Synthesis and biological activities in vitro and in vivo of glycosylated human interleukin-1α, neoglyco IL-1α, coupled with N-acetylneuraminyl-galactose

In order to study the effect of glycosylation on its biological activities, and to develop IL-1α with less deleterious effects, recombinant human IL-1α was chemically coupled with N-acetylneuraminic acid (α1-6) galactose (Neu5Ac-Gal). Glycosylated IL-1α (Neu5Ac-Gal-IL-1α) was purified by anion-exchange chromatography and average number of carbohydrate molecules introduced per molecule of IL-1α was 2.5. Neu5Ac-Gal-IL-1α exhibited reduced activities about 1/15-fold compared to IL-1α in all the activities performed in vitro. Binding affinities of Neu5Ac-Gal-IL-1α to Type I and Type II IL-1 receptors were decreased to 1/15 and 1/10, respectively. Neu5Ac-Gal-IL-1α exhibited reduction in activities in vivo, including induction of serum amyloid A and NO$_x$, and down-regulation of serum glucose. However, Neu5Ac-Gal-IL-1α exhibited comparable activity to IL-1α in improvement of the recovery of peripheral white blood cells from myelosuppression in 5-fluorouracil-treated mice. In addition, tissue level of Neu5Ac-Gal-IL-1α was relatively high compared to IL-1α. These results indicate that coupling with Neu5Ac-Gal enabled us to develop neoIL-1α with selective activities in vivo. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development of glycosylated human interleukin-1 alpha, neoglyco IL-1 alpha, by coupling with D-galactose monosaccharide: biological activities in vitro

In the previous study, galactose with C9 spacer was chemically coupled to human recombinant (rh) IL-1 alpha in order to study the effect of glycosylation on its activities, and to develop IL-1 with less deleterious effects. In this study we examined a variety of IL-1 activities in vitro, including proliferative effect on T cells, antiproliferative effect on myeloid leukemic cells and melanoma cells, stimulatory effects on IL-6 synthesis by melanoma cells and PGE2 synthesis by fibroblast cells Galactose-introduced IL-1 alpha (Gal-IL-1 alpha) exhibited reduced activities from 10 to 10000 times compared with unmodified IL-1 alpha in all the activities performed in vitro. The competitive binding of 125I-IL-1 alpha to mouse T cells and pre-B cells with unlabeled IL-1 alpha s suggests a decrease in binding affinities of Gal-IL-1 alpha to both type I and type II IL-1 receptors. Therefore, reduced activities of Gal-IL-1 alpha are due, at least partially, to the decrease in their receptor binding affinities.     

Experimental autoimmune encephalomyelitis is exacerbated by IL-1 alpha and suppressed by soluble IL-1 receptor.

To assess the role of IL-1 in the development of experimental autoimmune encephalomyelitis (EAE), the effects of in vivo treatment with IL-1 alpha or an IL-1 antagonist on the clinical course of EAE were evaluated. First, Lewis rats were immunized with guinea pig myelin in CFA and treated for 19 consecutive days with i.p. injections of recombinant human IL-1 alpha. Clinical signs of paralysis in the IL-1 alpha-treated groups were of longer duration and of greater severity compared to placebo injected controls. In addition, more weight loss was observed in the IL-1 alpha-treated groups compared to controls. This enhanced weight loss was not due to IL-1 alpha injections alone as CFA-treated rats injected with IL-1 alpha did not lose weight when compared to placebo injected, CFA-treated controls. Second, soluble mouse rIL-1R (sIL-1R), which binds both IL-1 alpha and IL-1 beta, was given as an IL-1 antagonist. Treatment of guinea pig myelin/CFA immunized rats with sIL-1R for 13 consecutive days significantly delayed the onset of EAE, reduced the severity of paralysis and weight loss, and shortened the duration of disease. Treatment with sIL-1R was most effective in reducing EAE if administered for 15 consecutive days immediately after immunization. Shortened 5-day treatment regimens spanning days 1 to 5, days 6 to 10, or days 11 to 15 after immunization were less effective in reducing EAE. These data suggest that IL-1 may initiate or promote inflammation within the central nervous system. In addition, specifically blocking the biological activity of IL-1 in vivo by soluble receptors may prove beneficial for the treatment of autoimmune or inflammatory diseases.     

