Development of inflammation in proteoglycan-induced arthritis is dependent on Fc gamma R regulation of the cytokine/chemokine environment

FcgammaRs are specialized cell surface receptors that coordinately regulate immune responses. Although FcgammaR expression is a prerequisite for the development of several immune complex-mediated diseases, the mechanism responsible for FcgammaR-dependent regulation in autoimmunity remains unclear. Therefore, we assessed FcgammaR-dependent regulation of inflammation in proteoglycan-induced arthritis (PGIA) using FcgammaR(-/-) mice. FcgammaRIIb(-/-) mice developed arthritis at an earlier time point and with a greater severity than wild-type (WT) mice. In gamma-chain(-/-) (FcgammaRI(-/-) and FcgammaRIII(-/-)) mice, no clinical or histological evidence of inflammation was observed. Exacerbation of arthritis in FcgammaRIIb(-/-) mice correlated with enhanced PG-specific Ab production, but did not significantly affect PG-specific T cell priming. In gamma-chain(-/-) mice, the absence of arthritis did not correlate with serum Ab responses, as PG-specific Ab production was normal. Although PG-specific T cell proliferation was diminished, spleen cells from gamma-chain(-/-) mice successfully adoptively transferred arthritis into SCID mice. Our studies indicated that the mechanism responsible for FcgammaR regulation of PGIA development was at the level of inflammatory cytokine and beta-chemokine expression within the joint. FcgammaRIIb regulated the development of PGIA by controlling the initiation of cytokine and chemokine expression within the joint before the onset of arthritis, whereas the expression of FcgammaRI and or FcgammaRIII controlled cytokine and chemokine expression late in the development of PGIA during the onset of disease. These results suggest that FcgammaRs are critical for the development of inflammation during PGIA, possibly by maintaining or enhancing inflammatory cytokine and beta-chemokine production.     

CD11b/CD18-dependent interactions of neutrophils with intestinal epithelium are mediated by fucosylated proteoglycans

CD11b/CD18-mediated adhesive interactions play a key role in regulating polymorphonuclear leukocytes (PMN)) migration across intestinal epithelium. However, the identity of epithelial ligands for migrating PMN remains obscure. In this study we investigated the role of carbohydrates in mediating adhesive interactions between T84 intestinal epithelial cells and CD11b/CD18 purified from PMN. Fucoidin, heparin/heparin sulfate, N-acetyl-D-glucosamine, mannose-6-phosphate, and laminarin were found to inhibit adhesion of T84 cells to CD11b/CD18. The most potent inhibitory effects were observed with fucoidin (50% inhibition at 1-5 x 10(-8) M). Binding assays demonstrated that fucoidin directly bound to CD11b/CD18 in a divalent cation- and sulfation-dependent fashion that was blocked by anti-CD11b mAbs. Experiments employing CD11b/CD18 as a probe to blot T84 cell fucosylated proteins purified via fucose-specific lectin column revealed several candidate CD11b/CD18 binding proteins with molecular masses of 95, 50, 30, 25, and 20 kDa. Fucosidase treatment of T84 cells resulted in significantly reduced cell adhesion to CD11b/CD18, while no inhibition was observed after neuraminidase treatment. Finally, significant inhibition of T84 cell adhesion to CD11b/CD18 was observed after blocking cell proteoglycan synthesis with p-nitrophenyl-beta-D-xylopyranoside. These findings implicate epithelial cell surface proteoglycans decorated with sulfated fucose moieties as ligands for CD11b/CD18 during PMN migration across mucosal surfaces.     

Atractis marquezi n. sp. (Nematoda: Atractidae) and a revision of Atractis dujardin, 1845, sensu Baker, 1987

Atractis marquezi n. sp. from the large intestine of the tortoise Geochelone nigrita is described and illustrated. Atractis Dujardin, 1845, sensu Baker, 1987, is revised to contain only those atractids having a lagonoid spicule. The remaining species of Atractis sensu Baker, 1987, are assigned to Cyrtosomum, Pseudatractis, and Klossinemella. A. marquezi represents the 12th species to be assigned to this genus and is distinguished from other Neotropical species by the distribution pattern of caudal papillae of the male: 2 pairs precloacal, 2 pairs adcloacal, and 6 pairs postcloacal.     

