Reversal of refractory septic shock with drotrecogin alpha (activated).

OBJECTIVE: We previously reported that early continuous veno-venous hemodiafiltration (CVVHDF) enables rapid identification of a subgroup of patients with "refractory" septic shock and a 100% risk of death. The objective of this study was to investigate whether early administration of drotrecogin alpha (activated) (DrotAA) to this selected subgroup of septic patients at extremely high risk of death would significantly improve prognosis. METHOD: Prospective observational study in a medical intensive-care unit of a University Hospital. Twenty-three patients with refractory septic shock were included. "Refractory" shock was defined as persistent circulatory failure despite adequate circulatory support, associated with persisting lactic acidosis despite early CVVHDF. Response to CVVDHF was assessed after 6 h of this continuous procedure. Patients selected by this strategy received DrotAA infusion for four days. RESULTS: The 28-day mortality rate of the 23 patients was 39%. No difference was observed at inclusion between survivors and nonsurvivors. In patients who finally survived, 12 h of DrotAA infusion was associated with a significant decrease in lactic acidosis and in norepinephrine dose. CONCLUSION: DrotAA therapy was associated with unexpectedly high 28-day survival in patients with "refractory" septic shock.

Important B-cell epitopes for neutralization of human immunodeficiency virus type 1 Tat in serum samples of humans and different animal species immunized with Tat protein or peptides.

The Tat regulatory protein of human immunodeficiency virus type 1 (HIV-1) is secreted by infected cells and plays a key role in viral pathogenesis and replication. Tat protein has been proposed as a target antigen for vaccine design since anti-Tat antibodies may interfere with virus spread and disease progression. The aim of this study was to analyse the serum antibody response of mice, rabbits, macaques and humans immunized with recombinant Tat, synthetic Tat, Tat toxoid or Tat peptides and to examine the biological properties of these antibodies in terms of Tat-induced transactivation and HIV-1 replication. Only sera with antibody specificity to both N-terminal and basic functional domains were able to inhibit extracellular Tat-dependent transactivation significantly in vitro. Antibodies from a human subject immunized with Tat also reduced HIV-1 replication in acutely infected T cells and blocked reactivation of virus replicating low levels in chronically infected cells by exogenous Tat. These results demonstrate that immunization with Tat protein or a combination of synthetic Tat peptides elicits the production of Tat-neutralizing serum antibodies and suggest that Tat vaccination could be used to block in vivo extracellular Tat autocrine/paracrine transactivation of HIV-1 replication.

Immunity under the skin: potential application for topical delivery of vaccines.

With the technological advances in biomedical sciences and the better understanding of how the immune system works, new immunisation strategies and vaccine delivery options, such sprays, patches, and edible formulations have been developed. This has opened up the possibility of administering vaccines without the use of needles and syringes. Already topical immunisation is a reality and it has the potential to make vaccine delivery more equitable, safer, and efficient. Furthermore, it would increase the rate of vaccine compliance and greatly facilitate the successful implementation of worldwide mass vaccination campaigns against infectious diseases. This review gives a brief account of the latest developments of application of candidate vaccine antigens onto bare skin and describes some of our recent observations using peptide and glycoconjugate vaccines as immunogens.

Increased sterol biosynthesis in tobacco calli resistant to a triazole herbicide which inhibits demethylation of 14α-methyl sterols.

The γ-keto triazole derivative 4,4-dimethyl-1-(2-methoxyphenyl)-1-(1,2,4-triazol-1-yl)-1-penten-3-one is toxic to Nicotiana tabacum L. cv. Xanthi plants or cell cultures. Analysis of the sterol composition of treated wild-type plant material demonstrates that this herbicide is an inhibitor of the C-14α-methyl demethylation process in sterol biosynthesis. Selection experiments, consisting of screening large populations of microcalli derived from UV-mutagenized tobacco protoplasts for resistance to a lethal dose (1 mg · 1(-1)) of the γ-keto triazole, have resulted in the recovery of two groups of resistant calli. In the first group, selected calli show a sterol composition in the absence or presence of the inhibitor very similar to that of wild-type sensitive calli, whereas in the second group the main feature of the selected calli is a new sterol profile. These calli present an overproduction of sterols with a concomitant esterification of overproduced metaolites, just as it was demonstrated for calli previously selected in our laboratory for resistance to LAB 170250F, a triazole fungicide (Maillot-Vernier et al., 1991, Mol. Gen. Genet. 231, 33-40).