IL-1 alpha,innate immunity,and skin carcinogenesis: the effect of constitutive expression of IL-1 alpha in epidermis on chemical carcinogenesis

Tumor promoters such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) are proinflammatory agents, and their mechanism of action in epithelial carcinogenesis has been linked to the release of IL-1 alpha and the induction of chronic inflammation in skin. To test the role of IL-1 alpha and inflammation in models of cutaneous carcinogenesis, we used our previously described FVB/N transgenic mice overexpressing 17-kDa IL-1 alpha in the epidermis under the keratin 14 (K14) promoter. Strikingly, the K14/IL-1 alpha mice were completely resistant to papilloma and carcinoma formation induced by a two-stage DMBA/TPA protocol, while littermate controls developed both tumor types. K14/IL-1 alpha mice crossed with the highly sensitive TG.AC mice, constitutively expressing mutant Ha-Ras, also failed to develop papillomas or carcinomas. When the K14/IL-1 alpha transgene was bred onto a recombinase-activating gene-2-deficient background, the resistance persisted, indicating that innate, but not acquired, mechanisms may be involved in the resistance to the initiation/promotion model. As an alternative approach, a complete carcinogenesis protocol using repetitive application of DMBA alone was applied. Surprisingly, although the IL-1 alpha mice still did not develop papillomas, they did develop carcinomas de novo at an accelerated rate compared with controls. We conclude that constitutive IL-1 alpha expression rendered FVB mice completely resistant to carcinomas that required evolution from prior papillomas, but facilitated carcinomas that did not evolve from papillomas, as in the complete carcinogenesis protocol. Thus, the role of IL-1 alpha and, by extension that of other proinflammatory factors, in epithelial carcinogenesis are more complex than previously appreciated. These mice may provide a mechanism to investigate the validity of these models of human skin tumorigenesis.     

Structural analysis of trisialylated biantennary glycans isolated from mouse serum transferrin. Characterization of the sequence Neu5Gc(alpha 2-3)Gal(beta 1-3)[Neu5Gc(alpha 2-6)]GlcNAc(beta 1-2)Man

Five variants of mouse serum transferrin (mTf, designated mTf-I to mTf-V) with respect to carbohydrate composition have been isolated by DEAE-cellulose chromatography in the following relative percentages: mTf-I: 0.55; mTf-II: 0.79; mTf-III: 71.80; mTf-VI: 21. 90 and mTf-V: 4.96. The primary structures of the major glycans from mTf-III and mTf-IV were determined by methylation analysis and 1H-nuclear magnetic resonance (NMR) spectroscopy. All glycans possessed a common trimannosyl-N,N'-diacetylchitobiose core. From the glycovariant mTf-III two isomers of a conventional biantennary N-acetyllactosamine type were isolated, in which two N-glycolylneuraminic acid (Neu5Gc) residues are linked to galactose either by a (alpha 2-6) or (alpha 2-3) linkage. A subpopulation of this glycovariant contains a fucose residue (alpha 1-6)-linked to GlcNAc-1. The structure of the major glycan found in variant mTf-IV contained an additional Neu5Gc and possessed the following new type of linkage: Neu5Gc(alpha 2-3)Gal(beta 1-3)[Neu5Gc(alpha 2-6)]GlcNAc(beta 1-2 )Man(alpha 1-3). In addition to this glycan, a minor compound contained the same antennae linked to Man(alpha 1-6). In fraction mTf-V, which was found to be very heterogeneous by (1)H NMR analysis, carbohydrate composition and methylation analysis suggested the presence of tri'-antennary glycans sialylated by Neu5Gc alpha-2,6- and alpha-2, 3-linked to the terminal galactose residues. In summary, mTf glycans differed from those of other analyzed mammalian transferrins by the presence of Neu5Gc and by a Neu5Gc(alpha 2-6)GlcNAc linkage in trisialylated biantennary structures, reflecting in mouse liver, a high activity of CMP-Neu5Ac hydroxylase and (alpha 2-6)GlcNAc sialyltransferase.     

