Pathological role of excessive DNA as a trigger of keratinocyte proliferation in psoriasis.

Psoriasis is characterized by excessive growth and aberrant differentiation of epidermal keratinocytes due to persistent inflammation. However, the underlying mechanism that triggers immune activation in psoriasis is not clear. In this study, we explored excessive DNA as a potential trigger of psoriasis using cultured human keratinocytes and psoriatic skin tissues. We demonstrated that human genomic DNA fragments induced tumour necrosis factor (TNF)-α expression, hyperproliferation and over-expression of heparin-binding epidermal-like growth factor (HB-EGF) and transforming growth factor (TGF)-α, accompanied by defective expression of keratins 1 and 10 in cultured normal human epidermal keratinocytes, which have a similar phenotype to that of keratinocytes in psoriatic skin lesions. In psoriatic lesions, we found high levels of double-stranded (ds)DNA fragments, accompanying keratinocytes expressing Ki-67, HB-EGF and TNF-α. In addition, we showed that 1,25-dihydroxyvitamin D3 inhibited genomic DNA fragment-induced TNFA and interleukin-1ß (IFNB) expression in human keratinocytes, and an intact function of cathelicidin anti-microbial peptide (CAMP) was required for this effect. These results suggest that excessive dsDNA fragments probably act as a risk factor for immune activation in psoriasis, and the active form of vitamin D can prevent genomic DNA-mediated skin inflammation via CAMP.

Periostin levels correlate with disease severity and chronicity in patients with atopic dermatitis.

BACKGROUND: Recent findings indicate that periostin, an extracellular matrix protein induced by T helper 2 cytokines, plays a critical role in the pathogenesis of atopic dermatitis (AD). OBJECTIVES: To determine whether serum periostin level is associated with clinical phenotype in adult patients with AD. METHODS: An enzyme-linked immunosorbent assay was performed to determine serum periostin levels in 257 adult patients with AD, 66 patients with psoriasis vulgaris (PV) as a disease control and 25 healthy controls. Serum periostin levels were analysed together with clinical characteristics and laboratory parameters, including thymus and activation-regulated chemokine (TARC), lactate dehydrogenase (LDH), blood eosinophil count and total IgE. Immunohistochemical analysis evaluated the expression of periostin in association with various clinical phenotypes of AD. The effect of treatment on serum periostin level was also assessed. RESULTS: Serum periostin was significantly higher in patients with AD than in patients with PV and healthy controls. Periostin level was found to be positively correlated with disease severity, TARC level, LDH level and eosinophil count, but not with IgE level. Higher serum periostin level was observed in patients with extrinsic AD compared with patients with intrinsic AD; the positive correlation of disease severity disappeared in patients with intrinsic AD. Robust expression of periostin was detected in the dermis of patients with AD with erythroderma, lichenification and, to a lesser extent, scaly erythema. Serial measurement of serum periostin revealed decreased levels of periostin after treatment for AD. CONCLUSIONS: Periostin may play a critical role in disease severity and chronicity in the pathogenesis of AD.

Two cases of wheat-dependent anaphylaxis induced by aspirin administration but not by exercise.

BACKGROUND: Aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) have been reported to enhance the symptoms of wheat-dependent exercise-induced anaphylaxis (WDEIA). In contrast to many reports on WDEIA, there have been only a few reports of wheat-dependent aspirin-induced anaphylaxis not induced by the combination of wheat and exercise. METHODS: Two patients with wheat-dependent anaphylaxis underwent provocation tests to clarify the cause of their symptoms. Skin-prick testing (SPT) was also performed with and without administration of aspirin. Specific IgE antibody to wheat, gluten, and omega-5 gliadin were examined. RESULTS: In the provocation tests, anaphylactic reactions were not induced by wheat or aspirin alone or by the combination of wheat and exercise, but were induced by the combination of wheat and aspirin. An increase in the blood histamine level was detected after provocation in both patients. Pretreatment with aspirin enhanced the SPT reactions to wheat and gluten in both patients. Specific IgE antibodies to wheat and gluten were expressed in the serum of both patients, and specific omega-5 gliadin IgE antibody was detected in the serum of one patient. CONCLUSIONS: We present two cases of specific wheat-dependent anaphylaxis induced by aspirin but not by exercise. We suggest that pretreatment with aspirin under controlled conditions is useful to confirm the diagnosis of food allergy when a challenge test with food alone or with food and exercise fails to induce positive reactions.

