Effect of Group A Streptococcal Cysteine Protease on Invasion of Epithelial Cells

Cysteine protease of group A streptococci (GAS) is considered an important virulence factor. However, its role in invasiveness of GAS has not been investigated. We demonstrated in this study that two strains of protease-producing GAS had the ability to invade A-549 human respiratory epithelial cells. Isogenic protease mutants were constructed by using integrational plasmids to disrupt the speB gene and confirmed by Southern hybridization and Western immunoblot analyses. No extracellular protease activity was produced by the mutants. The mutants had growth rates similar to those of the wild-type strains and produced normal levels of other extracellular proteins. When invading A-549 cells, the mutants had a two- to threefold decrease in activity compared to that of the wild-type strains. The invasion activity increased when the A-549 cells were incubated with purified cysteine protease and the mutant. However, blockage of the cysteine protease with a specific cysteine protease inhibitor, E-64, decreased the invasion activity of GAS. Intracellular growth of GAS was not found in A-549 cells. The presence or absence of protease activity did not affect the adhesive ability of GAS. These results suggested that streptococcal cysteine protease can enhance the invasion ability of GAS in human respiratory epithelial cells.     

Identification of Candida albicans by specific primers of polymerase chain reaction and DNA probes

Candida albicans is a pathogenic yeast. Two sets of universal primers were used for specific identification of Candida albicans with PCR-amplified ribosomal DNA internal transcribed spacers (ITS). Among the species of Candida, the amplified ITSI and ITSII of DNA fragments were similar in size. The PCR product was purified and labeled with digoxigenin and used as DNA probe in the detection with target DNA of Candida albicans by hybridization. Two sets of specific primers (CA1 and CA2 to amplify ITSI, CA3 and CA4 to amplify ITSII) were designed by alignment of ribosomal ITS sequence of pathogenic Candida albicans with other species to detect C. albicans by PCR. The sensitivity of PCR using the specific primers to detect pure culture of C. albicans was 0.1 ng (about 10(3)-10(4) cells). If the yeast cells were mixed with two other strains, there was a 10-fold decrease in sensitivity (1 ng or 10(4)-10(5) cells) under the same PCR conditions.     

Nonencapsulated Neisseria meningitidis strain produces amylopectin from sucrose: altering the concept for differentiation between N. meningitidis and N. polysaccharea

Neisseria meningitidis is the causative agent of meningococcal sepsis and meningitis. Neisseria polysaccharea is a nonpathogenic species. N. polysaccharea is able to use sucrose to produce amylopectin, a starch-like polysaccharide, which distinguishes it biochemically from the pathogenic species N. meningitidis. The data presented here indicate that this may be an insufficient criterion to distinguish between these two species. The nonencapsulated Neisseria strain 93246 expressed a phenotype of amylopectin production similar to that of N. polysaccharea. However, strain 93246 reacted with N. meningitidis serotype 4 and serosubtype P1.14 monoclonal antibodies and showed the N. meningitidis L1(8) lipo-oligosaccharide immunotype. Further analyses were performed on four genetic loci in strain 93246, and the results were compared with 7 N. meningitidis strains, 13 N. polysaccharea strains, and 2 N. gonorrhoeae strains. Three genetic loci, opcA, siaD, and lgt-1 in strain 93246, were the same as in N. meningitidis. Particularly, the siaD gene encoding polysialyltransferase responsible for biosynthesis of N. meningitidis group B capsule was detected in strain 93246. This siaD gene was inactivated by a frameshift mutation at the poly(C) tract, which makes strain 93246 identical to other nonencapsulated N. meningitidis strains. As expected, the ams gene encoding amylosucrase, responsible for production of amylopectin from sucrose, was detected in strain 93246 and all 13 N. polysaccharea strains but not in N. meningitidis and N. gonorrhoeae strains. These data suggest that strain 93246 is nonencapsulated N. meningitidis but has the ability to produce extracellular amylopectin from sucrose. The gene for amylopectin production in strain 93246 was likely imported from N. polysaccharea by horizontal genetic exchange. Therefore, we conclude that genetic analysis is required to complement the traditional phenotypic classification for the nonencapsulated Neisseria strains.     

Anti-tumor immunoglobulin M increases lung metastasis in an experimental model of malignant melanoma

Cancer metastasis involves distinct steps that depend on complicated tumor–host interactions. The hematogenous dissemination of tumor cells may be facilitated by factors that promote the arrest and adherence of cancer cells in capillaries. We examined whether anti-tumor monoclonal immunoglobulin M (IgM) antibodies promoted the hematogenous dissemination of B16 melanoma cells in syngeneic mice. IgM monoclonal antibodies were generated that selectively bind to B16 melanoma cells as compared to syngeneic fibroblasts, lymphocytes or Lewis lung carcinoma cells. Incubation of B16-BL6 or B16-F0 melanoma cells with these IgM anti-tumor antibodies significantly increased the number of lung colonies as compared with control antibodies. Moreover, intraperitoneal injection of specific antibody also significantly increased lung colonization. All anti-tumor antibodies promoted the aggregation of B16 melanoma cells. A chemically generated immunoglobulin G (IgG)-like fragment of an anti-tumor IgM antibody displayed greatly reduced tumor aggregation and, in contrast to intact IgM, did not significantly increase lung colonization of B16 melanoma cells. Neither intact IgM nor the IgG-like fragment enhanced the in vitro invasiveness of B16 melanoma cells across Matrigel-coated membranes. Our results, therefore, suggest that besides their beneficial anti-tumor effects, anti-tumor IgM antibodies may also promote the hematogenous dissemination of cancer cells. This revised version was published online in July 2006 with corrections to the Cover Date.     