Differential biological activities of human interleukin-1 alpha and interleukin-1 beta

We examined the biological effects induced by both human recombinant interleukin-1 alpha (IL-1 alpha) and beta (IL-1 beta) in five different cell types of human, rat and mouse origin. IL-1 alpha and beta preparations were standardized in terms of biological activity in the EL-4/CTLL bioassay and, in parallel, employed to stimulate PGE2 secretion in human fibroblasts, mesangial cells (MC), C57B1/6 mouse MC, DBA/2 mouse macrophages and Sprague Dawley rat MC. In addition, the co-mitogenic effects of IL-1 alpha and beta were determined in freshly prepared Sprague Dawley rat thymocytes. No significant differences in IL-1 alpha and beta concentration dependent PGE2 production were detectable in the different cell types (MC, fibroblasts and macrophages) of human or mouse origin. Incubation of Sprague Dawley rat MC with both IL-1 alpha and beta resulted in a concentration dependent production of PGE2. However, in contrast to mouse or human MC the potency of IL-1 beta to induce PGE2 in Sprague Dawley rat MC was 26-fold higher compared to IL-1 alpha. In addition, the potency of IL-1 beta to enhance co-stimulated proliferation of Sprague Dawley thymocytes was 200-fold higher than that of equal biological activities of IL-1 alpha. When we tested the additive effects on Sprague Dawley cells, increasing IL-1 beta concentrations added to a fixed IL-1 alpha concentration resulted in a cumulative rise in both, PGE2 secretion by MC and thymocyte proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of IL-1 alpha and IL-1 beta on tumorigenicity patterns and invasiveness

In this study, we show that distinct compartmentalization patterns of the IL-1 molecules (IL-1alpha and IL-1beta), in the milieu of tumor cells that produce them, differentially affect the malignant process. Active forms of IL-1, namely precursor IL-1alpha (pIL-1alpha), mature IL-1beta (mIL-1beta), and mIL-1beta fused to a signal sequence (ssIL-1beta), were transfected into an established fibrosarcoma cell line, and tumorigenicity and antitumor immunity were assessed. Cell lines transfected with pIL-1alpha, which expresses IL-1alpha on the membrane, fail to develop local tumors and activate antitumor effector mechanisms, such as CTLs, NK cells, and high levels of IFN-gamma production. Cells transfected with secretable IL-1beta (mIL-1beta and ssIL-1beta) were more aggressive than wild-type and mock-transfected tumor cells; ssIL-1beta transfectants even exhibited metastatic tumors in the lungs of mice after i.v. inoculation (experimental metastasis). In IL-1beta tumors, increased vascularity patterns were observed. No detectable antitumor effector mechanisms were observed in spleens of mice injected with IL-1beta transfectants, mock-transfected or wild-type fibrosarcoma cells. Moreover, in spleens of mice injected with IL-1beta transfectants, suppression of polyclonal mitogenic responses (proliferation, IFN-gamma and IL-2 production) to Con A was observed, suggesting the development of general anergy. Histologically, infiltrating mononuclear cells penetrating the tumor were seen at pIL-1alpha tumor sites, whereas in mIL-1beta and ssIL-1beta tumor sites such infiltrating cells do not penetrate inside the tumor. This is, to our knowledge, the first report on differential, nonredundant, in vivo effects of IL-1alpha and IL-1beta in malignant processes; IL-1alpha reduces tumorigenicity by inducing antitumor immunity, whereas IL-1beta promotes invasiveness, including tumor angiogenesis, and also induces immune suppression in the host.     