Spauligodon loboi n. sp. (Nematoda: Pharyngodonidae) parasite of Liolaemus spp. (Iguania: Liolaemidae) from northwestern Argentina

Three-hundred and forty-nine specimens of Spauligodon loboi n. sp. (Nematoda, Pharyngodonidae) were found in the large intestines of 55 of 225 adult specimens representing 5 species of Liolaemus collected in 11 localities of northwestern Argentina. Prevalence of infection was 24% (mean intensity = 6.3 +/- 3.4, range = 2-28). Spauligodon loboi n. sp. differs from other neotropical species in that the filamentous portion of the tail of males is spiny, whereas that of females is smooth. A key to the species of Spauligodon in the Neotropical Realm is provided.     

Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication

Hepatitis C virus (HCV) replication appears to be restricted to the human hepatoma cell line Huh-7, indicating that a favorable cellular environment exists within these cells. Although adaptive mutations in the HCV nonstructural proteins typically enhance the replicative capacity of subgenomic replicons in Huh-7 cells, replication can only be detected in a subpopulation of these cells. Here we show that self-replicating subgenomic RNA could be eliminated from Huh-7 clones by prolonged treatment with alpha interferon (IFN-alpha) and that a higher frequency of cured cells could support both subgenomic and full-length HCV replication. The increased permissiveness of one of the cured cell lines allowed us to readily detect HCV RNA and antigens early after RNA transfection, eliminating the need for selection of replication-positive cells. We also demonstrate that a single amino acid substitution in NS5A is sufficient for establishing HCV replication in a majority of cured cells and that the major phosphate acceptor site of subtype 1b NS5A is not essential for HCV replication.     

Button osteoma: its etiology and pathophysiology

The present study investigates a circumscribed bony overgrowth on the cranial vault, known as button osteoma (BtO) and referred to here as button lesion (BtL). We discuss its anthropological implications. Data on its histology, location, and population distribution (by age, race, and gender) are provided. Microscopically, BtL is composed of well-organized dense lamellated bone which is poorly vascularized and with very few osteocytes. It forms a dome-shaped roof over an underlying diploeized area which includes the ectocranial table. The frequency of BtL is similar in modern (37.6%) and archaeological (41.1%) populations, in blacks, whites, males, and females, and correlates with age. It is rare in nonhuman primates. Fifty-five percent of the human skulls studied by us had BtL only on the parietal, 23.6% on the frontal, and 3.6% on the occipital bones. Fifteen percent had BtL on both the frontal and parietal bones. No lateral preference was found. Most skulls with BtL (64.1%) had only one lesion, 20.4% had two BtL, and 15.4% demonstrated multiple BtL. The average number of button osteomas on an affected skull was 1.97. The frequency of large osteomas (0.5-1.0 cm) was similar in young and old age groups. The demographic characteristics of BtL, mainly its high frequency among ancient and modern populations, its independence of sex and race, its scarcity in other primates, and the fact that its macro- and microstruture are indicative of an hamartoma (and not an osteoma or exostosis) suggest an evolutionary background to the phenomenon.     

Determination of the affinity of each component of a composite quaternary transition-state analogue complex of creatine kinase

