Deposition of complement protein C3b on mixed self-assembled monolayers carrying surface hydroxyl and methyl groups studied by surface plasmon resonance

Since complement activation is recognized as a common response of the host defense system when an artificial medical device is applied to a patient, great effort has been devoted to studies on the interaction of the complement system with artificial materials. However, some uncertainties remain, partially because of the lack of well characterized surfaces and suitable analytic methods for study of the surface phenomena that occur on artificial materials under physiologic conditions. In this study, we employed self-assembled monolayers (SAMs) and the surface plasmon resonance (SPR) technique to study interactions of the serum complement with well characterized surfaces. Self-assembled monolayers carrying various concentrations of hydroxyl groups were prepared using 11-mercapto-1-undecanol (C11-OH) and one of n-nonanethiol, n-dodecanethiol, and n-hexadecanethiol. The amount of NHS deposition on the SAMs increased with increasing C11-OH content of the SAMs, and the amount of anti-C3b antibody immobilization formed on the NHS deposition layers increased with increasing C11-OH content of the SAMs. These results clearly demonstrate that a large amount of C3b, produced through the activation of the complement system, binds covalently to and is adsorbed by hydroxyl-group-rich surfaces. The combination of SAMs and the SPR technique is suitable for studying the interaction of the complement system with solid surfaces, and the results should give basic information needed for a rational design of biocompatible surfaces on synthetic materials.     

Mevalonic acidemia: First case of Japan

Summary Mevalonic acidemia is a rare metabolic disorder due to mevalonate kinase deficiency which affects the biosynthesis of cholesterol and nonsterol isoprenes. We report the first case of Japan. The clinical course is characterized by intrauterine growth retardation, postnatal growth failure, intractable diarrhea, liver dysfunctions and death at three months of age. Dysmorphic features including triangular face, protrusion of forehead, hypertelorism, low set ears and micrognathism were noted. High mevalonic acid level was found by GC/MS.     

Evidence for increased expression of eotaxin and monocyte chemotactic protein-4 in atopic dermatitis

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease associated with tissue eosinophilia and the activation of T lymphocytes. The novel eosinophil chemoattractants, eotaxin and monocyte chemotactic protein (MCP)-4, are up-regulated at sites of allergic inflammation, yet their contribution to the pathophysiologic mechanisms of AD remains to be determined. OBJECTIVE: We sought to investigate the expression of eotaxin and MCP-4 in acute and chronic lesions from patients with AD and to determine their relationship to the numbers of resident inflammatory cells. METHODS: With use of in situ hybridization, the expression of eotaxin and MCP-4 messenger RNA (mRNA) in skin biopsy specimens from patients with acute and chronic AD skin lesions was compared with that of uninvolved skin from these patients and skin from healthy volunteers. RESULTS: There was a constitutive expression of eotaxin and MCP-4 mRNA in skin biopsy specimens from healthy subjects. Positive signal for chemokine mRNA was observed both within the epidermis and inflammatory cells (macrophages, eosinophils, and T cells) of the subepidermis in AD skin lesions. Within the subepithelium acute and chronic skin lesions exhibited a significant increase in the numbers of eotaxin and MCP-4 mRNA-positive cells compared with uninvolved skin (P <.01), whereas the numbers of eotaxin and MCP-4 mRNA-positive cells were significantly higher in chronic AD compared with acute AD skin lesions (P <.005, P <.001, respectively). Correlations were observed between the expression of eotaxin and MCP-4 mRNA and the presence of eosinophils and macrophages, respectively, in AD lesions (r(2) = 0.84, r(2) = 0.94). CONCLUSION: There is an increased expression of eotaxin and MCP-4 in acute and chronic lesions, suggesting that these chemotactic factors play a major role in the pathophysiologic mechanisms of AD.     

Protease inhibitors (gebexate mesylate and ulinastatin) stimulate intracellular chemiluminescence in human neutrophils.