Identification of disialic acid-containing glycoproteins in mouse serum: a novel modification of immunoglobulin light chains, vitronectin, and plasminogen

Serum glycoproteins are involved in various biologic activities, such as the removal of exogenous antigens, fibrinolysis, and metal transport. Some of them are also useful markers of inflammation and disease. Although the amount of sialic acid increases following inflammation, little attention has been paid to the presence of linkage-specific epitopes in serum, especially the alpha2,8-linkage. In a previous study, we demonstrated that four components in mouse serum contain alpha2,8-linked disialic acid (diSia), based on immunoreactivity with monoclonal antibody 2-4B, which is specific to N-glycolylneuraminic acid (Neu5Gc)alpha2-->(8Neu5Gc alpha2-->)(n-1), n > or = 2 [Yasukawa et al., (2005) Glycobiology, 15, 827-837]. In this study, we purified three components, 30-, 70-, and 120-kDa gp, and identified them as an immunoglobulin (Ig) light chain, vitronectin, and plasminogen, respectively, using matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy analyses. Modifications of these proteins with alpha2,8-linked diSia were chemically confirmed by fluorometric C7/C9 analyses and mild acid hydrolysates-fluorometric anion-exchange chromatography analyses. We also demonstrated that the IgG, IgM, and IgE light chains are commonly modified with alpha2,8-linked diSia. In addition, both mouse and rat vitronectin contained diSia, and the amount of disialylation in vitronectin dramatically decreased after hepatectomy. These results indicate that a novel diSia modification of serum glycoproteins is biologically important for immunologic events and fibrinolysis.     

Production of interleukin-1 alpha and interleukin-2 by mononuclear cells from children with growth delay in relation to the degree of growth hormone deficiency: effects of substitutive treatment

Interleukin-1 alpha (IL-1 alpha) and interleukin-2 (IL-2) levels were measured by radioimmunoassay in samples of conditioned medium from mononuclear cells taken from 20 normal subjects (14 adults ranging in age from 20 to 45 years and 6 children ranging in age from 3 to 11 years) and from 49 children with growth delay. Cultures were performed with 10(6) cells/ml in medium containing 1% normal human serum and 4.8 g/l phytohemagglutinin M. The incubation was performed for 48 h in an atmosphere containing 5% CO2. In normal subjects, the production of IL-1 alpha was 38.5 +/- 9.8 fmol/ml of conditioned medium (mean +/- SEM) in 14 adults and 41.6 +/- 3.0 fmol/ml in 6 children. The production of IL-2 was 46.9 +/- 6.5 and 57.3 +/- 10.5 fmol/ml, respectively. In the 16 patients with growth hormone (GH) deficiency studied before treatment, the production of ILs was significantly decreased in relation to the degree of deficiency. We observed a positive correlation between the production of IL-1 alpha and the values of insulin-like growth factor I but not with serum GH values. IL-1 alpha production was normalized after 15 days of substitutive GH therapy and IL-2 was normalized after 3 months of therapy. In 10 other patients with GH deficiency (4 with total and 6 with partial isolated GH deficiency) studied after long-term GH treatment (5 months or more), the mean of IL production was not significantly different from that of GH-deficient children treated for 3 months.(ABSTRACT TRUNCATED AT 250 WORDS)     

IL-10, but not IL-4, suppresses infection-stimulated bone resorption in vivo

Periapical bone resorption occurs following infection of the dental pulp and is mediated mainly by IL-1alpha in the murine model. The production and activity of IL-1alpha is modulated by a network of regulatory cytokines, including those produced by Th1 (pro-inflammatory) and Th2 (anti-inflammatory) subset T cells. This study was designed to assess the functional role of the Th2-type cytokines IL-4 and IL-10 in infection-stimulated bone resorption in vivo. The dental pulps of the first molars were exposed and infected with a mixture of four common endodontic pathogens, and bone destruction was determined by micro-computed tomography at sacrifice on day 21. The results demonstrate that IL-10(-/-) mice had significantly greater infection-stimulated bone resorption in vivo compared with wild-type mice (p < 0.001), whereas IL-4(-/-) exhibited no increased resorption. IL-10(-/-) had markedly elevated IL-1alpha production within periapical inflammatory tissues (>10-fold) compared with wild type (p < 0.01), whereas IL-4(-/-) exhibited decreased IL-1alpha production (p < 0.05). IL-10 also suppressed IL-1alpha production by macrophages in a dose-dependent fashion in vitro, whereas IL-4 had weak and variable effects. We conclude that IL-10, but not IL-4, is an important endogenous suppressor of infection-stimulated bone resorption in vivo, likely acting via inhibition of IL-1alpha expression.     

