Vertebrate host protective immunity drives genetic diversity and antigenic polymorphism in Schistosoma mansoni.

Schistosomes are gonochoric blood parasites with a complex life cycle responsible for a disease of considerable medical and veterinary importance in tropical and subtropical regions. Understanding the evolution of schistosome genetic diversity is clearly of fundamental importance to interpreting schistosomiasis epidemiology and disease transmission patterns of this parasite. In this article, we investigated the putative role of the host immune system in the selection of male genetic diversity. We demonstrated the link between genetic dissimilarity and the protective effect among male worms. We then compared the proteomes of three male clones with different genotypes and differing by their capacity to protect against reinfection. The identified differences correspond mainly to antigens known or supposed to be involved in the induction of protective immunity. These results underline the role played by host immune system in the selection of schistosome genetic diversity that is linked to antigenic diversity. We discuss the evolutionary consequences in the context of schistosome infection.

Changes in brain morphology in patients with obstructive sleep apnoea.

BACKGROUND: Obstructive sleep apnoea (OSA) is a common disease that leads to daytime sleepiness and cognitive impairment. Attempts to investigate changes in brain morphology that may underlie these impairments have led to conflicting conclusions. This study was undertaken to aim to resolve this confusion, and determine whether OSA is associated with changes in brain morphology in a large group of patients with OSA, using improved voxel-based morphometry analysis, an automated unbiased method of detecting local changes in brain structure. METHODS: 60 patients with OSA (mean apnoea hypopnoea index 55 (95% CI 48 to 62) events/h, 3 women) and 60 non-apnoeic controls (mean apnoea hypopnoea index 4 (95% CI 3 to 5) events/h, 5 women) were studied. Subjects were imaged using T1-weighted 3-D structural MRI (69 subjects at 1.5 T, 51 subjects at 3 T). Differences in grey matter were investigated in the two groups, controlling for age, sex, site and intracranial volume. Dedicated cerebellar analysis was performed on a subset of 108 scans using a spatially unbiased infratentorial template. RESULTS: Patients with OSA had a reduction in grey matter volume in the right middle temporal gyrus compared with non-apnoeic controls (p<0.05, corrected for topological false discovery rate across the entire brain). A reduction in grey matter was also seen within the cerebellum, maximal in the left lobe VIIIb close to XI, extending across the midline into the right lobe. CONCLUSION: These data show that OSA is associated with focal loss of grey matter that could contribute to cognitive decline. Specifically, lesions in the cerebellum may result in both motor dysfunction and working memory deficits, with downstream negative consequences on tasks such as driving.

Nocturnal non-invasive nasal ventilation in stable hypercapnic COPD: a randomised controlled trial.

BACKGROUND: Sleep hypoventilation has been proposed as a cause of progressive hypercapnic respiratory failure and death in patients with severe chronic obstructive pulmonary disease (COPD). A study was undertaken to determine the effects of nocturnal non-invasive bi-level pressure support ventilation (NIV) on survival, lung function and quality of life in patients with severe hypercapnic COPD. METHOD: A multicentre, open-label, randomised controlled trial of NIV plus long-term oxygen therapy (LTOT) versus LTOT alone was performed in four Australian University Hospital sleep/respiratory medicine departments in patients with severe stable smoking-related COPD (forced expiratory volume in 1 s (FEV1.0) <1.5 litres or <50% predicted and ratio of FEV1.0 to forced vital capacity (FVC) <60% with awake arterial carbon dioxide tension (PaCO2) >46 mm Hg and on LTOT for at least 3 months) and age <80 years. Patients with sleep apnoea (apnoea-hypopnoea index >20/h) or morbid obesity (body mass index >40) were excluded. Outcome measures were survival, spirometry, arterial blood gases, polysomnography, general and disease-specific quality of life and mood. RESULTS: 144 patients were randomised (72 to NIV + LTOT and 72 to LTOT alone). NIV improved sleep quality and sleep-related hypercapnia acutely, and patients complied well with therapy (mean (SD) nightly use 4.5 (3.2) h). Compared with LTOT alone, NIV (mean follow-up 2.21 years, range 0.01-5.59) showed an improvement in survival with the adjusted but not the unadjusted Cox model (adjusted hazard ratio (HR) 0.63, 95% CI 0.40 to 0.99, p = 0.045; unadjusted HR 0.82, 95% CI 0.53 to 1.25, p = NS). FEV1.0 and PaCO2 measured at 6 and 12 months were not different between groups. Patients assigned to NIV + LTOT had reduced general and mental health and vigour. CONCLUSIONS: Nocturnal NIV in stable oxygen-dependent patients with hypercapnic COPD may improve survival, but this appears to be at the cost of worsening quality of life. TRIAL REGISTRATION NUMBER: ACTRN12605000205639.

