Role of Moraxella catarrhalis outer membrane protein CD in bacterial cell morphology and autoaggregation

Moraxella catarrhalis, an important pathogen in the human respiratory tract, causes otitis media and lower respiratory tract infections. M. catarrhalis outer membrane protein CD (OMPCD) is a major heat-modifiable OMP with demonstrable potential as a vaccine candidate. The gene encoding OMPCD of M. catarrhalis strains was subjected to nucleotide sequence analysis and then inactivated by insertional mutagenesis. The ompCD mutant strains exhibited a modest growth defect in comparison with the wild-type strains. In optical microscopy and scanning/transmission electron microscopy examinations, regarding morphology, the cell size and cell wall of the ompCD mutant strains were significantly larger and thinner, respectively, than those of the wild-type strain. Furthermore, the ompCD mutant strains exhibited significant autoaggregation and increased surface hydrophobicity, in addition to a reduction in the adherence to HEp-2 cells, compared to the wild-type strains. Strains repaired by replacing the mutated ompCD gene exhibited phenotypic characteristics very similar to those of the wild-type strains. These results indicate that M. catarrhalis OMPCD, in addition to its functions related to bacterial growth and adherence to human epithelial cells, plays a very important role in bacterial physiology and pathogenesis, including aspects such as stabilizing bacterial cell morphology and preventing autoaggregation by reducing surface hydrophobicity.     

Validation of a reference control for an SYBR-Green fluorescence assay-based real-time PCR for detection of bovine herpesvirus 5 in experimentally exposed bovine embryos

The objective of this study was to optimize an internal control to improve SYBR-Green-based qPCR to amplify/detect the BoHV-5 US9 gene in bovine embryos produced in vitro and experimentally exposed to the virus. We designed an SYBR-Green-based binding assay that is quick to perform, reliable, easily optimized and compares well with the published assay. Herein we demonstrated its general applicability to detect BoHV-5 US9 gene in bovine embryos produced in vitro experimentally exposed to BoHV-5. In order to validate the assay, three different reference genes were tested; and the histone 2a gene was shown to be the most adequate for normalizing the qPCR reaction, by considering melting and standard curves (p < 0.05). On the other hand, no differences were found in the development of bovine embryos in vitro whether they were exposed to BoHV-5 reference and field strains comparing to unexposed embryos. The developed qPCR assay may have important field applications as it provides an accurate BoHV-5 US9 gene detection using a proven reference gene and is considerably less expensive than the TaqMan qPCR currently employed in sanitary programs.     

Increase in ovarian 15-hydroxyeicosatetraenoic acid during ovulation in the gonadotropin-primed immature rat

The ovarian level of 15-hydroxyeicosatetraenoic acid (15-HETE) was measured by RIA during ovulation in gonadotropin-primed immature Wistar rats. The ovulatory process was initiated in 25-day-old rats by a 10-IU injection of hCG sc 2 days after the animals had been primed with 10 IU PMSG, sc. Ovarian follicles begin to ovulate 10 h after hCG. At 0 h after hCG, the ovarian 15-HETE level was 0.6 +/- 0.2 ng/mg ovarian protein. At 6 h the 15-HETE level increased sharply to 27.3 +/- 4.2 ng/mg protein and reached a peak of 50.0 +/- 9.8 ng/mg protein at 10 h. Ovarian 15-HETE decreased significantly between 10-16 h after hCG (when ovulation was essentially completed). The pattern of secretion of this 15-lipoxygenase product was reciprocal to the pattern of secretion of leukotriene-B4 by the rat ovary. Ovarian 15-HETE production and ovulation were inhibited in a dose-dependent manner when indomethacin was administered sc 1 h after hCG in doses ranging from 0.10-10.0 mg/rat. In contrast, the synthesis of ovarian prostaglandins-E and -F was inhibited by a dose of indomethacin as low as 0.0316 mg/rat, but this dose did not significantly affect the ovarian 15-HETE level or the ovulation rate. Therefore, the ovulation rate was more closely correlated with the ovarian 15-HETE level (P less than 0.001) than with the ovarian levels of either prostaglandin-E or -F (0.10 greater than P greater than 0.05). The results suggest that products of the 15-lipoxygenase pathway of arachidonic acid metabolism may be important in the biochemical events of mammalian ovulation.     

Cytotoxic Chemotherapy Successfully Induces Durable Complete Remission in 2 Patients with Mosquito Allergy Resulting from Epstein-Barr Virus-Associated T-/Natural Killer Cell Lymphoproliferative Disease

Recent findings indicate that Epstein-Barr virus (EBV)-infected T-/natural killer (NK) cells play an important role in the pathogenesis of mosquito allergy, and most patients with mosquito allergy die early in life if not properly treated. Over the last 7 years, we have been using combination chemotherapy and allogeneic stem cell transplantation for the treatment of EBV-associated T-/NK cell lymphoproliferative disease (LPD) in which chronic active EBV infection and mosquito allergy were included. As of this writing, we have successfully treated 2 patients with mosquito allergy with chemotherapy in which EBV-infected T-/NK cells were eradicated. The findings suggest the possible role of chemotherapy in the treatment of EBV-associated T-/NK cell LPD.     

Interferon-gamma production by human cord blood monocyte-derived dendritic cells

Interferon (IFN)-gamma is produced by T cells and natural killer cells and activates monocytes and dendritic cells (DCs). Recently, IFN-gamma has been shown to be produced by mouse DCs following stimulation with interleukin (IL)-12, which is markedly augmented by the addition of IL-18. We here analyzed whether human DCs secrete IFN-gamma in response to IL-12 and/or IL-18. Human immature DCs, generated from cord blood CD14(+) monocytes by treating with granulocyte-macrophage colony-stimulating factor and IL-4, were incubated with IL-12 and/or IL-18 and assayed for IFN-gamma production. IL-12, but not IL-18, weakly induced IFN-gamma production, while IL-12 together with IL-18 induced high levels of IFN-gamma production. Similar results were obtained with mature DCs, although levels of IFN-gamma production were less than those in immature DCs. Also with mature and immature DCs, IL-12 upregulated the expression of IL-18 receptor alpha (Ralpha), and costimulation with IL-12 and IL-18 upregulated CD40 expression. Anti-IL-18Ralpha antibody abrogated both the IFN-gamma induction and the CD40 upregulation by IL-12 plus IL-18. These findings suggest that IL-12 upregulates IL-18Ralpha expression on human DCs and acts synergistically with IL-18 to induce high levels of IFN-gamma, which subsequently enhances CD40 expression on DCs in an autocrine manner.     

Successful therapy with argatroban for superior mesenteric vein thrombosis in a patient with congenital antithrombin deficiency

A 38-year-old woman was admitted with superior mesenteric vein (SMV) thrombosis, which was refractory to anticoagulation therapy. The plasma antithrombin activity was decreased and hardly compensated by concentrated antithrombin preparation due to high consumption rate. However, successful anticoagulation was achieved by administration of direct thrombin inhibitor, argatroban. Family studies of antithrombin activity revealed that she had type I congenital antithrombin deficiency. A novel heterozygous mutation in the gene for antithrombin (single nucleotide T insertion at 7916 and 7917, Glu 272 to stop in exon 4) was identified. Argatroban administration would be effective in the treatment of congenital antithrombin deficiency with SMV thrombosis.