alpha 2,3-Sialylation of terminal GalNAc beta 1-3Gal determinants by ST3Gal II reveals the multifunctionality of the enzyme. The resulting Neu5Ac alpha 2-3GalNAc linkage is resistant to sialidases from Newcastle disease virus and Streptococcus pneumoniae

Enzymatic alpha 2,3-sialylation of GalNAc has not been described previously, although some glycoconjugates containing alpha 2,3-sialylated GalNAc residues have been reported. In the present experiments, recombinant soluble alpha 2,3-sialyltransferase ST3Gal II efficiently sialylated the X(2) pentasaccharide GalNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc, globo-N-tetraose GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc, and the disaccharide GalNAc beta 1-3Gal in vitro. The purified products were identified as Neu5Ac alpha 2-3GalNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc, Neu5Ac alpha 2-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc, and Neu5Ac alpha 2-3GalNAc beta 1-3Gal, respectively, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, enzymatic degradations, and one- and two-dimensional NMR-spectroscopy. In particular, the presence of the Neu5Ac alpha 2-3GalNAc linkage was firmly established in all three products by a long range correlation between Neu5Ac C2 and GalNAc H3 in heteronuclear multiple bond correlation spectra. Collectively, the data describe the first successful sialyltransfer reactions to the 3-position of GalNAc in any acceptor. Previously, ST3Gal II has been shown to transfer to the Gal beta 1-3GalNAc determinant. Consequently, the present data show that the enzyme is multifunctional, and could be renamed ST3Gal(NAc) II. In contrast to ST3Gal II, ST3Gal III did not transfer to the X(2) pentasaccharide. The Neu5Ac alpha 2-3GalNAc linkage of sialyl X(2) was cleaved by sialidases from Arthrobacter ureafaciens and Clostridium perfringens, but resisted the action of sialidases from Newcastle disease virus and Streptococcus pneumoniae. Therefore, the latter two enzymes cannot be used to differentiate between Neu5Ac alpha 2-3GalNAc and Neu5Ac alpha 2-6GalNAc linkages, as has been assumed previously.     

The role of MIP-1 alpha in the development of systemic inflammatory response and organ injury following trauma hemorrhage

Although MIP-1alpha is an important chemokine in the recruitment of inflammatory cells, it remains unknown whether MIP-1alpha plays any role in the development of systemic inflammatory response following trauma-hemorrhage (T-H). C57BL/6J wild type (WT) and MIP-1alpha-deficient (KO) mice were used either as control, subjected to sham operation (cannulation or laparotomy only or cannulation plus laparotomy) or T-H (midline laparotomy, mean blood pressure 35 +/- 5 mmHg for 90 min, followed by resuscitation) and sacrificed 2 h thereafter. A marked increase in serum alpha-glutathione transferase, TNF-alpha, IL-6, IL-10, MCP-1, and MIP-1alpha and Kupffer cell cytokine production was observed in WT T-H mice compared with shams or control. In addition lung and liver tissue edema and neutrophil infiltration (myeloperoxidase (MPO) content) was also increased following T-H in WT animals. These inflammatory markers were markedly attenuated in the MIP-1alpha KO mice following T-H. Furthermore, compared with 2 h, MPO activities at 24 and 48 h after T-H declined steadily in both WT and KO mice. However, normalization of MPO activities to sham levels within 24 h was seen in KO mice but not in WT mice. Thus, MIP-1alpha plays an important role in mediating the acute inflammatory response following T-H. In the absence of MIP-1alpha, acute inflammatory responses were attenuated; rapidly recovered and less remote organ injury was noted following T-H. Thus, interventions that reduce MIP-1alpha levels following T-H should be useful in decreasing the deleterious inflammatory consequence of trauma.     