Genetic study and further biochemical characterization of a tobacco mutant that overproduces sterols.

A genetic and biochemical characterization is presented of a tobacco mutant that was previously shown to have an increased sterol content with an accumulation of biosynthetic intermediates. We first show that a precise regulation of the membrane sterol composition occurs in this mutant, via a selective esterification process. Indeed, sterols representing the usual end-products of the biosynthetic pathway are preferably integrated into the membranes as free sterols, whereas most of the intermediates pool is esterified and stored in cytoplasmic lipid droplets. It is further demonstrated that overproduction of sterols by the LAB1-4 mutant is due to a single nuclear and semi-dominant mutation. Finally, increase of biosynthesis and esterification of unusual sterols are shown to be responsible for the resistance of LAB1-4 calli to LAB170 250F, the triazole pesticide used to select this mutant. However, differentiated LAB1-4 tissues do not express the resistance trait, suggesting that sterol biosynthesis might not be the only site of action for the triazole at the plant level.

In Vitro Selection of Calli Resistant to a Triazole Cytochrome-P-450-Obtusifoliol-14-Demethylase Inhibitor from Protoplasts of Nicotiana tabacum L. cv Xanthi.

The selection of biochemical mutants has been undertaken in order to elucidate regulatory and functional aspects of sterol biosynthesis in plants. 2-(4-Chlorophenyl)-3-phenyl-1-(1H-1,2,4- triazol-1-yl)-2,3-oxidopropane (LAB170250F), an experimental fungicide of the triazole family, was used as a selective agent. Indeed, this compound is a strong inhibitor of the cytochrome-P-450-obtusifoliol-14-demethylase in sterol biosynthesis. The selection strategy consisted of screening large populations of microcalli derived from ultraviolet-mutagenized protoplasts of Nicotiana tabacum L. cv Xanthi for resistance to a lethal concentration of LAB170250F. The best selective conditions were first determined, i.e. strength of the selection pressure as well as the time and duration of its application in the developmental process from protoplast to whole plant. Selection experiments resulted in the recovery of 40 resistant calli. These calli were divided into three classes according to the modification of their sterol content in response to LAB170250F. Some of these calli might be impaired in sterol biosynthesis, but most have a sterol profile identical to that of the control calli. This suggests that the toxic properties of LAB170250F are due to the parallel inhibition of sterol biosynthesis and of at least one additional unidentified target in the plant cell.

The steady-state level of potato sucrose synthase mRNA is dependent on wounding, anaerobiosis and sucrose concentration.

The steady-state level of potato sucrose synthase (SSase) mRNA was investigated in different plant organs and in response to certain stimuli. SSase mRNA is mainly present in developing tubers, but its presence is not restricted to this organ and the transcript is found at detectable levels in all the tissues analysed, except leaves. Wounding results in a decrease in SSase mRNA, but this can be overcome by incubation under anaerobic conditions. In leaves and petioles, an increase in sucrose concentration leads to an increase in SSase mRNA.

Biochemical characterization of a sterol mutant plant regenerated from a tobacco callus resistant to a triazole cytochrome-P-450-obtusifoliol-14-demethylase inhibitor.

We report here, for the first time, the biochemical characterization of a plant mutant impaired in sterol biosynthesis. A fertile plant was regenerated from a tobacco callus resistant to LAB170250F, a potent inhibitor of the cytochrome-P450-obtusifoliol-14-demthylase. The resistant callus and the leaves from the regenerated plant are characterized by profound qualitative and quantitative changes in their sterol content. Self-fertilization of this plant yielded seeds with the same biochemical features, indicating that the new phenotype is of mutational origin.

Molecular cloning and sequencing of sucrose synthase cDNA from potato (Solanum tuberosum L.): preliminary characterization of sucrose synthase mRNA distribution.

A cDNA library constructed from poly(A)+ RNA from potato tuber in lambda gt11 was screened for sucrose synthase using a maize sucrose synthase cDNA probe. The longest 2.7-kb insert was sequenced. An ATG located within the sequence CTGCAATGG starts an open reading frame of 805 codons. The nucleotide sequence, when compared to the maize sucrose synthase cDNA sequence, exhibits about 70% identity and the deduced amino acid sequence about 75%. Three amino acid regions are about 90% homologous and two of them could be important for the protein function. Expression studies show that transcription of the potato sucrose synthase gene is at least ten fold higher in tubers compared to photosynthetically active tissues.