The effect of protease inhibitors on the intracellular production of free radicals was investigated by measuring chemiluminescence (CL) elicited from phagocytosed luminol-bound microspheres (Lumispheres) in human neutrophils stimulated with formylmethionyl-leucyl-phenylalanine (fMLP), interleukin-8 (IL-8), phorbol 12-myristate 13-acetate, or diacylglycerol. Both gabexate mesylate (Foy) and ulinastatin (Miraclid), urinary trypsin inhibitor, increased intracellular CL in a dose dependent manner. Compared to control buffer without protease inhibitor, gabexate mesylate (322 micrograms/ml) caused about a 10-fold increase in intracellular CL in stimulated neutrophils, and ulinastatin (3100 U/ml) a twofold increase in neutrophils stimulated with fMLP or IL-8. When the protease inhibitors were added to the cell suspension after the phagocytosis of lumispheres, CL responses rapidly increased again to the level which was observed when both protease inhibitors and neutrophil stimulants were incubated simultaneously. In contrast, extracellular release of oxygen metabolites from stimulated neutrophils, assayed by a conventional measurement of luminol-dependent CL, was reduced by the protease inhibitors in a dose dependent fashion. When luminol-unbound microspheres were incubated with neutrophils stimulated by fMLP in luminol solution, extracellular CL was almost completely inhibited by gabexate mesylate. These results indicate that the protease inhibitors enhance the generation of intracellular CL and suppress the extracellular release of free radicals.     

Down-regulation of antigen-specific antibody production by TCR antagonist peptides in vivo

The efficacy of TCR antagonist peptides in inhibition of antigen-specific antibody production and T cell responses in vivo was evaluated. Among amino acid-substituted analogs of a peptide corresponding to residues 119 - 133 of bovine beta-lactoglobulin (p119 - 133), pR124Q and pD129S, prepared by substitution of Gln and Ser for Arg(124) and Asp(129), respectively, have been shown to display TCR antagonist activity for three out of four distinct p119 - 133-specific T cell clones and for polyclonal T cells derived from p119 - 133-immunized C57BL / 6 mice. Both pD129S and pR124Q inhibited in vivo priming and subsequent activation of T cells by p119 - 133 when co-injected with p119 - 133 into mice, as shown by the decreased proliferation of T cells in response to p119-133 in vitro. pD129S significantly inhibited production of anti-p119 - 113 antibodies of IgG1, IgG2b and IgE isotype in vivo when co-injected into mice together with p119 - 133 at the time of the first immunization. However, pR124Q was totally ineffective in inhibition of the antibody responses. Anti-p119 - 133 antibodies from p119 - 133-immunized mice could bind to pR124Q but not to pD129S, suggesting that the difference in cross-reactivity is responsible for the different effect of these two peptides on specific antibody production. Our findings demonstrate that a single TCR antagonist peptide can inhibit antigen-specific polyclonal antibody production when this antagonist peptide does not cross-react with the antibody elicited in response to an antigenic peptide.     

Endogenous peroxidase activity in primary culture of human thyroid cells. Localization and measurement

Intracellular localization of and an assay method for endogenous peroxidase (PO) activity were studied using primary culture of thyroid cells obtained from patients with hyperthyroidism. PO activity was visualized by cytochemical reaction and was located mainly in perinuclear cisternae and rough endoplasmic reticulum. With increased culture time, the number of cells showing positive PO activity and amount of the enzyme reaction product in individual cells showed a parallel decrease. For measurement of PO activity, cultured thyroid cells were frozen and thawed and then incubated with citric acid buffer solution containing o-phenylenediamine (opd) and hydrogen peroxide. After incubation, the optical density (OD) of the solution colorized by endogenous peroxidase was measured at 405 nm using a microplate reader. About 1 X 10(4) cells were sufficient for assay of PO activity. Using the above method to assay PO activity and sandwich enzyme immunoassay for thyroglobulin (TG), chronological changes in the PO activity and TG concentration in the culture medium were examined. Although the cells showed no decrease in number, PO activity and TG concentration decreased chronologically. When the ratio of PO activity to TG concentration was calculated, in 3 cases the ratio was almost constant, and in the remaining two, it decreased chronologically. The present biochemical method thus seems useful for determining peroxidase activity of cultured thyroid en masse.     

Uneven distribution of enzymatic alterations on the liver cell surface in experimental extrahepatic cholestasis of rat.