IL-1alpha but not IL-1beta-induced prostaglandin synthesis is inhibited by corticotropin-releasing factor

Interleukin (IL)-1alpha and IL-1beta share low amino acid homology, but exhibit a very similar array of biological activities. The authors previously showed negative regulation of IL-1alpha-induced prostaglandin (PG) production by corticotropin releasing factor (CRF). In this study, the authors compared the effect of CRF on IL-1alpha- and IL-1beta-induced PG synthesis. IL-1alpha (100 U/ml) increased prostacyclin (PGI2) (measured as 6-keto PGF1alpha[6K]) synthesis in endothelial cells and the production of PGE2in fibroblasts. The PG response to IL-1alpha was suppressed by simultaneous exposure to CRF (2.5x10(-11)-2.5x10(-8) M) in both cell types. IL-1alpha enhanced both phospholipase A2(PLA2) and prostaglandin H synthase (PGHS) activities, and the two effects were completely abrogated by CRF. IL- 1beta (100 U/ml) was as active as IL-1alpha in triggering release of PGI2 from endothelial cells and PGE2 from fibroblasts. However, CRF (2.5x10(-11)-2.5x10(-8) M) failed to alter the IL-1beta-induced PG synthesis in both cell types. Following IL-1beta PGHS activity, and to a lesser extent PLA2 activity, were enhanced, however CRF only inhibited PGHS and not PLA2 activity. It is concluded that although IL-1alpha and IL-1beta usually produce similar biological effects, here they seem to act via different mechanisms. The different regulation of IL-1alpha and IL-1beta pro-inflammatory activities by CRF may attribute special precision and specificity to the neuroendocrine-immune control of inflammatory processes.     

Membrane-associated IL-1 contributes to chronic synovitis and cartilage destruction in human IL-1 alpha transgenic mice

IL-1 molecules are encoded by two distinct genes, IL-1alpha and IL-1beta. Both isoforms possess essentially identical activities and potencies, whereas IL-1alpha, in contrast to IL-1beta, is known to act as a membrane-associated IL-1 (MA-IL-1) and plays an important role in a variety of inflammatory situations. The transgenic (Tg) mouse line (Tg1706), which was generated in our laboratory, overexpresses human IL-1alpha (hIL-1alpha) and exhibits a severe arthritic phenotype characterized by autonomous synovial proliferation with subsequent cartilage destruction. Because the transgene encoded Lys(64) to Ala(271) of the hIL-1alpha amino acid sequence, Tg mice may overproduce MA-IL-1 as well as soluble IL-1alpha. The present study investigated whether MA-IL-1 contributes to synovial proliferation and cartilage destruction in the development of arthritis. Flow cytometric analysis revealed that both macrophage-like and fibroblast-like synoviocytes constitutively produce MA-IL-1. D10 cell proliferation assay revealed MA-IL-1 bioactivity of paraformaldehyde-fixed synoviocytes and the further induction of endogenous mouse MA-IL-1 via autocrine mechanisms. MA-IL-1 expressed on synoviocytes triggered synoviocyte self-proliferation through cell-to-cell (i.e., juxtacrine) interactions and also promoted proteoglycan release from the cartilage matrix in chondrocyte monolayer culture. Interestingly, the severity of arthritis was significantly correlated with MA-IL-1 activity rather than with soluble IL-1alpha activity or concentration of serum hIL-1alpha. Moreover, when the Tg1706 line was compared with the Tg101 line, which selectively overexpresses the 17-kDa mature hIL-1alpha, the severity of arthritis was significantly higher in the Tg1706 line than in the Tg101 line. These results suggest that MA-IL-1 contributes to synoviocyte self-proliferation and subsequent cartilage destruction in inflammatory joint disease such as rheumatoid arthritis.     

