The Secreted Larval Acidic Proteins (SLAPs) of Onchocerca spp. are encoded by orthologues of the alt gene family of Brugia malayi and have host protective potential

Onchocerca volvulus is a tissue-dwelling, vector-borne nematode parasite of humans and the causative agent of onchocerciasis, or 'River Blindness'. Resistance to infection is associated with immune responses to the infective, third-stage (L3) larvae. The antigens of greatest interest for their vaccine potential are surface and secreted molecules. We have previously identified a family of Secreted Larval Acidic Proteins (SLAPs) from the L3 larvae of O. volvulus by biosynthetic labelling. Here, we provide further characterisation of these molecules following cloning and expression of the corresponding cDNAs. Using protein sequencing, we show that SLAPs are members of the alt gene family, first described in the lymphatic filarial parasite, Brugia malayi. Ov-ALT-1 and Ov-ALT-2 correspond with 20 and 18kDa SLAPs. Both proteins are highly acidic and related by sequence, differing chiefly in an 8-amino acid deletion from Ov-ALT-2. By immunochemistry, we confirm that Ov-ALTs are highly stage-specific, being expressed exclusively in late L2 and L3 larvae during growth in the vector. They are synthesised and stored in the glandular oesophagus. Secretion is triggered by the resumption of development in the definitive host and occurs via the pseudocoelom and cuticle. Serological responses in humans to recombinant Ov-ALT-1 indicate that the level of IgG production may be governed by the force of transmission but does not overtly reflect infection status. Immunisation of mice with recombinant Ov-ALT-1 resulted in a modest level of protection against challenge with O. volvulus L3 larvae (P = 0.036). We conclude that Ov-ALT genes, like those of other filariae, are of interest from the standpoint of parasite transmission and infectivity. They may also offer promise as components of a future sub-unit vaccine should the means to enhance protection be achieved.     

Application of influence diagrams to prostate intensity-modulated radiation therapy plan selection

The purpose is to incorporate clinically relevant factors such as patient-specific and dosimetric information as well as data from clinical trials in the decision-making process for the selection of prostate intensity-modulated radiation therapy (IMRT) plans. The approach is to incorporate the decision theoretic concept of an influence diagram into the solution of the multiobjective optimization inverse planning problem. A set of candidate IMRT plans was obtained by varying the importance factors for the planning target volume (PTV) and the organ-at-risk (OAR) in combination with simulated annealing to explore a large part of the solution space. The Pareto set for the PTV and OAR was analysed to demonstrate how the selection of the weighting factors influenced which part of the solution space was explored. An influence diagram based on a Bayesian network with 18 nodes was designed to model the decision process for plan selection. The model possessed nodes for clinical laboratory results, tumour grading, staging information, patient-specific information, dosimetric information, complications and survival statistics from clinical studies. A utility node was utilized for the decision-making process. The influence diagram successfully ranked the plans based on the available information. Sensitivity analyses were used to judge the reasonableness of the diagram and the results. In conclusion, influence diagrams lend themselves well to modelling the decision processes for IMRT plan selection. They provide an excellent means to incorporate the probabilistic nature of data and beliefs into one model. They also provide a means for introducing evidence-based medicine, in the form of results of clinical trials, into the decision-making process.     

Learning in escape/avoidance tasks in female rats does not vary with reproductive condition

To determine whether the development of novel stimulus-response associations by the mother during the periparturient period is attributable to a general facilitation of learning produced by the hormonal milieu during that period, learning ability under various reproductive conditions was assessed in two tasks unrelated to the periparturitional situation. The two tasks, selected because they equalized the various groups for motivation and performance variables, were acquisition of a water-maze escape (including two reversals), and acquisition and retention of an unsignalled shuttlebox shock avoidance. The groups tested in the water maze were a midpregnant group, an immediately prepartum group, and an immediately postpartum group. In the shuttlebox, the same conditions (different rats) were compared, together with a nonpregnant estrus condition, and a nonpregnant diestrus condition. The results of both experiments indicate that although learning occurred, the characteristics of acquisition and retention were not influenced by reproductive condition.     