The feasibility of using auto-titrating continuous positive airway pressure to treat obstructive sleep apnoea after acute tetraplegia.

STUDY DESIGN: A prospective cohort with acute tetraplegia. OBJECTIVES: Obstructive sleep apnoea (OSA) is common within weeks of tetraplegia. This study aimed at determining the feasibility of auto-titrating continuous positive airway pressure (CPAP) to treat OSA after acute tetraplegia. SETTING: The Victorian Spinal Cord Service, Melbourne, Australia. METHODS: Participants underwent full, portable polysomnography. Those with an apnoea hypopnoea index of more than 10 events per hour were defined as having OSA and were offered treatment with CPAP. Treatment adherence was objectively monitored, and measures of quality of life, sleepiness and functional outcomes were determined at enrollment and 3 months later at study conclusion. RESULTS: A total of 44 patients were admitted to our Spinal Cord Service over 9 months, and 19 participated. Fourteen of them had OSA and seven were adherent with therapy for 3 months. Compared with those who did not have OSA, and with those with OSA who were not adherent with CPAP, those who adhered to CPAP were older (mean (s.d.) age 54 years (13) versus non-adherent 28 years (15) and no OSA 29 years (10)) and heavier (body mass index (BMI) 32.5 (11.7), 24.1 (3.7) and 20.6 (3.1), respectively). CPAP-adherant patients and those without OSA showed a 50% or greater improvement in their state sleepiness over the 3 months. Patients with OSA who did not tolerate CPAP had no improvement in sleepiness. CONCLUSION: Auto-titrating CPAP is a feasible treatment for OSA in acute tetraplegia. Intensive clinical support was required initially, and a tolerance of therapy for at least 4 h for one of the first 3 days was predictive of good CPAP usage. SPONSORSHIP: Transport Accident Commission.

A conserved retinoid X receptor (RXR) from the mollusk Biomphalaria glabrata transactivates transcription in the presence of retinoids.

Retinoid X receptors (RXR) are members of the nuclear receptor superfamily of ligand-activated transcription factors that have been characterized in a wide variety of metazoan phyla. They act as heterodimer partners of other nuclear receptors, and in vertebrates also activate transcription as homodimers in the presence of a ligand, 9-cis retinoic acid. In order to test the hypothesis that retinoic acid signaling pathways involving RXRs are present in the Lophotrochozoa, we have sought to isolate conserved members of this family from the platyhelminth parasite Schistosoma mansoni and its intermediate host, the mollusk Biomphalaria glabrata. Here we report that an RXR ortholog from B. glabrata (BgRXR) is better conserved, compared with mouse RXRalpha, both in the DNA-binding domain (89% identity) and in the ligand-binding domain (LBD) (81% identity), than are arthropod homologs. In EMSA, BgRXR binds to the direct repeat response element DR1 as a homodimer or as a heterodimer with mammalian RARalpha, LXR, FXR or PPARalpha. When transfected alone into mammalian cell lines, BgRXR transactivated transcription of a reporter gene from the Apo-A1 promoter in the presence of 9-cis retinoic acid or DHA. Constructs with the Gal4 DNA binding domain fused to the hinge and LBDs of BgRXR were used to show that ligand-dependent activation of transcription by BgRXR required its intact AF-2 activation domain, and that the LBD can form homodimers. Finally, the binding of 9-cis retinoic acid preferentially protected the LBD of BgRXR from degradation by trypsin in a proteolysis protection assay. Our results show that BgRXR binds and is activated by retinoids and suggest that retinoid signaling pathways are conserved in the Lophotrochozoa. The nucleotide sequence reported in this paper has been submitted to the GenBank/EBI Data Bank with accession no. AY048663.

