A rabbit polyclonal antiserum exhibiting a specific recognition pattern for Mycobacterium tuberculosis proteins was used to screen an M. tuberculosis genomic library constructed in the expression vector lambda gtll. One clone, denominated C1:10, expressed M. tuberculosis-specific determinants as part of a large fusion protein with ,I-galactosidase. The gene for this protein has been sequenced, and it encodes a protein of 134 amino acids (13.8 kDa) which did not display significant homology with any of the previously reported proteins in the data bases. Hybridization studies with restriction fragments of the cloned sequence revealed that it was not present in the genomes of related mycobacteria, namely, M. bovis, M. bovis BCG, M. flavescens, M. fortuitum, M. phlei, and M. vaccae. These findings suggest that we have detected a gene, or a fragment therefrom, unique for M. tuberculosis whose nucleotide and amino acid sequences could be useful tools in the design of an improved vaccine or a diagnostic method of greater accuracy for tuberculosis. * Corresponding author. focused our studies on those proteins exclusively present in M. tuberculosis and not in M. bovis BCG. Here, we describe the results obtained when TB40 serum, a rabbit serum raised against one of these M. tuberculosis-specific proteins, was used to screen a lambda gtll genomic library. MATERIALS AND METHODS Bacterial strains and vectors. The following mycobacterial strains were obtained from the Trudeau Mycobacterial Collection (TMC): M. tuberculosis (TMC 102, strain H37Rv), M. bovis (TMC 410), M. bovis BCG (TMC 1011, substrain Pasteur), M. phlei (ATCC 11758), M. vaccae (TMC 1526), M. flavescens (ATCC 14474), and M. fortuitum (TMC 1529). Bacteriophage lambda gtll and Escherichia coli Y1088, Y1089, and Y1090 were provided by Amersham (Amersham, United Kingdom). E. coli DH5a, E. coli XL1-Blue, and phagemid vector Bluescript were purchased from Stratagene (La Jolla, Calif.). M13mpl8 was from Pharmacia (Uppsala, Sweden). Sonic extracts. Mycobacteria were grown on Sauton's medium, harvested, and sonicated as described by Janicki et al. (16) with minor modifications. Briefly, the bacilli were sonicated by subjecting them to 15-min pulses in a Branson sonicator at 0°C, followed by a 5-min rest period between pulses, and the process was repeated four times. The sonicate was then centrifuged at 150,000 x g for 1 h at 4°C. The supernatant was removed, and the protein content was determined by the method of Lowry et al. (20). This material was stored in aliquots at-70°C until needed. Antiserum and immunological analysis. Polyclonal antisera raised against purified M. tuberculosis proteins have been produced by us and described elsewhere (25). Briefly, 150 ,ug of the MTP40 protein, which had been isolated from polyacrylamide gels under nonreducing conditions, was mixed volume for volume with incomplete Freund adjuvant and injected subcutaneously into rabbits on days 0, 20, and 45. The rabbits were bled on days 30, 45, and 60. The antiserum obtained after this immuni...
Background: Recombinant adeno-associated virus (rAAV) is widely used in the neuroscience field to manipulate gene expression in the nervous system. However, a limitation to the use of rAAV vectors is the time and expense needed to produce them. To overcome this limitation, we evaluated whether unpurified rAAV vectors secreted into the media following scalable PEI transfection of HEK293T cells can be used in lieu of purified rAAV. Methods:We packaged rAAV2-EGFP vectors in 30 different wild-type and mutant capsids and subsequently collected the media containing secreted rAAV. Genomic titers of each rAAV vector were assessed and the ability of each unpurified virus to transduce primary mixed neuroglial cultures (PNGCs), organotypic brain slice cultures (BSCs) and the mouse brain was evaluated.Results: There was~40-fold wide variance in the average genomic titers of the rAAV2-EGFP vector packaged in the 30 different capsids, ranging from a low~4.7 × 10 10 vector genomes (vg)/mL for rAAV2/5-EGFP to a high of2 .0 × 10 12 vg/mL for a capsid mutant of rAAV2/8-EGFP. In PNGC studies, we observed a wide range of transduction efficiency among the 30 capsids evaluated, with the rAAV2/6-EGFP vector demonstrating the highest overall transduction efficiency. In BSC studies, we observed robust transduction by wild-type capsid vectors rAAV2/6, 2/8 and 2/9, and by capsid mutants of rAAV2/1, 2/6, and 2/8. In the in vivo somatic brain transgenesis (SBT) studies, we found that intra-cerebroventricular injection of media containing unpurified rAAV2-EGFP vectors packaged with select mutant capsids resulted in abundant EGFP positive neurons and astrocytes in the hippocampus and forebrain of non-transgenic mice. We demonstrate that unpurified rAAV can express transgenes at equivalent levels to lysate-purified rAAV both in vitro and in vivo. We also show that unpurified rAAV is sufficient to drive tau pathology in BSC and neuroinflammation in vivo, recapitulating previous studies using purified rAAV. Conclusions: Unpurified rAAV vectors secreted into the media can efficiently transduce brain cells in vitro and in vivo, providing a cost-effective way to manipulate gene expression. The use of unpurified virus will greatly reduce costs of exploratory studies and further increase the utility of rAAV vectors for standard laboratory use.
Mice harbouring a null deletion mutation in the IFNgamma receptor gene were used to study the role of IFNgamma responsiveness during experimental systemic candidiasis of mucosal or haematogenous origin. After intravenous (i.v.) or intranasal (i.n.) challenge with Candida albicans the progression of infection and concomitant cellular and antibody anti-C. albicans immune responses were analysed. During the week following i.v. challenge, the rate of C. albicans multiplication in kidneys, liver and spleen was faster in IFNgammaR (-/-) than IFNgammaR (+/+) mice. As a result, IFNgammaR (-/-) mice perished earlier than IFNgammaR (+/+) mice when challenged with equal numbers of live yeast cells. However, the overall susceptibility of the two mouse strains, in terms of survival against different C. albicans challenge doses over a 60-day period, was similar. No differences were found in the cellular anti-C. albicans response generated by i.v. challenge in both mouse strains. In contrast the kinetics and strength of the serum anti-C. albicans antibody responses were markedly different. Significantly stronger, predominantly IgG2a antibody responses accompanied the eventual control of C. albicans infection in IFNgammaR (-/-) mice. Following intranasal challenge, there was no difference in the rate of C. albicans clearance from the lungs of IFNgammaR (-/-) and IFNgammaR (+/+) mice. However, 48 h after challenge, large, conspicuous abscesses appeared in the lungs, liver, kidneys and spleen of IFNgammaR (-/-) mice. These abscesses were characterised by the presence of C. albicans and abundant neutrophilic infiltrates, but very few macrophages. No such abscesses developed in i.n. challenged IFNgammaR (+/+) mice. In both mouse strains, i.n. challenge induced strong systemic anti-C. albicans cellular responses, but relatively low titre systemic antibody responses. Mucosal anti-C. albicans antibody responses were detected in IFNgammaR (+/+), but not IFNgammaR (-/-) mice. Splenic adherent macrophages obtained from IFNgammaR (-/-) mice exhibited a significantly lower candidacidal activity than those of IFNgammaR (+/+) mice, and as expected, were not responsive to IFNgamma. In summary, these data suggest that IFNgamma has a role in limiting C. albicans multiplication during the early stages of infection, as well as in preventing the development of C. albicans-associated abscesses. Activation of macrophages by IFNgamma might be pivotal in mediating this role.
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