Trans-sialidase recombinant protein mixed with CpG motif-containing oligodeoxynucleotide induces protective mucosal and systemic trypanosoma cruzi immunity involving CD8+ CTL and B cell-mediated cross-priming

The Trypanosoma cruzi trans-sialidase (TS) is a unique enzyme with neuraminidase and sialic acid transfer activities important for parasite infectivity. The T. cruzi genome contains a large family of TS homologous genes, and it has been suggested that TS homologues provide a mechanism of immune escape important for chronic infection. We have investigated whether the consensus TS enzymatic domain could induce immunity protective against acute and chronic, as well as mucosal and systemic, T. cruzi infection. We have shown that: 1) TS-specific immunity can protect against acute T. cruzi infection; 2) effective TS-specific immunity is maintained during chronic T. cruzi infection despite the expression of numerous related TS superfamily genes encoding altered peptide ligands that in theory could promote immune tolerization; and 3) the practical intranasal delivery of recombinant TS protein combined with a ssDNA oligodeoxynucleotide (ODN) adjuvant containing unmethylated CpG motifs can induce both mucosal and systemic protective immunity. We have further demonstrated that the intranasal delivery of soluble TS recombinant Ag combined with CpG ODN induces both TS-specific CD4(+) and CD8(+) T cells associated with vaccine-induced protective immunity. In addition, optimal protection induced by intranasal TS Ag combined with CpG ODN requires B cells, which, after treatment with CpG ODN, have the ability to induce TS-specific CD8(+) T cell cross-priming. Our results support the development of TS vaccines for human use, suggest surrogate markers for use in future human vaccine trials, and mechanistically identify B cells as important APC targets for vaccines designed to induce CD8(+) CTL responses.     

Metabolism of sulfur compounds by whole filaments and heterocysts of Anabaena variabilis.

Filaments of the heterocyst-forming cyanobacterium Anabaena variabilis reproduced 35SO4(2)-, incorporating 35S into cysteine, methionine, glutathione, sulfolipid, and several unidentified metabolites. The majority of the incorporated label accumulated in reduced glutathione. Heterocysts isolated from labeled filaments contained the same major labeled products. Isolated, metabolically active heterocysts were unable to reduce 35SO4(2)-, but were able to incorporate 35S2- into cysteine and glutathione. The results suggest that the initial activation of SO4(2)- occurs in vegetative cells and that some reduced forms, possibly including S20, are translocated into heterocysts.     

Interaction of CK1δ with γTuSC ensures proper microtubule assembly and spindle positioning

Casein kinase 1δ (CK1δ) family members associate with microtubule-organizing centers (MTOCs) from yeast to humans, but their mitotic roles and targets have yet to be identified. We show here that budding yeast CK1δ, Hrr25, is a γ-tubulin small complex (γTuSC) binding factor. Moreover, Hrr25''s association with γTuSC depends on its kinase activity and its noncatalytic central domain. Loss of Hrr25 kinase activity resulted in assembly of unusually long cytoplasmic microtubules and defects in spindle positioning, consistent with roles in regulation of γTuSC-mediated microtubule nucleation and the Kar9 spindle-positioning pathway, respectively. Hrr25 directly phosphorylated γTuSC proteins in vivo and in vitro, and this phosphorylation promoted γTuSC integrity and activity. Because CK1δ and γTuSC are highly conserved and present at MTOCs in diverse eukaryotes, similar regulatory mechanisms are expected to apply generally in eukaryotes.     

Angiogenesis Is Induced and Wound Size Is Reduced by Electrical Stimulation in an Acute Wound Healing Model in Human Skin

