In Vitro and In Vivo Interactions of Haemophilus ducreyi with Host Phagocytes

We investigated the phagocytosis of Haemophilus ducreyi both in vitro and in vivo. Human granulocyte and monocyte phagocytosis of opsonized and nonopsonized, fluorescence-labeled H. ducreyi was assessed by flow cytometry. Both Escherichia coli and noncapsulated H. influenzae were included as controls. The maximal percentage of granulocytes taken up by H. ducreyi was 35% after 90 min. In contrast, 95% of H. influenzae bacteria were phagocytosed by granulocytes after 30 min. These results indicated that H. ducreyi phagocytosis was slow and inefficient. Bacterial opsonization by using specific antibodies increased the percentage of granulocytes phagocytosing H. ducreyi from 24 to 49%. The nonphagocytosed bacteria were completely resistant to phagocytosis even when reexposed to granulocytes, indicating that the H. ducreyi culture comprised a mixture of phenotypes. The intracellular survival of H. ducreyi in granulocytes, in monocytes/macrophages, and in a monocyte cell line (THP-1) was quantified after application of gentamicin treatment to kill extracellular bacteria. H. ducreyi survival within phagocytes was poor; approximately 11 and <0.1% of the added bacteria survived intracellularly after 2 and 20 h of incubation, respectively, while no intracellular H. influenzae bacteria were recovered after 2 h of incubation with phagocytes. The role of phagocytes in the development of skin lesions due to H. ducreyi was also studied in vivo. Mice that were depleted of granulocytes and/or monocytes and SCID mice, which lacked T and B cells, were injected intradermally with approximately 10⁶ CFU of H. ducreyi. Within 4 days of inoculation, the granulocyte-depleted mice developed lesions that persisted throughout the experimental period. This result reinforces the importance of granulocytes in the early innate defense against H. ducreyi infection. In conclusion, H. ducreyi is insufficiently phagocytosed to achieve complete eradication of the bacteria. Indeed, H. ducreyi has the ability to survive intracellularly for short periods within phagocytic cells in vitro. Since granulocytes play a major role in the innate defense against H. ducreyi infection in vivo, bacterial resistance to phagocytosis probably plays a crucial role in the pathogenesis of chancroid.     

Hepatitis C virus seroconversion rate in established blood donors

The results of hepatitis C virus (HCV) antibody test of 237, 813 blood donations collected from 143, 815 donors by the West Midlands Blood Transfusion Centre in 1993 were analyzed retrospectively in order to determine the seroconversion rate among established previously anti-HCV negative donors. Three hundred sixteen (0.22%; 1 in 455) donors were positive by the enzyme linked immunosorbent assay (ELISA) screening test and 34 (0.024%; 1 in 4, 230) donors were positive by ELISA and the Recombinant Immuno Blot Assay (RIBA). Three donors previously negative for HCV antibody reacted positively by both tests. The annual seroconversion rate was calculated as one in 35, 937 donors. This figure argues against limitation of HCV antibody screening to new blood donors. A further 45 donors negative on previous screening reacted positively by ELISA and were indeterminate by RIBA. Unexpectedly, lapsed blood donors first tested for HCV antibody in 1993 had high positive reaction rates by ELSA and RIBA, which was significantly (P < 0.001) higher than those of new donors. RIBA-positive reaction rate among ELISA-positive donors was significantly higher amongst males than females (P < 0.0011. © 1995 Wiley-Liss, Inc.     

Sequences of two kinetoplast minicircle DNAs of Trypanosoma (Nannomonas) congolense

Random minicircle DNA molecules were released from isolated kinetoplast network DNA of Trypanosoma congolense by BamHI digestion and cloned into plasmid pUC19. The sequences of two cloned minicircles (958 bp and 964 bp) were determined. Both minicircles contain the 13 bp sequence, 5'-GGGGTTGGTGTAA-3', thought to be the replication origin of minicircles in other trypanosomatids. The two minicircles have extensive homology in the 120 bp preceeding, and the 20 bp following, this 13-mer but only scattered homology elsewhere. Both possess tandem repeats downstream of the 13-mer. Comparison of these minicircles with minicircle sequences from other trypanosomatids reveals that they have the same general sequence organization as the others although only the 13-mer and its flanking regions are homologous.     

A synthetic nanofibrillar matrix promotes in vivo-like organization and morphogenesis for cells in culture

The purpose of this study was to design a synthetic nanofibrillar matrix that more accurately models the porosity and fibrillar geometry of cell attachment surfaces in tissues. The synthetic nanofibrillar matrices are composed of nanofibers prepared by electrospinning a polymer solution of polyamide onto glass coverslips. Scanning electron and atomic force microscopy showed that the nanofibers were organized into fibrillar networks reminiscent of the architecture of basement membrane, a structurally compact form of the extracellular matrix (ECM). NIH 3T3 fibroblasts and normal rat kidney (NRK) cells, when grown on nanofibers in the presence of serum, displayed the morphology and characteristics of their counterparts in vivo. Breast epithelial cells underwent morphogenesis to form multicellular spheroids containing lumens. Hence the synthetic nanofibrillar matrix described herein provides a physically and chemically stable three-dimensional surface for ex vivo growth of cells. Nanofiber-based synthetic matrices could have considerable value for applications in tissue engineering, cell-based therapies, and studies of cell/tissue function and pathology.     