The effect of bile duct ligation on enzyme activities in the subfractions of the rat liver plasma membrane was investigated. Two subfractions were isolated from the rat liver plasma membrane by homogenization and subsequent centrifugation in a discontinuous sucrose gradient. The light subfraction contained fragments of the bile canalicular surface and the heavy fraction contained fragments of the sinusoidal and lateral surfaces of the hepatocyte. Bile duct ligation decreased NaK ATPase and Mg-ATPase activity in the light subfraction, whereas it had no significant effect on these enzyme activities in the heavy fraction. Leucyl-β-naphthylamidase activity was reduced and alkaline phosphatase activity was increased in both subfractions. These facts suggested that ligation of the bile duct led to loss of the secretory polarity of the liver cell. The in vitro effects of some bile acids on the membranebound enzymes in the light subfraction were investigated, and a possible involvement of chenodeoxycholic acid in the alteration of enzyme activities in the bile canalicular membrane was suggested.     

IgE antibody to fish gelatin (type I collagen) in patients with fish allergy

BACKGROUND: Most children with anaphylaxis to measles, mumps, and rubella vaccines had shown sensitivity to bovine gelatin that was included in the vaccines. Recently, it was found that bovine type I collagen, which is the main content in the gelatin, is a major allergen in bovine gelatin allergy. Fish meat and skin also contain type I collagen. OBJECTIVE: The present study was designed to investigate IgE antibody to fish gelatin in children with fish allergy. METHODS: Serum samples were taken from patients in 3 groups: (1) 10 patients with fish allergy and specific IgE to fish meat; (2) two patients with allergies to both fish meat and bovine gelatin and specific IgE to fish meat and bovine gelatin; and (3) 15 patients with atopic dermatitis and specific IgE to fish meat. Various fish gelatins (type I collagen) were prepared from fish skin. IgE antibody to fish gelatin was analyzed by using ELISA and immunoblotting. RESULTS: Of 10 patients with fish allergy, 3 had specific IgE to fish gelatin. Of two patients with fish allergy and bovine gelatin allergy, all had specific IgE to fish gelatin. Of 15 patients with atopic dermatitis and specific IgE to fish meat, 5 had specific IgE to fish gelatin. Furthermore, IgE from pooled serum of the patients reacted with both the alpha1 and alpha2 chains of fish type I collagen in immunoblots. There is cross-reactivity among gelatins from various fishes, but there is little cross-reactivity between fish and bovine gelatins. CONCLUSION: Some fish-sensitive patients possessed IgE antibody to fish gelatin. Fish gelatin (type I collagen) might be an allergen in subjects with fish allergy.     

Grading score system: A method for evaluation of the degree of senescence in Senescence Accelerated Mouse (SAM)

For evaluation of the degree of senescence in SAM-P, accelerated senescence prone mouse, formerly called SAM or prone series or P-series, consisting of SAM-P/1, SAM-P/2, SAM-P/3 and SAM-P/4 corresponding to P-1, P-2, P-3 and P-4 series, respectively, in the previous reports, and in SAM-R, accelerated senescence resistant mouse, formerly called resistant series or R-series, consisting of SAM-R/1, SAM-R/2 and SAM-R/3 corresponding to R-1, R-2 and R-3 series, respectively, in the previous reports, the grading score system was adopted. The items to be examined in this system include 11 categories selected from the clinical signs and gross lesions considered to be associated with the aging process. The degree of the senescence in each category was graded from 0 to 4 according to the detailed criteria devised in our laboratory. After 8 months of age each mouse was examined every 4 months, and some of the mice were examined after 2 months of age.In almost all categories, the grading score and incidence began to increase from 4 or 6 months of age and continued to increase with advancing age in both SAM-P and SAM-R. The increase, however, was more marked in SAM-P than in SAM-R. The slow but steady increase in the SAM-R levelled out at 24 months of age and was comparable to that of 12 months of age in SAM-P. In both SAM-P/1 at 8 months of age and SAM-R/2 at 12 months of age, there was a significant reverse correlation between total score of this grading score system and length of residual life after examination.Systematic and extensive studies using the grading score system showed that if the validity of the system is, based on “irreversibility” and “universality” of the changes in