To determine whether a protective immune response could be elicited by oral delivery of a recombinant bacterial vaccine, tetanus toxin fragment C (TTFC) was expressed constitutively in Lactococcus lactis and administered orally to C57 BL/6 mice. The antibody titers elicited were lower than those following intranasal immunization (a route already known to result in high-level systemic anti-TTFC immune responses) but the protective efficacy was the same order of magnitude. The serum antibody isotypes elicited were predominantly IgG1 and IgG2a. TTFC-specific fecal IgA responses could be detected following oral or intranasal immunization. Chemically killed lactococci administered via the intranasal route were also able to elicit serum antibody responses of similar levels and kinetics to those induced by live bacteria.
Trypanosomes of the species Trypanosoma brucei reproduce primarily by binary fission, but the frequency of enzyme electrophoretic variants in natural populations of T. brucei has provided indirect evidence for the existence of a sexual cycle. These studies, coupled with studies of restriction fragment length polymorphisms of genes encoding glycolytic enzymes, have also provided evidence for T. brucei being diploid. Here we report direct evidence of gene exchange between two different clones of trypanosomes after mixed infection and full cyclical development in the tsetse fly vector.
To determine if the food-grade bacterium Lactococcus lactis holds promise as a vaccine antigen delivery vector we have investigated whether this bacterium can be made to produce high levels of a heterologous protein antigen. A regulated expression system has been developed which may be generally suitable for the expression of foreign antigens (and other proteins) in L. lactis. The system utilizes the fast-acting T7 RNA polymerase to transcribe target genes, and provides the first example of the successful use of this polymerase in a Gram-positive bacterium. When the performance of the expression system was characterized using tetanus toxin fragment C (TTFC) up to 22% of soluble cell protein was routinely obtained as TTFC. Mice immunized subcutaneously with L. lactis expressing TTFC were protected from lethal challenge with tetanus toxin. These results show for the first time that L. lactis is able to express substantial quantities of a heterologous protein antigen and that this organism can present this antigen to the immune system in an immunogenic form.
Four shuttle vectors (pMIG 1, 2, 2H and 3) have been constructed based on the broad host-range plasmid pCK1. All the pMIG vectors possess a multiple cloning site containing 12 or more unique restriction enzyme sites, and are stably maintained at either high or low copy number in Lactococcus lactis and in Escherichia coli. By cloning the E. coli pUC replicon into one of these vectors a plasmid was constructed which can replicate to high copy number in recA strains of E. coli. The broad host-range of the pCK1 replicon may enable these cloning vectors to be used in a number of Gram-positive bacteria. One of these vectors was used to optimize an electroporation procedure for transformation of a commonly used plasmid-cured strain MG1363 of L. lactis which routinely yielded 1 x 10(7) to 5 x 10(7) transformants micrograms-1 supercoiled DNA using stored, snap-frozen cells. This transformation efficiency was obtained by growing the cells in medium containing the cell wall weakening agent glycine, to an upper limit of 2.5% w/v. Although growth of L. lactis strain MG1363 was inhibited by the use of 0.5 mol l-1 sucrose as an osmotic stabilizer, the presence of sucrose in the electroporation buffer was critical for high transformation efficiency. Other variables which were tested for their effect on the efficiency of transformation were cell concentration, DNA concentration, pulse time and field strength. These results provide a model procedure which can be followed to optimize conditions for the genetic transformation of various strains of L. lactis.
Thyroxine dose requirement increases during pregnancy and thus close monitoring of thyroid function with appropriate adjustment of thyroxine dose to maintain a normal serum TSH level is necessary throughout gestation. Within a joint endocrine-obstetric clinic, maternal hypothyroidism at presentation and in the third trimester may increase the risk of low birthweight and the likelihood for caesarean section. The latter observation was not due to a higher rate of emergency caesarean section nor to a lower threshold for performing elective caesarean section. A larger study with adjustments made for the various confounders is required to confirm this observation.
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