Crystallization and preliminary X-ray diffraction studies of a protective cloned 28 kDa glutathione S-transferase from Schistosoma mansoni.

Crystals of the recombinant 28 kDa glutathione S-transferase from Schistosoma mansoni have been obtained by the hanging-drop method of vapor diffusion from ammonium sulfate solutions. The successful crystallization of this enzyme required the presence of a reducing agent and S-hexylglutathione. The crystals belong to the cubic space group P4(1)32 (or P4(3)32), with unit cell dimensions a = 122.6 A and contain one molecule in the asymmetric unit. The crystals diffract to at least 2.8 A resolution and are suitable for X-ray crystallographic structure analysis.

Cloning and characterization of the gene encoding Schistosoma mansoni glutathione peroxidase.

Antioxidant enzymes are thought to play a crucial role in the survival of the parasite, Schistosoma mansoni, during its migration through the tissues of the definitive host. We recently cloned the cDNA encoding one such enzyme, glutathione peroxidase (Gpx). In order to elucidate the regulation of expression of this gene, we describe the cloning and characterization of a Gpx gene of S. mansoni. An initial screen of a lambda EMBL4 genomic library using the corresponding cDNA sequence as a probe yielded 14 positive clones, two of which have so far been analyzed in detail. The complete Gpx gene contains five introns, four of which, located at the 5' end, are extremely short (30-51 bp) and the last of which is approximately 6 kb long. We present the sequence of the gene including 73 bp at the 5' end, the complete sequence to 137 bp downstream from the penultimate exon, 164 bp upstream and 131 bp downstream from the last 3' exon. The potential mRNA cap site is situated 219 bp upstream from the ATG start codon. All intron/exon junctions correspond to the conventional eukaryotic splice signal. Analysis of the 5' flanking region revealed the presence of a potential TATA box at--26 bp from the cap site, but no CAAT-like element is present. Southern blot analysis showed a unique Gpx gene organisation in the S. mansoni genome.

Antigenic properties of Schistosoma mansoni aminopeptidases: evolution during the development in mammalian hosts.

The involvement in the immunity to schistosomiasis of an aminopeptidase activity of schistosomula, 20-day-old and adult worms of Schistosoma mansoni has been studied. This activity, hydrolysing leucine-p-nitroanilide and alanine-p-nitroanilide at pH 7.4 was immunoprecipitated by various infected rat and human sera. These antigenic enzymes were expressed at day 28 after infection in the rat and seemed to be specific for the Schistosoma species. Several aminopeptidase activities were found after analysis by isoelectric focusing of the adult extract but only the pI 8.3 peak was antigenic. Three antigenic peaks were demonstrated after AcA 34 Ultrogel filtration. The biological relevance of these antigenic enzymes in schistosomiasis is discussed.

In vitro synthesis of a 28 kilodalton antigen present on the surface of the schistosomulum of Schistosoma mansoni.

Adult Schistosoma mansoni proteins were fractionated on polyacrylamide slab gels, recovered by electrophoretic elution and used for immunization of Fischer rats. Three antisera recognizing, respectively, 28, 78 and 85 kDa antigens were obtained. The 28 kDa antigen was found among the in vitro translation products from adult worm RNA, and among the 125I-labelled surface antigens of S. mansoni schistosomula. The isoelectric point of the 28 kDa antigen was 6.3-6.5. The 28 kDa antiserum mediated a cytotoxic activity against schistosomula when used in an in vitro assay in the presence of a purified eosinophil cell population.