High frequency of low-level microsatellite instability in early colorectal cancer.

Molecular events in early colorectal cancers (CRCs) have not been well elucidated because of the low incidence of early CRCs in clinical practice. Therefore, we studied 104 sporadic early CRCs with invasion limited to submucosa compared with 116 advanced CRCs. Loss of heterozygosity as well as microsatellite instability (MSI) status was examined. A significantly high frequency of low-level MSI (MSI-L) phenotype was detected in early CRCs (51.0%) compared with advanced CRCs (25.9%; P = 0.0001). In early and advanced CRCs, samples with MSI-L phenotype differed from microsatellite stable (MSS) phenotype with respect to loss of heterozygosity at 1p32 and 8p12-22. MSI-L is a frequent genetic event in early CRCs and may be a novel pathway in colorectal carcinogenesis distinct from both MSI-H and MSS.

The major plasma kallikrein inhibitor of guinea pig plasma.

A plasma kallikrein inhibitor in guinea pig plasma (KIP) was purified to homogeneity. KIP is a single chain protein and the apparent molecular weight is estimated to be 59,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In amino acid composition, KIP is similar to human and mouse alpha 1-proteinase inhibitors and mouse contrapsin. KIP forms an equimolar complex with plasma kallikrein in a dose- and time-dependent fashion. The association rate constants for the inhibition of guinea pig plasma kallikrein by KIP, alpha 2-macroglobulin, C1-inactivator and antithrombin III were 2.5 +/- 0.3.10(4), 2.4 +/- 0.4.10(4), 6.6 +/- 0.5.10(4) and 9.1 +/- 0.6.10(2), respectively. Comparison of the association rate constants and the normal plasma concentrations of the four inhibitors demonstrates that KIP is ten-times as effective as alpha 2-MG and other two inhibitors are marginally effective in the inhibition of kallikrein. KIP inhibits trypsin and elastase rapidly, and thrombin and plasmin slowly, but is inactive for chymotrypsin and gland kallikrein. These results suggest that KIP is the major kallikrein inhibitor in guinea pig plasma and the proteinase inhibitory spectrum is unique to KIP in spite of the molecular similarity to alpha 1-proteinase inhibitor.

A protease-like permeability factor in the guinea pig skin. 1. Partial purification and characterization.

A permeability factor was extracted in a latent form from guinea pig skin and separated by ammonium sulfate fractionation into the pseudoglobulin fraction (30--50% saturation). The activation of the latent form of the permeability factor seemed to be caused in the desalting step by gel filtration with Sephadex G-50. The factor was partially purified by streptomycin treatment and column chromatography using hydroxyapatite, diethylaminoethyl cellulose and Sephadex G-75, in this order. Gel filtration showed that its molecular weight was approx. 35000. Its permeability activity was heat stable at 61 degrees C for 60 min at neutral pH, resistant at pH 5--10 and at ionic strengths from deionized water to 1 M NaCl at 4 degrees C. Its activity was transient and suppressed by guinea pig serum, but insensitive to an anti-histamic agent (triprolidine). Furthermore, its permeability activity was inhibited by diisopropylfluorophosphate, soybean trypsin inhibitor and leupeptin, and completely adsorbed by soybean trypsin inhibitor affinity column. These findings suggested that the permeability factor was a serine-type protease.

Different effects of concanavalin A and its succinylated derivative on superoxide release in peritoneal macrophages.

