Protection against Leptospira interrogans sensu lato challenge by DNA immunization with the gene encoding hemolysin-associated protein 1

The use of DNA constructs encoding leptospiral proteins is a promising new approach for vaccination against leptospirosis. In previous work we determined that immunization with hemolysis-associated protein 1 (Hap1) (LipL32) expressed by adenovirus induced significant protection against a virulent Leptospira challenge in gerbils. To avoid the use of the adenovirus vector, we checked for clinical protection against lethal challenge by DNA vaccination. A DNA vaccine expressing Hap1 was designed to enhance the direct gene transfer of this protein into gerbils. A challenge was performed 3 weeks after the last immunization with a virulent strain of serovar canicola. Our results show that the cross-protective effect with pathogenic strains of Leptospira, shared by Hap1, could be mediated by the DNA plasmid vector. This finding should facilitate the design and development of a new generation of vaccines against bacteria, particularly Leptospira interrogans sensu lato.     

Strain differences in mouse cellular responses to Mycobacterium lepraemurium and BCG subcutaneous infections. I. Analysis of cell surface phenotype in local granulomas.

C57BL/6, BALB/c and CBA mice were subcutaneously infected with either Mycobacterium lepraemurium (MLM) or BCG, and studied for bacillary growth, granuloma size of infected footpads and draining lymph nodes (DLN), and DLN cell surface phenotype. Whereas, BCG-infected mice controlled the infection and developed early and large granulomas, MLM-infected mice exhibited major strain variations in their resistance to the infection, as well as in the granuloma size and kinetics. C57BL/6 mice, highly resistant, displayed early and regressive granulomas; BALB/c mice showed lower resistance and early granulomas that grew continuously; CBA mice, highly susceptible, developed late, soft, phagocyte-rich granulomas. Important strain differences in lymph node lymphocyte subset distribution could be observed prior to any infection: C57BL/6 mice displayed higher B cell percentages than both of the other strains and BALB/c mice showed the highest CD4/CD8 ratios, followed by CBA and C57BL/6 mice. BCG and MLM infections both induced similar changes of these parameters in all three strains: that is a decrease of the B cell percentage and a decrease of the CD4/CD8 ratio, and the strain differences observed in uninfected mice persisted. On the other hand, DLN cells stimulated by the infecting bacillus and interleukin 2 also displayed an increase of the CD8 T cell percentage as compared with normal lymph node cells, but this phenomenon was much less pronounced in BALB/c mice, whether infected by MLM or BCG, and in MLM-infected CBA mice, than in BCG- or MLM-infected C57BL/6 (B6) mice. Thus the ability of C57BL/6 mice to generate an early and persistent CD8 T cell response to mycobacteria may contribute to their resistance to MLM.     

Study of blood group B antigen with a specific monoclonal antibody (anti-B, b-183).

A murine anti-B monoclonal antibody was obtained by the hybridoma technique. This antibody called anti-B (b-183) is of IgM nature; it is capable of agglutinating normal B, B3, Bx, cis AB and some acquired B red cells. Its association constant is 1.1 X 10(8) l/mol, and appears high compared to those of the monoclonal anti-A. This monoclonal anti-B was used to determine the number of B sites on B3 and Bx red cells.     

Diagnosis of arylsulfatase A deficiency in intact cultured cells using a fluorescent derivative of cerebroside sulfate

A fluorescent derivative of cerebroside sulfate (12-(1-pyrene)dodecanoyl-sphingosylgalactosyl-0-3-sulfate (P12-sulfatide) has been synthesized as a potential substrate for the determination of cerebroside sulfatidase (or arylsulfatase A) activity. It was administered into cultured human skin fibroblasts and thereby utilized for the diagnosis of arylsulfatase A deficiency. Cultured skin fibroblasts from normal individuals and healthy persons suffering from a pseudoarylsulfatase A deficiency (PD) degraded the P12-sulfatide, while in cells derived from a metachromatic leukodystrophy (MLD) patient it remained essentially intact. This contrasts with in vitro determinations of enzymatic activity, where the MLD or PD-derived arylsulfatase A exhibit similar deficiency, in spite of a profoundly different clinical course. Administration of the fluorescent sulfatide into the intact cells permitted a sensitive and rapid diagnosis of MLD and its distinction from the PD-phenomenon. This might be of particular importance for cases in which a rapid diagnosis is required and for prenatal diagnosis of fetuses from families afflicted with both MLD and pseudo-deficiency mutant genes.     