Use of interleukin 15 to enhance interferon-gamma production by antigen-specific stimulated lymphocytes from rhesus macaques

The enzyme-linked immune spot (ELISPOT) assay is receiving increased attention as a means for quantifying antigen-specific CD8 T-cell responses in rhesus macaques. Further improving the sensitivity of this assay could aid in the evaluation of vaccine candidates and/or immune therapeutic candidates. Interleukin (IL)-15 has been demonstrated to stimulate expansion of human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) and to regulate homeostatic proliferation of CD8+ memory cells. We evaluated the in vitro effect of IL-15 to increase the detection of interferon-gamma (IFN-gamma) production by antigen-specific stimulated lymphocytes from a group of rhesus macaques exposed to simian-human immunodeficiency virus (SHIV) and a second group infected with SIVmac251, before and after antiretroviral treatment (ART). Results from these studies demonstrate that the presence of IL-15 during stimulation in a peptide-based ELISPOT assay greatly enhanced IFN-gamma production in both SHIV and simian immunodeficiency virus (SIV)-infected macaques. IFN-gamma production was mainly mediated by CD8 lymphocytes. The optimal concentrations of IL-15 that give enhancement of IFN-gamma production to specific antigen, without a significant increase in the spontaneous IFN-gamma release, ranged from 0.5 to 2.5 ng/ml. The mean number of IFN-gamma spots was increased 3.1- to 3.6-fold in response to SIV gag or HIV env peptide pools, respectively, in peripheral blood mononuclear cells (PBMC) from SHIV-infected macaques. Similarly, in SIV-infected macaques, IL-15 increased the mean number of IFN-gamma spots 2.7-fold in response to both SIV gag and env peptide pools. In samples obtained after ART in the same macaques, the increase factor was 2.5 for SIV gag and 1.8 for the env peptide pools. Thus, the sensitivity of the ELISPOT assay can be enhanced by addition of IL-15. This modified assay will be useful for detection of low frequencies of IFN-gamma producing cells in rhesus macaques.     

Lipoxygenase-3 (ALOXE3) and 12(R)-lipoxygenase (ALOX12B) are mutated in non-bullous congenital ichthyosiform erythroderma (NCIE) linked to chromosome 17p13.1.

We report the identification of mutations in lipoxygenase-3 (ALOXE3) and 12(R)-lipoxygenase (ALOX12B) genes in non-bullous congenital ichthyosiform erythroderma (NCIE) linked to chromosome 17. Linkage disequilibrium analysis of six families affected by NCIE permitted us to reduce a recently reported interval of 8.4 cM on chromosome 17p13.1 to a 600 kb region around the marker D17S1796, which contains LOX genes. LOX products have long been implicated in skin disorders. Two point mutations and one deletion were found in ALOXE3 and three point mutations were found in ALOX12B in these consanguineous families from the Mediterranean basin. ALOXE3 and ALOX12B are two genes which are physically linked and functionally related. They are separated by 38 kb, have one more exon than the other LOX genes and are mainly expressed in epithelial cells including keratinocytes. Although the main substrate(s) of the two enzymes is (are) still unknown, the products of ALOX12B obtained in experimental systems have been demonstrated to be of R-chirality. It seems likely that the product of one of these enzymes may be the substrate of the other, and that they belong to the same metabolic pathway.     

How self-initiated memorized movements become automatic: a functional MRI study

We used functional magnetic resonance imaging (fMRI) and dual tasks to investigate the physiology of how movements become automatic. Normal subjects were asked to practice some self-initiated, self-paced, memorized sequential finger movements with different complexity until they could perform the tasks automatically. Automaticity was evaluated by having subjects perform a secondary task simultaneously with the sequential movements. Our secondary task was a letter-counting task where subjects were asked to identify the number of times a target letter from the letter sequences was seen. Only the performances that achieved high accuracy in both single and dual tasks were considered automatic. The fMRI results before and after automaticity was achieved were compared. Our data showed that for both conditions, sequential movements activated similar brain regions. No additional activity was observed in the automatic condition. There was less activity in bilateral cerebellum, presupplementary motor area, cingulate cortex, left caudate nucleus, premotor cortex, parietal cortex, and prefrontal cortex during the automatic stage. These findings suggest that most of the motor network participates in executing automatic movements and that it becomes more efficient as movements become more automatic. Our results do not provide evidence for any area to become more activated for automatic movements.     

Prenatal detection of cri du chat syndrome on uncultured amniocytes using fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization (FISH) with a chromosome-region-specific DNA probe was used prospectively on uncultured amniocyte interphase cells to detect an unbalanced chromosome abnormality that resulted in cri du chat or 5p – syndrome. Confirmation was performed by routine cytogenetics.