The effects of tetravalent conconavalin A and its succinylated derivative on the intracellular production of superoxide anion (O-2) and its release into cell exterior of peritoneal macrophages were observed. Both tetravalent concanavalin A and its succinylated derivative induced marked enhancement of intracellular reduction of nitroblue tetrazolium, which could be inhibited by alpha-methyl-D-glucoside. The extent of activation of nitroblue tetrazolium reduction induced by both types of the lectin paralleled the activation ratio of oxygen consumption. There was little difference in the extent of intracellular O-2 production induced by two types of the lectin. Nitroblue tetrazolium reduction was not affected significantly by pretreatment with colchicine, rotenone or malonate, inhibitors of the cytoskeletal system and of the electron transport system. In contrast, tetravalent concanavalin A induced a higher rate of superoxide release than did succinylated divalent concanavalin A, which lacks the cross-linking activity of surface glycoproteins. These results indicate that superoxide production following oxygen consumption and superoxide release into cell exterior are controlled independently by a separate membrane mechanism and that superoxide production system is not essentially dependent on the involvement of the cytoskeletal system.

Effect of concanavalin A and its succinylated derivative on the oxidative metabolism of guinea pig peritoneal macrophages.

The effect of concanavalin A and its succinylated derivative on the metabolic regulation of guinea pig peritoneal macrophages was observed. The binding of tetravalent concanavalin A to the surface glycoproteins of macrophages caused a marked increase in the rate of oxygen consumption due to the activation of the hexose monophosphate shunt. Divalent succinylated concanavalin A, also induced a similar change in the rate of oxygen metabolism. The metabolic change induced by these two types of lectin was reversibly inhibited by alpha-methyl-D-glucoside, a haptenic inhibitor of these lectins, and was temperature dependent (observed at above 15 degrees C). It is suggested that the binding of these lectins to the surface glycoproteins, and not their cross-linking into caps, is required for the activation of oxygen metabolism of macrophages, and that highly fluid state of the plasma membrane seems to be an essential requirement for the transduction of glycoprotein perturbation on the macrophage surface into cellular interior via transmembrane control mechanism.

Interstitial-tissue localization of high-molecular-weight kininogen in guinea-pig skin.

A rabbit antibody against the light-chain of guinea-pig high-molecular-weight (HMW) kininogen, which was specific to HWM kininogen and did not recognize low-molecular-weight kininogen, was prepared. This antibody demonstrated the presence of HMW kininogen antigen at the interstitial-tissue space in the guinea-pig skin by means of immunohistochemistry. The interstitial-tissue HMW kininogen antigen was extracted from the skin. This antigen molecule in the skin extract behaved identically as HWM kininogen of plasma in slab-polyacrylamide gel electrophoresis under the presence of sodium dodecyl sulfate followed by immunoblotting. Therefore, it was concluded that HMW kininogen was present in the interstitial-tissue fluid in the skin. The amount of HMW kininogen in the skin extract was quantified by a sandwich enzyme-linked immunosorbent assay with the anti-light-chain antibody and a goat anti-guinea-pig HMW kininogen antibody. On the assumption that the interstitial-tissue volume is 50 ml/100 g wet skin tissue, the average concentration of HMW kininogen in the interstitial-tissue fluid of the skin was calculated to be 23% of the plasma concentration. On the other hand, the proportion of intravascular HMW kininogen (derived from blood remaining in the vessels of the harvested skin) in relation to the total HMW kininogen in the skin extract was quantified by measuring the radio-labelled HMW kininogen which had been injected intravenously as a tracer of the intravascular HMW kininogen. About 5% of the total HMW kininogen in the skin extract was calculated to be derived from the intravascular blood volume of the skin, indicating that the majority of the HMW kininogen in the skin extract was derived from the extravascular-tissue space.

A protease-like permeability factor in the guinea pig skin. 2. In vitro activation of the latent form permeability factor by weakly acidic phosphate buffer.

Conditions for the in vitro activation of the latent form of a protease-like permeability factor in the pseudoglobulin fraction from guinea pig skin were examined. (1) The factor was activated by dialysis against 67 mM phosphate buffer at pH 5.8--6.4, not at pH 7.0--8.0. (2) High salt concentration (200 mM or greater phosphate buffer or 67 mM phosphate buffer containing 200 mM or greater KCl or NaCl) prevented the activation at pH 6.2. (3) High osmotic pressure (sucrose at 1 M) did not affect activation at pH 6.2. (4) Reconversion of the activated permeability factor into an inactive form was not observed under high salt conditions, under which the latent permeability factor was stable in its own form. (5) The molecular size of the latent permeability factor was estimated as approx. 80 000 by Sephadex G-100 gel filtration at high salt concentration.