Detection of the Neu5 Ac (alpha 2,3) Gal (beta 1,4) GlcNAc sequence with the leukoagglutinin from Maackia amurensis: light and electron microscopic demonstration of differential tissue expression of terminal sialic acid in alpha 2,3- and alpha 2,6-linkage

The Maackia amurensis leukoagglutinin has been shown to react specifically with the Neu5Ac (alpha 2,3) Gal sequence of asparagine-linked complex type oligosaccharides. We report here the preparation of Maackia amurensis lectin-gold complexes and their application for light and electron microscopic detection of the Neu5 Ac (alpha 2,3) Gal sequence in various tissues. The use of the lectin directly gold labeled was superior to a two-step cytochemical affinity technique using a fetuin-gold complex. The Maackia amurensis lectin-gold staining was inhibited by pre-incubation of the lectin-gold complexes with 50 mM alpha 2,3 sialyllactose, whereas alpha 2,6 sialyllactose up to concentrations of 1 M had no effect, thus demonstrating the high specificity of the histochemical staining. In addition to N-glycanase-sensitive asparagine-linked oligosaccharides, beta-elimination-sensitive serine/threonine-linked oligosaccharides could be detected. Data are presented which show that cellular staining patterns obtained with Maackia amurensis lectin-gold complexes may differ from those with elderberry bark lectin-gold, which detects the Neu5 Ac (alpha 2,6) Gal/GalN Ac sequence. Electron microscopic double labeling for direct study of the differential distribution of the Neu5 Ac (alpha 2,3) Gal and Neu5 Ac (alpha 2,6) Gal sequences is reported. Therefore, the availability of two sialic acid binding lectins with different linkage specificity for histochemistry provides the first opportunity to study tissue and cell type expression of these terminal sequences of glycoproteins.     

Circulating FGF-23 is regulated by 1alpha,25-dihydroxyvitamin D3 and phosphorus in vivo

Fibroblast growth factor-23 (FGF-23), a novel phosphate-regulating factor, was elevated in hypophosphatemic patients with X-linked hypophosphatemic rickets/osteomalacia and also in patients with chronic kidney disease. These observations suggested the pathophysiological importance of FGF-23 on phosphate homeostasis. However, regulation of FGF-23 production is still unclear. We investigated effects of both dietary phosphorus and 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) on circulating FGF-23 in vivo Administration of. 1alpha,25(OH)(2)D(3) dose-dependently increased serum FGF-23 in thyroparathyroidectomized rats without correlating with serum inorganic phosphorus or serum parathyroid hormone. On the other hand, vitamin D receptor null mice had very low serum FGF-23 and did not respond to the 1alpha,25(OH)(2)D(3) administration. These observations suggested 1alpha,25(OH)(2)D(3) directly or indirectly regulates circulating FGF-23. Serum FGF-23 had a strong correlation with serum inorganic phosphorus controlled by dietary phosphorus in 5/6 nephrectomized rats. High phosphate diet elicited a 5-fold increase in serum FGF-23 compared with sham-operated rats, whereas serum FGF-23 did not correlate with serum calcium or serum creatinine in 5/6 nephrectomized rats. Administration of 1alpha,25-dihydroxyvitamin D(3) also elicited a severalfold increase in serum FGF-23 in the uremic rats. Taken together, this shows that both serum phosphorus and 1alpha,25(OH)(2)D(3) regulate circulating FGF-23 independent of each other. Therefore, we proposed there was a feedback loop existing among serum phosphorus, 1alpha,25(OH)(2)D(3), and FGF-23, in which the novel phosphate-regulating bone-kidney axis integrated with the parathyroid hormone-vitamin D(3) axis in regulating phosphate homeostasis.     

Detection of interleukin 1 alpha and 1 beta in rabbit tissues during endotoxemia using sensitive radioimmunoassays.