The effects of antioxidant supplementation during Percoll preparation on human sperm DNA integrity

The integrity of sperm DNA is crucial for the maintenance of genetic health. A major source of damage is reactive oxygen species (ROS) generation; therefore, antioxidants may afford protection to sperm DNA. The objectives of the study were, first, to measure the effects of antioxidant supplementation in vitro on endogenous DNA damage in spermatozoa using the single cell gel electrophoresis (comet) assay and, second, to assess the effect of antioxidant supplementation given prior to X-ray irradiation on induced DNA damage. Spermatozoa from 150 patients were prepared by Percoll centrifugation in the presence of ascorbic acid (300, 600 microM), alpha tocopherol (30, 60 microM), urate (200, 400 microM), or acetyl cysteine (5, 10 microM). DNA damage was induced by 30 Gy X-irradiation. DNA strand breakage was measured using the comet assay. Sperm DNA was protected from DNA damage by ascorbic acid (600 microM), alpha tocopherol (30 and 60 microM) and urate (400 microM). These antioxidants provided protection from subsequent DNA damage by X-ray irradiation. In contrast, acetyl cysteine or ascorbate and alpha tocopherol together induced further DNA damage. Supplementation in vitro with the antioxidants ascorbate, urate and alpha tocopherol separately has beneficial effects for sperm DNA integrity.      

Extensive study of DRB, DQA, and DQB gene polymorphism in 23 DR2-positive, insulin-dependent diabetes mellitus patients.

To gain insight into the HLA subregions involved in protection against insulin-dependent diabetes mellitus (IDDM) we investigated the polymorphism of HLA-DR and -DQ genes in 23 DR2 IDDM patients. Results show the following. (1) Fourteen patients (61%) possess the DRB1, DRB5, and DQB1 alleles found in DRw16/DQw5 healthy people. These data contrast with the 5% of DRw16 normally found in DR2 populations and are in agreement with former observations supporting that the DRw16 haplotype is not protective. (2) Nine DR2 patients, i.e., 39% versus 95% in published DR2 controls, possess the DRB alleles found in DRw15 unaffected people. Among them, six patients have also DQA1 and DQB1 alleles identical to those found in DRw15/DQw6 healthy individuals. These data confirm that the DRw15/DQw6 haplotype is protective but indicate that none of the DR or DQ alleles, alone or in association, confers an absolute protection. (3) Our most striking results concern the very high frequency of recombinant haplotypes among the DRw15 patients: 3 of 9. In these three patients recombinations led to the elimination of both DQB1 and DQA1 alleles usually associated with DRw15. This strongly suggests that the occurrence of IDDM in these DRw15 patients is due to the absence of the usual DQ product and thus reinforces the assumption that DQ rather than DR region is involved in the protection conferred by the DRw15/DQw6 haplotype. Finally, analysis of the non-DRw15 haplotypes in heterozygous patients showed that IDDM can occur in the absence of any DQ alpha beta heterodimer of susceptibility.     

Detection of N-(3-oxohexanoyl)-L-homoserine lactone in mice infected with Yersinia enterocolitica serotype O8

Yersinia enterocolitica synthesizes N-acyl-L-homoserine lactone (AHL) signal molecules via the LuxR-LuxI homologues YenR-YenI. In this study we checked two prototypes of mouse-virulent Y. enterocolitica serotype O8 strains WA-314 and 8081 for AHL production in vitro and in vivo (mouse infection model). We used thin-layer chromatography in combination with the Escherichia coli AHL biosensor to identify the AHL species produced. We detected only OHHL [N-(3-oxohexanoyl)-L-homoserine lactone] and not HHL (N-hexanoyl-L-homoserine lactone) produced by Y. enterocolitica O8 in culture supernatant or infected mouse tissue. This is the first report demonstrating AHL production by yersiniae during infection.     