An alkaline proteinase which has inflammatory activity isolated from guinea pig lymphoid cells.

A phosphate buffer extract of regional lymph node cells (more than 90% lymphocytes) of guinea pigs immunized with bovine immunoglobulin G contained acid and alkaline proteinase activities. The immunized cells contained approximately twice as much proteinase activities than the normal cells. The alkaline proteinase was purified and two forms could be separated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that one form labeled with [3H]diisopropylfluorophosphate was localized at Mr 28,000 in a single peak in the presence and absence of reducing agent. The enzyme was inhibited by diisopropylfluorophosphate, p-chloromercuribenzoate, N-ethylmaleimide and iodoacetic acid, and was also partially inhibited by phenylmethylsulfonyl fluoride. It was relatively heat-stable. Intradermal injection of the purified form into normal guinea pigs induced a mixed neutrophil-mononuclear infiltrate. The immune reaction was reduced in intensity with the diisopropylfluorophosphate-inhibited enzyme. These findings suggest that the proteinase probably participates in the inflammatory response of cellular hypersensitivity.

Purification of guinea-pig plasma prekallikrein. Activation by prekallikrein activator derived from guinea-pig skin.

Prekallikrein was purified from guinea-pig plasma. The prekallikrein appeared homogeneous as a single-chain protein on polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS) and beta-mercaptoethanol. The apparent molecular weight was 82 000 by SDS-polyacrylamide gel electrophoresis, 99 000 by gel filtration on a Sephadex G-150 column and 84 500 (protein part) by amino acid analysis. The isoelectric point was approx. 9.0. The purification method yielded 3.8 mg (A280 3.800) of prekallikrein from 500 ml of plasma. Kallikrein was generated from the prekallikrein by limited proteolytic action of a prekallikrein activator which was derived from guinea-pig skin. From analysis using SDS-polyacrylamide gel electrophoresis, the kallikrein has two fragments with apparent molecular weights of 52 000 and 40 000 which are linked by disulfide bond(s). The 40 000 molecular weight fragment was shown to incorporate [3H]diisopropylfluorophosphate. The kallikrein hydrolyzed the synthetic substrates containing the Phe-Arg sequence at the COOH-terminal, and it cleaved carbobenzyloxy-Phe-Arg-4-methylcoumaryl-7-amide more readily than Pro-Phe-Arg-methylcoumaryl-7-amide. The Km for the kallikrein with carbobenzyloxy-Phe-Arg-methylcoumaryl amide was 2 times 104 M. Also, the kallikrein showed negligible activities on peptide-methylcoumaryl amide-substrate for alpha-thrombin, Factor Xa or plasmin.

The Hageman factor-dependent system in the vascular permeability reaction.

The mechanism by which the Hageman factor-dependent system induces vascular permeability has been analyzed. The Mr-28,000 active fragment of guinea pig Hageman factor (beta-HFa), injected intradermally, induces an increase in local vascular permeability. Inhibition of vascular permeability resulted from pretreatment of the beta-HFa with immunopurified anti-Hageman factor F(ab')2 antibody at concentrations of 10(-6)-10(-7) M as well as by incubation with corn and pumpkin seed inhibitors of beta-HFa. To determine whether prekallikrein and kallikrein participated in the permeability induced by beta-HFa, circulating prekallikrein was depleted by intra-arterial injections of anti-prekallikrein F(ab')2 antibody. This resulted in about 80% diminution of the vascular permeability response to beta-HFa, without affecting the permeability reaction to bradykinin. Soybean trypsin inhibitor (10(-6) M), injected at the same cutaneous site as the beta-HFa, inhibited the vascular permeability response to beta-HFa by more than 90%. This concentration of soybean inhibitor blocked more than 90% of the activity of guinea pig plasma kallikrein, but did not inhibit the amidolytic capacity of beta-HFa. The permeability activity of beta-HFa (but not its amidolytic activity) was augmented 10-fold by simultaneous injection of a synthetic kinin potentiator, SQ 20,881 (Glu-Tyr-Pro-Arg-Pro-Gln-Ile-Pro-Pro-OH), and was almost completely inhibited by the simultaneous injection of a kinin-destroying enzyme, carboxypeptidase B. These results support the hypothesis that the greatest proportion of vascular permeability induced by beta-HFa is produced by the activation of prekallikrein followed by the release of kinin in the cutaneous tissue. These data offer the first in vivo evidence that the Hageman factor-dependent system by itself can induce inflammatory changes.