Cloning of the gene encoding a Schistosoma mansoni antigen homologous to human Ro/SS-A autoantigen.

A cDNA library was constructed from the mRNA of adult worms of Schistomsoma mansoni in the expression vector lambda gt11 and screened with a rabbit antiserum raised against a 60-65-kDa electroeluted adult worm fraction. Two overlapping clones were selected and a partial nucleotide sequence was deduced (1172 bp). The full-length sequence was obtained by the amplification of the 5' end of first strand cDNA using PCR. The overall mRNA size was 1335 nt including a 25 nt 5' non-coding region and a 131 nt untranslated region with the poly(A) tail. The predicted amino acid sequence of 393 aa (45 kDa) has 52% identity with the human Ro/SS-A autoantigen, which is considered to be the human calreticulin. As for the human Ro/SS-A, the protein encoded by the cDNA described here contains a hydrophobic leader sequence and a carboxyl terminal sequence, HDEL consensus signal sequence for retention in the ER. An antiserum raised against the fusion protein of one clone recognized a 58-kDa antigen in homogenates of cercariae and of adult worms. The expression of the protein in the pGEX-2T fusion system allowed us to show the presence of specific antibodies in S. mansoni infected patients' sera and in the sera of patients with systemic lupus erythematosus, reflecting a cross-immunoreactivity between the S. mansoni protein and the human calreticulin autoantigen.

Tissue localization and stage-specific expression of the phospholipid hydroperoxide glutathione peroxidase of Schistosoma mansoni.

The tissue localization and the stage-specific expression of the phospholipid hydroperoxide glutathione peroxidase of Schistosoma mansoni (SmPHGSHpx) have been determined. An antiserum raised against the C-terminal region of the predicted protein sequence was used for immunocytochemical investigations. The native protein is expressed only in female and egg vitelline cells and is practically absent from male worm tissue. Western blot data confirmed these results and showed the complete absence of SmPHGSHpx from cercariae. However, Northern blotting indicated the presence of the corresponding mRNA at all life-cycle stages investigated. The sequence determination of the 5' flanking region of the SmPHGSHpx gene revealed the presence of an extended TATA box (5'-TAAATA-3') at -32, a possible CAAT box at -75 and a putative monomeric estrogen response element 5'-GGTCAA-3' at position -486. In addition, direct and inverted repeat elements are present.

Translation of Schistosoma mansoni antigens in Xenopus oocytes microinjected with mRNA from adult worms.

Oocytes from Xenopus laevis microinjected with RNA isolated from Schistosoma mansoni adult worms translated antigens recognized by sera from infected rats, humans, and from immunized rabbits. The pattern of immunoprecipitated proteins analysed by SDS-polyacrylamide gel electrophoresis was species specific in rats. Serum from infected Fischer rats recognized antigens of 20, 27 and several bands in the 50-60 kDa range whereas serum from infected Brown Norway rats also immunoprecipitated major bands at 29, 43 and 100 kDa. Human infection sera gave a very variable pattern of immunoprecipitation not apparently dependent on the patients' age. At least 20 different antigenic species could be identified ranging from 14 to 150 kDa. Some S. mansoni antigenic proteins could be isolated from the membrane fraction of the oocytes whereas notably the 29 kDa band was present mainly in the soluble fraction. N-Glycosylation of S. mansoni antigens occurred as evidenced by the effects of tunicamycin treatment and concanavalin A binding. A multiple series of bands between 50 and 60 kDa, present in the membrane fraction, were glycosylated and secreted from the oocytes. Monoclonal antibodies to larval stage surface antigens failed to immunoprecipitate oocyte translation products, but sera absorbed with live schistosomula identified at least three putative surface antigens of 100, 43 and 29 kDa. However, the 29 kDa molecule was neither synthesized into membranes, nor secreted from oocytes.

