study demonstrates glomerular deposition of C3d in patients with lupus nephritis and reveals a significant correlation between mesangial C3d deposition and disease activity.     

Lipopolysaccharides of Bacteroides intermedius (Prevotella intermedia) and Bacteroides (Porphyromonas) gingivalis induce interleukin-8 gene expression in human gingival fibroblast cultures.

Lipopolysaccharides (LPS) prepared from Bacteroides intermedius (Prevotella intermedia) and Bacteroides (Porphyromonas) gingivalis by hot phenol-water extraction induced interleukin-8 (IL-8) mRNA in normal human gingival fibroblast cultures, as demonstrated by Northern (RNA) blot analysis. IL-8 mRNA levels began to increase after a 2-h exposure, reached a maximum after 12 h, and then dropped to the unstimulated level at 48 h. IL-8 mRNA levels were also enhanced in a dose-dependent manner. By contrast, LPS specimens from various Salmonella species with S and R chemotypes and bacterial [corrected] and synthetic lipid A preparations did not increase IL-8 mRNA levels in fibroblasts. Although recombinant human IL-1 alpha induced IL-8 mRNA expression in fibroblast cultures, an antiserum to recombinant human IL-1 alpha did not decrease the IL-8 mRNA accumulation induced by B. intermedius LPS. Fibroblasts primed with natural human gamma interferon (IFN-gamma) expressed higher IL-8 mRNA levels upon stimulation with B. intermedius LPS, but not with Salmonella LPS, compared with nontreated cells. Natural human IFN-beta exhibited a similar priming effect on the fibroblasts, and antiserum to IFN-beta added to the cultures together with B. intermedius LPS decreased the IL-8 mRNA levels. Therefore, endogenous IFN-beta enhanced IL-8 mRNA production in response to B. intermedius LPS in fibroblasts.     

Bioactive bone cement: effects of phosphoric ester monomer on mechanical properties and osteoconductivity

A new bioactive bone cement, designated GBC, has been developed. It consists of polymethyl methacrylate (PMMA) as an organic matrix and bioactive glass beads as an inorganic filler. The bioactive beads, consisting of MgO--CaO--SiO(2)--P(2)O(5)--CaF(2) glass, have been newly designed, and a novel PMMA powder was selected. The purpose of the present study was to evaluate the effects on mechanical properties and osteoconductivity of adding a phosphoric ester (PE) monomer to the cement as an adhesion-promoting agent. Four kinds of cements were prepared: GBC, GBC with PE (designated GBC/PE), a cement consisting of the same PMMA used in GBC with apatite- and wollastonite-containing glass-ceramic (AW-GC) powder (designated AWC), and AWC with PE (designated AWC/PE). Each filler was added to the cement at 70 wt %. Adding PE to either GBC or AWC resulted in increases in the bending strength and decreases in the Young's modulus compared with the unmodified cements. Cements were packed into the intramedullar canals of rat tibiae to evaluate osteoconductivity as determined by an affinity index. Rats were sacrificed at 4 and 8 weeks after operation. The affinity index (length of bone in direct contact with the cement expressed as a percentage of the total length of the cement surface) was calculated for each cement. Adding PE to either GBC or AWC resulted in significant increases in the affinity index compared with the unmodified cements. The affinity index for GBC was significantly higher than that of AWC, and that for GBC/PE was also significantly higher than that of AWC/PE. The affinity indices for each cement increased significantly with time up to 8 weeks. Our study revealed that the higher osteoconductivity of GBC/PE was due to the large alkyl group in the PE monomer, to the hydrophilicity of the phosphoric acid in the PE monomer, and to the higher bioactivity of the bioactive glass beads at the cement surface. GBC/PE shows promise as an alternative bone cement with improved properties compared with conventional PMMA bone cement.     

Effect of decay-accelerating factor on the assembly of the classical and alternative pathway C3 convertases in the presence of C4 or C3 nephritic factor

Decay-accelerating factor (DAF) is a 70,000 MW membrane protein that regulates the complement system on the cell surface. In the present study, we found that DAF had no effect on the classical pathway C3 and C5 convertases that had been stabilized by C4 nephritic factor (C4NeF). In DAF-incorporated cells, however, the assembly of the classical pathway C3 convertase was markedly inhibited even in the presence of C4NeF. C3 nephritic factor (C3NeF) in the alternative pathway protected the C3 convertase from the action of DAF to some extent, while the generation of C3 convertase was also inhibited by DAF. These results indicate that under physiological conditions, DAF functions to inhibit the assembly of C3 convertases even in the presence of nephritic factors, although it has no or little effect on the stabilized convertases. Thus, it is likely that DAF plays an important role in protection of host cells from damage by autologous complement in patients with nephritic factors.     

The roles of soluble osteopontin using osteopontin-transgenic mice in vivo: proliferation of CD4+ T lymphocytes and the enhancement of cell-mediated immune responses.