Recombinant rabbit muscle creatine kinase (CK) was titrated with MgADP in 50 mM Bicine and 5 mM Mg(OAc)2, pH 8.3, at 30.0 degrees C by following a decrease in the protein's intrinsic fluorescence. In the presence of 50 mM NaOAc, but in the absence of added creatine or nitrate, MgADP has an apparent K(d) of 135 +/- 7 microM, and the total change in fluorescence on saturation (Delta%F) is 15.3 +/- 0.3%. Acetate was used as the anion in this experiment because it does not promote the formation of a CK.MgADP.anion.creatine transition-state analogue complex (TSAC) [Millner-White and Watts (1971) Biochem. J. 122, 727-740]. In the presence of 80 mM creatine, but no nitrate, the apparent K(d) for MgADP remains essentially unchanged at 132 +/- 10 microM, while Delta%F decreases slightly to 13.2 +/- 0.3%. In the presence of 10 mM nitrate, but no creatine, the apparent K(d) is once again essentially unchanged at 143 +/- 23 microM, but the Delta%F is markedly reduced to 4.2 +/- 0.2%. The presence of both 10 mM nitrate and 80 mM creatine during titration reduces the apparent K(d) for MgADP 10-fold to 13.7 +/- 0.7 microM, and Delta%F increases to 20.6 +/- 0.3%, strongly suggesting that the simultaneous presence of saturating levels of creatine and nitrate increases the affinity of CK for MgADP and promotes the formation of the enzyme*MgADP*nitrate*creatine TSAC. When the fluorescence of CK was titrated with MgADP in the presence of 80 mM creatine and fixed saturating concentrations of various anions, apparent K(d) values for MgADP of 132 +/- 10 microM, 25.2 +/- 1.3 microM, 18.8 +/- 0.9 microM, 13.7 +/- 0.7 microM, and 6.4 +/- 0.7 microM were observed as the anion was changed from acetate to formate to chloride to nitrate to nitrite, respectively. This is the same trend reported by Millner-White and Watts for the effectiveness of various monovalent anions in forming the CK.MgADP.anion.creatine TSAC. On titration of CK with MgADP in the presence of 80 mM creatine and various fixed concentrations of NaNO3, the apparent K(d) for MgADP decreases with increasing fixed concentrations of nitrate. A plot of the apparent K(d) for MgADP vs [NO3-] suggests a K(d) for nitrate from the TSAC of 0.39 +/- 0.07 mM. Similarly, titration with MgADP in the presence of 10 mM NaNO3 and various fixed concentrations of creatine gives a value of 0.9 +/- 0.4 mM for the dissociation of creatine from the TSAC. The data were used to calculate K(TDAC), the dissociation constant of the quaternary TSAC into its individual components, of 3 x 10(-10) M3. To our knowledge this is the first reported dissociation constant for a ternary or quaternary TSAC.     

Substrate specificity of the Plasmodium falciparum glycosylphosphatidylinositol biosynthetic pathway and inhibition by species-specific suicide substrates

The substrate specificities of the early glycosylphosphatidylinositol biosynthetic enzymes of Plasmodium were determined using substrate analogues of D-GlcN(alpha)1-6-D-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol (GlcN-PI). Similarities between the Plasmodium and mammalian (HeLa) enzymes were observed. These are as follows: (i) The presence and orientation of the 2'-acetamido/amino and 3'-OH groups are essential for substrate recognition for the de-N-acetylase, inositol acyltransferase, and first mannosyltransferase enzymes. (ii) The 6'-OH group of the GlcN is dispensable for the de-N-acetylase, inositol acyltransferase, all four of the mannosyltransferases, and the ethanolamine phosphate transferase. (iii) The 4'-OH group of GlcNAc is not required for recognition, but substitution interferes with binding to the de-N-acetylase. The 4'-OH group of GlcN is essential for the inositol acyltransferase and first mannosyltransferase. (iv) The carbonyl group of the natural 2-O-hexadecanyl ester of GlcN-(acyl)PI is essential for substrate recognition by the first mannosyltransferase. However, several differences were also discovered: (i) Plasmodium-specific inhibition of the inositol acyltransferase was detected with GlcN-[L]-PI, while GlcN-(2-O-alkyl)PI weakly inhibited the first mannosyltransferase in a competitive manner. (ii) The Plasmodium de-N-acetylase can act on analogues containing N-benzoyl, GalNAc, or betaGlcNAc whereas the human enzyme cannot. Using the parasite specificity of the later two analogues with the known nonspecific de-N-acetylase suicide inhibitor [Smith, T. K., et al. (2001) EMBO J. 20, 3322-3332], GalNCONH(2)-PI and GlcNCONH(2)-beta-PI were designed and found to be potent (IC(50) approximately 0.2 microM), Plasmodium-specific suicide substrate inhibitors. These inhibitors could be potential lead compounds for the development of antimalaria drugs.