Inter-species variation of schistosome 28-kDa glutathione S-transferases.

The 28-kDa glutathione S-transferase from Schistosoma mansoni (Sm28GST) is a candidate vaccine antigen. To evaluate the antigenic and phylogenetic variations between the 28-kDa GSTs from 4 species of schistosome, we have cloned and sequenced the 28-kDa GSTs from Schistosoma haematobium (Sh28GST) and Schistosoma bovis (Sb28GST). Sb28GST and Sh28GST are more similar to each other (97%) than to Sm28GST (90%) and particularly to the 28-kDa GST from Schistosoma japonicum (Sj28GST, 77%). Antisera directed against the major Sm28GST epitopes revealed differences in the recognition of the 28-kDa GSTs from the other schistosome species suggesting that these regions have been subjected to evolutionary pressure. The consequences of such species-specific epitopes on the development of a multi-species anti-schistosome vaccine are discussed.

Molecular cloning and tissue distribution of a 26-kilodalton Schistosoma mansoni glutathione S-transferase.

A Schistosoma mansoni cDNA library was constructed from the mRNA of adult worms in the expression vector lambda gt11 and screened with a rabbit antiserum raised against the 26-kDa S. mansoni glutathione S-transferase isoforms (Sm GST 26). Two clones were selected and the nucleotide sequences deduced. The predicted amino acid sequence, specified by these cDNAs, shows strong homology with a Schistosoma japonicum 26 kDa glutathione S-transferase and a lower level of homology with mammalian glutathione S-transferase class mu isoenzymes (EC 2.5.1.18). No significant homology score was found with a 28-kDa S. mansoni glutathione S-transferase (Sm GST 28). A study of the tissue distribution of the cloned Sm GST 26 by immunoelectron microscopy shows similarities to Sm GST 28 in that they are present in the tegument and in subtegumentary parenchymal cells. However, a major difference exists in the protonephridial region in which Sm GST 26 is present in the cytoplasmic digitations localized in the apical chamber delineated by the flame cell body, suggesting that Sm GST 26 may be actively excreted by adult worms.

Comparison of intravenous and inhaled terbutaline in the treatment of asthma.

In a double-blind crossover controlled study, intravenous (IV) or nebulized terbutaline was given to eight patients with moderately severe asthma on two separate days. Four incremental doses of terbutaline were given by each route to establish a maximal effect. Both routes of administration produced significant increases increases in FEV1, FVC, PEFR, MEF50% single-breath TLC, and effective pulmonary blood flow. A decrease in slope of alveolar argon plateau was observed with both routes, but helium responsiveness showed variable changes with no significant or consistent effect seen. There was no significant difference between responses to incremental doses and maximal response apart from pulse rate, which rose during IV treatment. These results showed that the IV route had no advantage in terms of effectiveness or site of action over the inhaled route. Since IV treatment can produce systemic side effects, inhaled bronchodilator therapy should be used as the route of choice.

Impact of different criteria for defining hypopneas in the apnea-hypopnea index.

OBJECTIVES: To explore the effect of using different scoring criteria for hypopneas in the scoring of polysomnographic studies: (1) by estimating the level of agreement between apnea-hypopnea index (AHI) scores derived from different scoring methods, and (2) by examining the effect on the point prevalence of disease using different threshold values of the AHI. DESIGN: Retrospective analysis of 48 diagnostic polysomnographic records. SETTING: Tertiary-hospital sleep-disorders clinic. MEASUREMENTS: AHIs were derived from three different methods for scoring hypopneas. The hypopnea definitions used incorporated different combinations and threshold values of respiratory signal changes in addition to differences in the requirement for associated oxygen desaturation or arousal. The level of agreement between different scoring methods was assessed by constructing Bland-Altman plots and calculating intraclass correlation coefficients (ICCs). kappa statistics were used to assess agreement between the different methods using varying thresholds of AHI to categorize sleep apnea (AHI > 5, AHI > 15, and AHI > 20). RESULTS: The random-effects ICC for the three methods was 0.89, suggesting that the different scoring methods tended to rank patients fairly consistently. However, the point prevalence of disease estimated by using different thresholds of AHI was found to vary depending on the method used to score sleep studies (kappa, 0.30 to 0.95). CONCLUSIONS: These findings have implications for case finding, population-prevalence estimates, and grading of disease severity for access to government-funded continuous positive airway pressure services. Guidelines for standardizing the measurement and reporting of sleep studies in clinical practice should be implemented.