Angiogenesis is critical for wound healing. Insufficient angiogenesis can result in impaired wound healing and chronic wound formation. Electrical stimulation (ES) has been shown to enhance angiogenesis. We previously showed that ES enhanced angiogenesis in acute wounds at one time point (day 14). The aim of this study was to further evaluate the role of ES in affecting angiogenesis during the acute phase of cutaneous wound healing over multiple time points. We compared the angiogenic response to wounding in 40 healthy volunteers (divided into two groups and randomised), treated with ES (post-ES) and compared them to secondary intention wound healing (control). Biopsy time points monitored were days 0, 3, 7, 10, 14. Objective non-invasive measures and H&E analysis were performed in addition to immunohistochemistry (IHC) and Western blotting (WB). Wound volume was significantly reduced on D7, 10 and 14 post-ES (p = 0.003, p = 0.002, p<0.001 respectively), surface area was reduced on days 10 (p = 0.001) and 14 (p<0.001) and wound diameter reduced on days 10 (p = 0.009) and 14 (p = 0.002). Blood flow increased significantly post-ES on D10 (p = 0.002) and 14 (p = 0.001). Angiogenic markers were up-regulated following ES application; protein analysis by IHC showed an increase (p<0.05) in VEGF-A expression by ES treatment on days 7, 10 and 14 (39%, 27% and 35% respectively) and PLGF expression on days 3 and 7 (40% on both days), compared to normal healing. Similarly, WB demonstrated an increase (p<0.05) in PLGF on days 7 and 14 (51% and 35% respectively). WB studies showed a significant increase of 30% (p>0.05) on day 14 in VEGF-A expression post-ES compared to controls. Furthermore, organisation of granulation tissue was improved on day 14 post-ES. This randomised controlled trial has shown that ES enhanced wound healing by reduced wound dimensions and increased VEGF-A and PLGF expression in acute cutaneous wounds, which further substantiates the role of ES in up-regulating angiogenesis as observed over multiple time points. This therapeutic approach may have potential application for clinical management of delayed and chronic wounds.     

TREATMENT OF HYPERLIPEMIC DIABETIC PATIENTS—Effects of Dextro-Thyroxine Therapy

Serum low-density lipoproteins of 12 hyperlipemic diabetic patients were lowered, in some cases to normal values, by administration of dextro-thyroxine. Accompanying reductions in serum total cholesterol were largely reflected in the low-density lipoprotein fraction. By contrast, high-density lipoprotein cholesterol concentrations remained relatively unchanged. Diabetic control by insulin or oral hypoglycemic agents was not detectably altered by dextro-thyroxine therapy for periods of 8 to 46 weeks. Therefore, use of the drug for treatment of diabetic hyperlipemia would appear to merit further investigation.     

Repair of degraded duplex DNA from prehistoric samples using Escherichia coli DNA polymerase I and T4 DNA ligase.

The most notable feature of DNA extracted from prehistoric material is that it is of poor quality. Amplification of PCR products from such DNA is consequently an exception. Here we present a simple method for the repair of degraded duplex DNA using the enzymes Escherichia coli DNA polymerase I and T4 DNA ligase. Adjacent sequences separated by nicks do not split up into intact strands during the denaturation step of PCR. Thus the target DNA is refractory to amplification. The proposed repair of nicked, fragmented ancient DNA results in an increase of amplification efficiency, such that the correct base order of the respective nuclear DNA segment can be obtained.     

Mitotic crossing over in chromosome I disomics of Aspergillus nidulans

Summary The frequency and pattern of homologous recombination in chromsome I disomics of Aspergillus nidulans is presented. Approximately 6% of randomly selected haploid breakdown sectors are recombinant. Most of these arise from double exchange events, one of which is located in the centromere region, the other distal on the left arm. Other marked regions are rarely involved in a recombination event. Reciprocal genotypes arise in approximately equal frequencies indicating that exchange results in reciprocally recombined non-sister chromatids at the four strand stage of mitosis. Possible theories for the extreme localisation of exchange events are discussed.     

A secondary effect of transformation in Rhizobium leguminosarum transgenic for Bacillus thuringiensis subspecies tenebrionisδ-endotoxin (cryIIIA) genes

 By introducing Bacillus thuringiensis subspecies tenebrionisδ-endotoxin genes (cryIIIA) into Rhizobium leguminosarum we have produced strains for the biological control of Sitona larvae. Comparisons between a transgenic and the parent strain show that transformation has induced changes not associated with the intended function of the transgene. Although growth rates in laboratory cultures are similar for both strains, the ability to compete for nodule occupancy is greater in the transgenic than in the non-transformed parent strain. This result demonstrates the importance of studying ecological and agronomic characters of transgenic micro-organisms that could have a bearing on the safety and success of their release into the environment, even if they are not thought to be connected with the transgenes introduced. Received: 20 April 1997 / Accepted: 2 June 1997