Activation of human Hageman factor by Pseudomonas aeruginosa elastase in the presence or absence of negatively charged substance in vitro.

Human Hageman factor, a plasma proteinase zymogen, was activated in vitro under a near physiological condition (pH 7.8, ionic strength I = 0.14, 37 degrees C) by Pseudomonas aeruginosa elastase, which is a zinc-dependent tissue destructive neutral proteinase. This activation was completely inhibited by a specific inhibitor of the elastase, HONHCOCH(CH2C6H5)CO-Ala-Gly-NH2, at a concentration as low as 10 microM. In this activation Hagemen factor was cleaved, in a limited fashion, liberating two fragments with apparent molecular masses of 40 and 30 kDa, respectively. The appearance of the latter seemed to correspond chronologically to the generation of activated Hageman factor. Kinetic parameters of the enzymatic activation were kcat = 5.8 x 10(-3) s-1, Km = 4.3 x 10(-7) M and kcat/Km = 1.4 x 10(4) M-1 x s-1. This Km value is close to the plasma concentration of Hageman factor. Another zinc-dependent proteinase, P. aeruginosa alkaline proteinase, showed a negligible Hageman factor activation. In the presence of a negatively charged soluble substance, dextran sulfate (0.3-3 micrograms/ml), the activation rate by the elastase increased several fold, with the kinetic parameters of kcat = 13.9 x 10(-3) s-1, Km = 1.6 x 10(-7) M and kcat/Km = 8.5 x 10(4) M-1 x s-1. These results suggested a participation of the Hageman factor-dependent system in the inflammatory response to pseudomonal infections, due to the initiation of the system by the bacterial elastase.

Activation of human plasma prekallikrein by Pseudomonas aeruginosa elastase in vitro.

Human plasma prekallikrein, precursor of the bradykinin-generating enzyme, was activated in a purified system under a near physiological condition (pH 7.8, ionic strength I = 0.14, 37 degrees C) by Pseudomonas aeruginosa elastase which is a tissue-destructive metalloproteinase. Compared with that, Pseudomonas aeruginosa alkaline proteinase poorly activated it with a rate as low as less than one-twentieth of that of elastase. The activation by elastase was blocked with a specific inhibitor of elastase, HONHCOCH(CH2C6H5)CO-Ala-Gly-NH2 (10 microM). Generation of kallikrein-like amidolytic activity was also observed in plasma deficient in Hageman factor by treatment with elastase, but was not in plasma deficient in prekallikrein. The kallikrein-like activity generated in Hageman factor deficient plasma as well as the generation process itself was indeed inhibited by anti-human prekallikrein goat antibody. These results suggest that the pathological activation of the kallikrein-kinin system might occur under certain clinical conditions in pseudomonal infections.

Pseudomonas aeruginosa alkaline proteinase might share a biological function with plasmin.