Threshold pressure training, breathing pattern, and exercise performance in chronic airflow obstruction.

The effects of six weeks of threshold pressure inspiratory muscle training (IMT) on inspiratory muscle performance, breathing pattern and exercise performance were studied in eight patients with severe airflow obstruction. The results indicated that IMT improved inspiratory muscle performance but did not affect exercise performance or breathing pattern during maximal exercise.

Intrathoracic manifestations of disseminated prostatic adenocarcinoma.

Four cases of disseminated adenocarcinoma of the prostate illustrating the clinical spectrum of intrathoracic involvement in this disease are presented. In two cases the presenting features of prostatic cancer were with lymphangitis carcinomatosa and an isolated pleural effusion, whereas two other cases developed intrathoracic metastases in the setting of previously known locally advanced prostatic cancer. In one this took the form of hilar and mediastinal lymphadenopathy and in the other that of pulmonary nodules. An immuno-cytochemical marker for prostatic specific antigen, a highly sensitive and specific tool for identifying prostatic epithelium, identified the prostate as the primary site of malignancy in the first two cases. Symptomatic and radiological responses were noted in all four cases after bilateral orchidectomy. Pulmonary metastases are common in the advanced stages of prostatic cancer but may also be present at the initial presentation with the disease even when the primary tumour is not clinically apparent. We recommend that (i) immuno-cytochemical stains for prostatic specific antigen are applied to all lung, pleural and mediastinal biopsy specimens showing adenocarcinoma in male patients, and (ii) all males with intrathoracic adenocarcinoma have prostatic aspiration cytology performed if the prostatic specific antigen stain is positive.

Chronic persistent cough: use of ipratropium bromide in undiagnosed cases following upper respiratory tract infection.

The clinical effects of inhaled ipratropium bromide were studied in 14 non-smoking patients with persistent post-viral infective cough employing a controlled double-blind, cross-over trial. Patients were selected if they demonstrated no apparent underlying cause for their persistent cough after appropriate radiological and respiratory function tests including methacholine reactivity and bronchoscopic examination. Inhaled ipratropium bromide (320 micrograms day-1) produced significantly less day and night time cough (P < 0.05) with overall clinical improvement in 12 cases, five of whom had total resolution of their cough. We conclude that ipratropium bromide is an effective treatment in non-smoking adults with protracted cough following clinical upper respiratory tract infection.

Clinical, pathologic, and microbiologic findings of foot abscess in neonatal pigs.

A longitudinal study was undertaken in a swine herd with an ever-present problem of foot abscess in suckling pigs reared on a woven-wire floor. Of 3,322 4-day-old pigs, 199 (6%) developed abscess lesions involving claws and accessory digits before weaning. Lesions were first detected in 4-day-old pigs; median and mean ages at onset were 10 and 11.3 days, respectively. At first detection, most pigs had only a single claw affected, but 39 pigs had at least 2 claws with abscesses. Hind limbs had more affected claws (140) than forelimbs (96). In the hind limbs, medial claws were most likely to have lesions, whereas the reverse was true for the forelimbs. Gross and microscopic examinations of affected claws indicated necrotic pododermatitis, with severe osteomyelitis, arthritis, and tenosynovitis. Bacteria isolated from foot abscess lesions included Actinomyces pyogenes, Staphylococcus spp, beta-hemolytic Streptococcus spp, Actinobacillus spp, Escherichia coli, Fusobacterium spp, Bacteroides spp, and Peptostreptococcus spp.