We generated transgenic mice expressing osteopontin (OPN) under the control of the alpha(1)-antitrypsin promoter. These mice (OPN-T mice) expressed OPN mRNA in liver and kidney, and released a large amount of plasma OPN, which increased after stimulation with turpentine oil. Before sensitization, the number of CD4+ T cells in lymph nodes was significantly higher in OPN-T than nontransgenic mice, and that in spleen was slightly higher, whereas that of CD8+ T cells was no different between OPN-T and nontransgenic mice. After sensitization, the CD4+ T cell numbers in spleen increased significantly, while there were almost no changes in the CD8+ T cells in lymph nodes and spleen. The intensity of contact hypersensitivity responses to 2,4-dinitrofluorobenzene (DNFB) was obviously enhanced in OPN-T mice. In the delayed-type hypersensitivity (DTH) model elicited by DNFB, the number of CD8+ T cells among DNFB-2,4,6-trinitrobenzenesulfonic acid (TNBS)-peritoneal exudate cells was significantly higher in OPN-T than nontransgenic mice, while there was almost no difference in that of CD4+ T cells. Adoptive transfer experiments revealed that the enhanced reactivity is carried by CD4+ and CD8+ T cells, respectively, although the ability of transferring DTH was significantly lower in CD8+ than in CD4+ T cells. The enhancement of CD8+ T cell migration was observed in OPN-T mice. These results suggest that OPN induces a proliferation of effector CD4+ and CD8+ cells in cell-mediated reactions and plays a role in the migration of CD8+ T cells.     

Measurement of antigen-specific mouse IgE by a fluorometric reverse (IgE-capture) ELISA

A reverse, or IgE-capture, enzyme-linked immunosorbent assay (ELISA) for measuring ovalbumin-specific IgE antibody in the serum of immunized mice has been developed. Microplate wells were first coated with a commercial anti-mouse IgE rat monoclonal antibody, and then incubated with two-fold serial dilutions of test sera with 10% normal mouse serum as diluent for the capturing of only IgE class molecules. Biotinylated ovalbumin and then beta-D-galactosidase-conjugated streptavidin were added and, finally, 4-methylumbelliferyl-beta-D-galactoside was used as the enzyme substrate. The fluorescence intensity of the reaction product (4-methylumbelliferone) was determined on a microplate fluorescence reader. The sensitivity of this assay was equal to that of passive cutaneous anaphylaxis (PCA). In contrast to indirect ELISAs this IgE-capture assay is free from competition by non-IgE antibodies. Furthermore, it requires much less antigen than the PCA assay.     

The antiallergic agent amoxanox suppresses SRS-A generation by inhibiting lipoxygenase

Amoxanox has potent antiallergic activity because it inhibits the release of chemical mediators such as histamine and leukotrienes. We studied the in vitro effect of amoxanox on arachidonic acid metabolism, including the lipoxygenase and cyclooxygenase pathways. Amoxanox inhibited calcium ionophore A23187-induced formation of 5-HETE, LTB4, SRS-A (LTC4, LTD4 and LTE4), and 12-HETE in rat peritoneal resident monocytes. These results indicate that amoxanox inhibits 5- and 12-lipoxygenases. The compound, however, did not affect the formation of TXB2 or 6-keto-PGF1 alpha in guinea pig lung fragments and PGE2 or PGF2 alpha in bovine seminal vesicles, suggesting that it did not inhibit cyclooxygenase. These results show that the antiallergic action of amoxanox is associated, at least in part, with the reduction of leukotrienes due to the inhibition of lipoxygenases.     

Expression profiling of cytokines and related genes in regenerating skeletal muscle after cardiotoxin injection: a role for osteopontin

To examine the roles of cytokines in muscle regeneration, we injected cardiotoxin into mouse tibialis anterior muscle and examined the expression profiles of cytokines and related genes in the regeneration process. Expression of 40, 64, and 7 genes among 522 genes spotted on a cytokine expression array were increased more than fivefold at 48 hours, 96 hours, and 7 days after toxin injection, respectively, when compared with those of the control muscle. Especially the levels of mRNA for chemokines and chemokine receptors, many of which are potent regulators of macrophages, were highly elevated 48 hours after injury. The expression of osteopontin (OPN), a versatile regulator of inflammation and tissue repair, was up-regulated more than 118-fold in regenerating muscle at 48 hours after injury. Northern blotting confirmed that the expression of OPN was highest at 48 hours after cardiotoxin injection and declined sharply thereafter. Immunohistochemistry showed that OPN was detected both in the cytoplasm of macrophages and in necrotic muscle infiltrated with macrophages. Our studies suggest OPN may serve as an adhesion molecule that promotes macrophage binding to necrotic fibers and may be an important mediator in the early phase of muscle regeneration.