Pseudomonas aeruginosa alkaline proteinase, which is a zinc-dependent bacterial endopeptidase, preferentially hydrolyzed Boc-Val-Leu-Lys-methylcoumarylamide (MCA) which was originally designed as a specific substrate of plasmin, a plasma serine proteinase. The hydrolytic capacity was resistant to tosyl-lysine chloromethylketone at a concentration as high as 1 mM, but was blocked by a treatment with metal chelator such as o-phenanthroline at the concentration of 5 mM. Kinetic parameters of the amidolytic reaction were Km = 21 microM, kcat = 0.067 s-1 and kcat/Km = 3190 M-1 s-1. A synthetic peptide inhibitor which bore a possible ligand for zinc atom at the carboxy terminal was designed. This inhibitor, Ac-Val-Leu-Lys-4-mercaptoanilide, blocked the amidolytic activity of the pseudomonal alkaline proteinase in a competitive manner with the dissociation constant (Ki) value of 24 microM. The results imply that P. aeruginosa alkaline proteinase must be an unusual zinc-dependent 'C (COOH)-type' endopeptidase, which hydrolyzes the peptide bond of certain amino acid residues at the carboxyl group side by specific recognition, like serine- and cysteine-proteinases. In comparison, P. aeruginosa elastase which is a typical 'N (NH2)-type' metalloproteinase did not hydrolyze all of the commercially available peptide-MCA substrates tested at the present study. P. aeruginosa alkaline proteinase also hydrolyzed natural substrates of plasmin, such as fibrin and fibrinogen, with similar specific activities to plasmin. The susceptible subunits of fibrinogen were the A-alpha and B-beta ones, in this order. P. aeruginosa alkaline proteinase also exhibited an anti-coagulant activity in human plasma attributed to the direct fibrinogenolytic function. Such potential anti-coagulant capacity of the P. aeruginosa alkaline proteinase might explain, at least partly, the most characteristic pathologic feature of the P. aeruginosa septicemia, hemorrhagic lesions with lacking thrombi (Fetzer, A.E. et al. (1967) Am. Rev. Respirat. Dis. 96, 1121-1130).

Primary structure of bovine Hageman factor (blood coagulation factor XII): comparison with human and guinea pig molecules.

A bovine Hageman factor cDNA was cloned from a liver cDNA library. The nucleotide sequence was analyzed and the amino-acid sequence was deduced. The sequence deduced was consistent with the partial amino-acid sequences of bovine Hageman factor protein. The sequences for three portions including the amino terminal had been previously reported (Fujikawa et al. (1977) Biochemistry 16, 2270-2278). In comparison with the primary structures of human and guinea pig Hageman factors, the putative domain structures were totally conserved. Each domain possessed high sequence homology with the human molecule (66-88%) and the guinea pig one (63-81%) except for the proline-rich region (less than 10%) which connects the amino-terminal five domains with a serine proteinase portion. Significant heterogeneities were observed among the three species around the essential cleavage sites for the conversion to the activated Hageman factors. Bovine Hageman factor has no suitable amino-acid sequence as the substrate for the trypsin-type proteinases at the proline-rich region in difference from the human and guinea pig molecules. Probably this is the reason why the beta-form activated Hageman factor (the proteinase moiety) is not liberated in the activation of the bovine molecule with trypsin or plasma kallikrein.

Role of proline residue in the channel-forming and catecholamine-releasing activities of the peptaibol, trichosporin-B-VIa.

Trichosporin-B-VIa (TS-B-VIa) has a Pro14-kinked helical structure which is considered to be important for the formation of peptaibol-type ion-channels in lipid bilayer membranes. TS-B-VIa and its analog [Aib14]TS-B-VIa with Pro-->Aib substitution at position 14, resulting in a straight helical structure, were tested for ion-channel-forming activity in planar lipid bilayer membranes and for ability to induce catecholamine secretion from cultured bovine adrenal chromaffin cells. Voltage-dependent multi-channel conductance, which is characteristic of TS-B-VIa, was also observed for [Aib14]TS-B-VIa. In single-channel measurements, current fluctuations induced by [Aib14]TS-B-VIa had a shorter life-time and showed fewer substates than those induced by TS-B-VIa. Catecholamine secretion induced by these peptides at low concentrations is completely Ca(2+)-dependent. At high concentrations, TS-B-VIa-induced secretion was partly independent of external Ca2+, but this was not the case for the analog. The differences of behavior can be explained in terms of the differences of hydrophobicity, and magnitude of dipole moment due to the conformational changes around position 14 and the C-terminal domain caused by the Pro-->Aib substitution.