Interleukin 1 (IL-1) is a primary mediator of a wide variety of immunologic and inflammatory responses, including reactions to microbial infections. To study this cytokine in an animal model, we have developed specific and sensitive radioimmunoassays for the quantitation of rabbit IL-1 alpha and IL-1 beta. The sensitivity (limit of detection at 95% confidence level) of our assay for IL-1 alpha and 1 beta was 20-40 and 40-80 pg/ml, respectively. Recovery of IL-1 from tissues ranged from 75 to 107%, with a mean of 95% for IL-1 alpha and 89% (range 19-98) for IL-1 beta. We employed these assays in in vivo and in vitro studies. In an in vivo model, we measured the amount of rabbit IL-1 alpha and 1 beta protein present in brain, kidney, liver, lung, muscle, and spleen at various times after the injection of endotoxin. IL-1 was found in all tissues studied but largely in the spleen; IL-1 levels were transient, reaching peak levels by 4 h after injection of endotoxin and rapidly decreasing to low levels by 24 h. In similar in vitro studies, IL-1 alpha levels reached peak elevation 6 h after addition of endotoxin, whereas IL-1 beta was maximal at 24 h. IL-1 alpha was detected in all tissues; IL-1 beta was observed primarily in lung, kidney, and spleen. These studies establish the presence of IL-1 in various tissues during endotoxemia.     

Increased production of IL-1alpha and TNF-alpha in lipopolysaccharide-stimulated blood from obese patients with non-alcoholic fatty liver disease

Enhanced pro-inflammatory cytokine production is considered a pathogenic factor in non-alcoholic fatty liver disease (NAFLD). Peripheral blood production of interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha) was studied in relation to the severity of histological changes of the liver in obese NAFLD patients. Basal levels in serum and production of IL-1alpha and TNF-alpha in peripheral blood cell cultures after stimulation with lipopolysaccharide (enzyme-linked immunoabsorbent assays) were measured in 11 patients with steatosis and 15 with steatohepatitis, who underwent gastrectomy with a gastro-jejunal anastomosis in roux and Y, and in 9 controls who underwent anti-reflux surgery. Production of IL-1alpha and TNF-alpha was 122 and 67% higher in patients with steatosis than control values, respectively. In patients with steatohepatitis, IL-1alpha production was 300 and 80% higher and that of TNF-alpha 110 and 26% higher, as compared with controls and steatosis patients, respectively. Production of IL-1alpha was positively correlated with that of TNF-alpha (r=0.78, p<0.0001). IL-1alpha and TNF-alpha production were both positively correlated with the degree of steatosis (r=0.68, p<0.001 and r=0.74, p<0.0001) and steatohepatitis (r=0.77 and r=0.75, p<0.0001) at liver biopsy, and with the homeostasis model assessment index (r=0.73, p<0.0001 and r=0.63, p<0.01), respectively. Basal serum IL-1alpha and TNF-alpha levels were comparable in the three groups studied. It is concluded that elevated production of IL-1alpha and TNF-alpha by in vitro stimulated whole blood cell cultures occurs in NAFLD obese patients, which might play a pathophysiological role upon inflammatory leukocyte infiltration of the liver.     

Interleukin-1alpha production in human corneal scars

We studied IL-1alpha level in corneal scars with/without neo-vascularization. A total of 27 patients underwent grafting for corneal scar. Recipients were grouped according to number of vascularized quadrants (0 to IV/IV): none (n = 12), one (n = 5), two (n = 4) and four (n = 6). Recipient corneas were collected during surgery and IL-1alpha measured by immunoassay. Controls were donor corneas unsuitable for transplantation. Graft rejection rate was calculated for each group. Mean IL-1alpha concentration in corneal scars was 6 +/- 3.93 pg/mm3; significantly higher as compared to controls (1.25 +/- 2.03 pg/mm3). IL-1alpha correlated well with amount of blood vessels, except in IV/IV scars: 5.17 +/- 3.65 pg/mm3 for 0/IV; 8.02 +/- 2.51 pg/mm3 for I/IV; 8.27 +/- 3.62 pg/mm3 for II/IV and 4.47 +/- 5.03 pg/mm3 for IV/IV corneal scars. Vascularization of corneal scar is associated with increased IL-1alpha level (in all but highly vascularized scars), indicating that IL-1alpha promotes early stages of vascularization. Graft rejection rate increases in patients with higher vascularization, independently of IL-1alpha level.