Dual effect of temperature on the human epithelial Na+ channel

The amiloride-sensitive epithelial sodium channel (ENaC) is the rate-limiting step for sodium reabsorption in the distal segments of the nephron, in the colon and in the airways. Its activity is regulated by intracellular and extracellular factors but the mechanisms of this regulation are not yet completely understood. Recently, we have shown that the fast regulation of ENaC by the extracellular [Na⁺], a phenomenon termed self-inhibition, is temperature dependent. In the present study we examined the effects of temperature on the single-channel properties of ENaC. Single-channel recordings from excised patches showed that the channel open probability (P _o, estimated from the number of open channels N·P _o, where N is the total number of channels) increased on average two- to threefold while the single-channel conductance decreased by about half when the temperature of the perfusion solution was lowered from ~30 to ~15 °C. The effects of temperature on the single-channel conductance and P _o explain the changes of the macroscopic current that can be observed upon temperature changes and, in particular, the paradoxical effect of temperature on the current carried by ENaC.     

Genetics of reovirus: the relationship of interference to complementation and reassortment of temperature-sensitive mutants at nonpermissive temperature.

Temperature-sensitive mutants of reovirus type 3 are capable of interfering with the replication of wild-type reovirus type 3. The interfering activity correlated with the ability of pairs of mutants to complement at 39°: Pairs of noninterfering mutants (tsD × tsE) yielded efficient complementation (indexes of 10–50); pairs of interfering mutants (including members of groups ts A, B, G) did not produce significant complementation (indexes ～ 1). The ability of pairs of mutants to reassort at 39° generally followed a similar pattern. Thus interference is an important property of ts mutants of reovirus and needs to be considered when genetic interactions are being studied at 39°.     

Thymic-dependent anti-hapten response in congenitally athymic (nude) mice immunized with DNP-thymosin.

Immunization of congenitally athymic (nu/nu) and adult thymectomized, irradiated bone marrow, reconstituted (TxBm) mice with DNP5-thymosin (dinitrophenylated-bovine thymosin fraction 5) was found to elcit IgM and IgG anti-DNP plaque-forming cells in these animals. Further studies indicated that this response was antigen specific and not due to polyclonal activation. Since the hormonal properties of the thymosin were retained following linkage with hapten and DNP-thymosin was immunogenic in CBA/N and CBA/N female X DBA/2 male)F1 male mice, animals previously shown to have an X-linked inability to respond to thymus-independent antigens, it was concluded that DNP-thymosin functions both as a hormone and as a T-dependent antigen in eliciting an immune response in nu/nu and TxBm mice. Additional support for this conclusion was provided by the demonstration that DNP-thymosin could specifically prime for and elicit an anamnestic response in nu/nu mice. These results indicate that further investigation of the immune activities of DNP-thymosin may provide valuable insight in characterizing the maturation of helper T cells and their subsequent interaction with B cells.     

Bacteremia and urinary tract infection associated with CDC group Vd biovar 2.

A case of urinary tract infection and bacteremia caused by CDC group Vd biovar 2 in a 23-year-old woman with Hodgkin's disease is described. This is the first report of CDC group Vd biovar 2 isolated from a clinical specimen and considered as a pathogen. Detailed antimicrobial susceptibility data are presented.     

Inherited prothrombotic defects in Budd-Chiari syndrome and portal vein thrombosis: a study from North India

We studied 57 patients with Budd-Chiari syndrome (BCS) and 48 with portal vein thrombosis (PVT) for underlying inherited prothrombotic defects such as protein C, protein S, and antithrombin III deficiencies. Genetic mutations for factor V Leiden, prothrombin gene 20210A, and methyltetrahydrofolate reductase (MTHFR) C677T were studied in 29 patients in each group. Inherited prothrombotic defects were detected in 16 (28%) of 57 patients with BCS and 7 (15%) of 48 patients with PVT. Factor V Leiden mutation was the most common prothrombotic defect in BCS (5/29 [17%]) followed by protein C deficiency (7/57 [12%]) and protein S deficiency (4/57 [7%]), whereas in PVT, protein C deficiency was the most common inherited prothrombotic defect (4/48 [8%]) followed by protein S deficiency (2/48 [4%]). The factor V Leiden mutation was detected in only 1 (3%) of 29 cases of PVT. The heterozygous MTHFR C677T mutation was detected in 7 (24%) of 29 patients with BCS and 6 (21%) of 29 patients with PVT. Antithrombin III deficiency, homozygous MTHFR C677T mutation, and prothrombin G20210A mutation were not detected in any patients.     

Abnormalities of cholesterol metabolism in autism spectrum disorders.

Although Smith-Lemli-Opitz Syndrome (SLOS), a genetic condition of impaired cholesterol biosynthesis, is associated with autism [Tierney et al., 2001; Am J Med Genet 98:191-200.], the incidence of SLOS and other sterol disorders among individuals with autism spectrum disorders (ASD) is unknown. This study investigated (1) the incidence of biochemically diagnosed SLOS in blood samples from a cohort of subjects with ASD from families in which more than one individual had ASD and (2) the type and incidence of other sterol disorders in the same group. Using gas chromatography/mass spectrometry, cholesterol, and its precursor sterols were quantified in 100 samples from subjects with ASD obtained from the Autism Genetic Resource Exchange (AGRE) specimen repository. Although no sample had sterol levels consistent with SLOS, 19 samples had total cholesterol levels lower than 100 mg/dl, which is below the 5th centile for children over age 2 years. These findings suggest that, in addition to SLOS, there may be other disorders of sterol metabolism or homeostasis associated with ASD.