The effect of adsorbed fibrinogen, fibronectin, von Willebrand factor and vitronectin on the procoagulant state of adherent platelets

Procoagulant (activated) platelets provide a site for assembly of the prothrombinase complex which can rapidly convert prothrombin into thrombin (a potent inducer of clot formation). Previously, we reported that adhesion of platelets to surfaces preadsorbed with blood plasma caused them to become procoagulant. In the present study we investigated the effect of adsorbed adhesion proteins (fibrinogen (Fg), fibronectin (Fn), von Willebrand factor (vWF) and vitronectin (Vn)) on the procoagulant activity of adherent platelets. Adsorbed Fn, vWF and Fg promoted platelet adhesion in the following order: Fn < vWF = Fg. However, these proteins promoted platelet activation (thrombin generation per adherent platelet) in the following order: Fg < Fn < vWF. Adsorption with a series of dilutions of normal plasma, serum, and plasmas deficient in or depleted of von Willebrand factor (de-vWF), fibronectin (de-Fn), vitronectin (de-Vn), or both vitronectin and fibronectin (de-VnFn) resulted in varied platelet adhesion, but little difference in platelet activation. However, preadsorption with dilute de-vWF plasma induced lower procoagulant activity than normal plasma. Preadsorption with normal plasma resulted in higher levels of platelet activation than preadsorption with Fg, suggesting that adsorption of plasma proteins other than Fg caused the high levels of activation observed for plasma preadsorbed surfaces.     

Abrupt temperature change triggers arthralgia in patients with juvenile rheumatoid arthritis.

BACKGROUND AND PURPOSE: Juvenile rheumatoid arthritis (JRA) is the most common form of arthritis in children and affects both quality of life and school attendance. Weather and temperature conditions are believed to affect joint pains; however, very few studies have investigated this issue. This study examined the association between joint pain in JRA patients and weather conditions. METHODS: The daily pain ratings of 52 patients previously diagnosed with JRA were recorded on visual analog scales over 4 months beginning January 1, 2004. These ratings were then compared with weather data to evaluate possible correlation between these two factors. RESULTS: Twenty nine patients kept daily records during the first 2 months. There was no positive correlation between weather parameters (such as temperature, humidity, and barometric pressure) and pain ratings. Interestingly, the pain rating significantly increased the day after the advent of a cold wave (sign test, p<0.01; Wilcoxon signed ranks test, p=0.001). The number of patients who experienced joint swelling was not related to weather conditions. Twenty one participants continued maintaining the diaries during the next 2 months. The patients reported higher pain levels in the first 2 months during the cold wave period than in the next 2 months when the cold wave period had ended (p<0.001). CONCLUSION: A dramatic weather change such as a sudden cold wave might influence the experience of joint pain.     

Synthesis and properties of thermotropic polyesters modified with a non-mesogenic rigid group

Four series of copolyesters, namely BB6-DMT, BB5-DMT, BB6-DMI and BB5-DMI series, were prepared by melt polycondensation of dimethyl 4,4′-bibenzoate (BB) with a dimethyl phthalate (DMT: dimethyl terephthalate or DMI: dimethyl isophthalate) and an alkanediol (1,6-hexanediol or 1,5-pentanediol). The homopolyesters poly(hexamethylene 4,4′-bibenzoate) (BB6) and poly(pentamethylene 4,4′-bibenzoate) (BB5) exhibit a smectic phase. The thermotropic liquid crystalline and crystalline properties of the copolyesters are significantly influenced by the presence of the non-mesogenic rigid phthalate unit. All BB6-DMT copolyesters remain crystalline. As x, the molar fraction of the phthalate units in the diacid units, ≧ 0.7 the mesophase of the BB6-DMT copolyesters is destroyed completely. For BB5-DMT copolyesters, the mesophase disappears as x ≧ 0.4, and the copolyesters become amorphous as 0.5 ≦ x ≦ 0.8. The mesophase and the crystallinity of the BB6-DMI copolyesters are destroyed completely as x > 0.5. The BB5-DMI copolyesters lose the mesophase as x ≧ 0.3, and become amorphous as x ≧ 0.4. The results indicate that the non-linear isophthalate unit destroys mesophase and crystallinity of the copolyesters to a greater extent than the para-linked terephthalate unit.