Binding of nucleosomes to a cell surface receptor: Redistribution and endocytosis in the presence of lupus antibodies

In the present study, we sought evidence for a surface nucleosome receptor in the fibroblastic cell line CV-1, and questioned whether anti-double-stranded (ds)DNA and/or anti-histone autoantibodies could recognized and influence the fate of cell surface-bound nucleosomes. ¹²⁵I-labeled mononucleosomes were shown to bind to the cell layer in a specific, concentration-dependent and a saturable manner. Scatchard analysis revealed the presence of two binding sites: a high-affinity site with a Kd of ～ 7nM and a low-affinity site (Kd ～ 400 nM) with a high capacity of 9 × 10⁷ sites. Visualization of bound mononucleosomes by fluorescence revealed staining on both the cell surface and the extracellular matrix (ECM). Purified mononucleosome-derived dsDNA (180–200 bp) was found to compete for binding of ¹²⁵I-mononucleosomes on the low-affinity site, to stain exclusively the ECM in immunofluorescence, and to precipitate three specific proteins of 43, 180 and 240 kDa from ¹²⁵-I-labeled cell lysates. Nucleosomes were found to precipitate not only the 180-kDa dsDNA-reactive component, but also a unique protein of 50 kDa, suggesting that this protein is a cell surface receptor for nucleosomes on these fibroblasts. Once bound on the cell surface, mononucleosomes were recognized and secondarily complexed by lupus anti-dsDNA or anti-histone antibodies (i.e. anti-nucleosome antibodies), thus forming immune complexes in situ. The presence of these complexing auto-antibodies was found dramatically to enhance the kinetics of mononucleosome internalization. Following the internalization of the nucleosome-anti-nucleosome complexes by immunofluorescence, we observed the formation of vesicles at the edge of the cells by 5–10 min which moved toward the perinuclear region by 20–30 min. By means of double-fluorescence labeling and proteolytic treatment, these fluorescent vesicles were shown to be in the cytoplasm, suggesting true endocytosis of nucleosome-anti-nucleosome immune complexes. As shown by confocal microscopy, at no stage of this endocytic process was there any indication that coated pits or coated vesicles participated. Co-distribution of the endocytic vesicles with regions rich in actin filaments and inhibition of endocytosis of nucleosome-anti-nucleosome complexes by disruption of the micro-filament network with cytochalasin D suggest a mechanism mediated by the cytoskeleton. Taken together, our data provide evidence for the presence of a surface nucleosome receptor. We also show that anti-dsDNA and anti-histone antibodies can form nucleosome-anti-nucleosome immune complexes in situ at the cell surface, and thus dramatically enhance the kinetics of nucleosome endocytosis.     

Thermal acclimation of neonates to prolonged cool exposure as regards sleep stages

The thermal responses of neonates during a cool acclimation period were studied with regard to sleep stages. Sleep stages, body temperatures and metabolic rate (VO2) were studied for seven neonates nursed in incubators and exposed to a cool temperature (thermoneutrality minus 2 degrees C) for 75 h. Each recording session lasted 3 h in the morning: firstly under thermoneutral baseline conditions, then during the first and last 3-h periods of the cool acclimation and finally during the last 3 h of a 24-h recovery period. Sleep structure was modified during the initial hours of cool exposure: the percentage of active sleep increased (AS: +13%, P = 0.028) at the expense of quiet sleep (QS: -11%, P = 0.043). This alteration in sleep structure persisted at the end of the acclimation period. Metabolic heat production only increased in the later period of cool acclimation. Throughout the cool exposure, VO2 increased more (P = 0.040) in QS (+33%) than in AS (+20%) so that by the end of the cool period, VO2 levels were similar in both sleep stages. During cool acclimation, the maintenance of homeothermy is related not only to a change in sleep organization but also to modifications in the thermoregulatory processes in both sleep stages. Considering the importance of AS/QS patterns in the neurobehavioral development of neonates, the present results could have clinical implications for the thermal management of neonates.