Design and Analysis of a Clinical Pressure Cuff Algometer, Compatible with an fMRI Machine.

Chronic pain affects both adolescent and adult Canadians. To study the effect of social rejection on pain management in adolescents with chronic pain, an algometer can be used in conjunction with functional Magnetic Resonance Imaging (fMRI) to measure brain activity in real time. The algometer uses an automated pneumatic control system that follows a customizable pain schedule, controlling the amount of airflow in and out of a pressure cuff wrapped around a human participant's thigh. Plastic components allow compatibility with an fMRI environment. Measurable pain stimuli allow repeatable pressure schedules to be administered with a standard deviation between trials of 300 Pa (2.25 mmHg). A Failure Mode Effects Analysis was used to reduce participant, researcher and facility harm, with multiple safety features incorporated into the design. Through the analysis of medical standards and studies, the algometer is shown to be biologically safe to use on research subjects within the suggested usage parameters of a maximum pressure of 42.6 kPa (320 mmHg) and a pressure application period of up to one hour. This makes it feasible for research studies using fMRI machines.

Assessment of interbreeding and introgression of farm genes into a small Scottish Atlantic salmon Salmo salar stock: ad hoc samples - ad hoc results?

An eclectic set of tissues and existing data, including purposely collected samples, spanning 1997-2006, was used in an ad hoc assessment of hybridization and introgression of farmed wild Atlantic salmon Salmo salar in the small Loch na Thull (LnT) catchment in north-west Scotland. The catchment is in an area of marine farm production and contains freshwater smolt rearing cages. The LnT S. salar stock was found to be genetically distinctive from stocks in neighbouring rivers and, despite regular reports of feral farm S. salar, there was no evidence of physical or genetic mixing. This cannot be completely ruled out, however, and low level mixing with other local wild stocks has been suggested. The LnT population appeared underpinned by relatively smaller effective number of breeders (Neb ) and showed relatively low levels of genetic diversity, consistent with a small effective population size. Small sample sizes, an incomplete farm baseline and the use of non-diagnostic molecular markers, constrain the power of the analysis but the findings strongly support the LnT catchment having a genetically distinct wild S. salar population little affected by interbreeding with feral farm escapes.

Marker genes for activation of the RNA interference (RNAi) pathway in the free-living nematode Caenorhabditis elegans and RNAi development in the ovine nematode Teladorsagia circumcincta.

The nematode Teladorsagia circumcincta is a major cause of parasitic gastroenteritis in sheep in temperate regions. The development of resistance to the major anthelmintic classes used for its control is a threat to small ruminant farming sustainability. Vaccination is a potential alternative control method for this nematode. Gene datasets can be exploited to identify potential vaccine candidates and these validated further by methods such as RNA interference (RNAi) prior to vaccine trials. Previous reports indicate that RNAi in parasitic nematodes is inconsistent and, to date, there are no internal controls that indicate activation of the RNAi pathway in response to double-stranded RNA (dsRNA). The present aims were to determine whether or not the transcription levels of potential marker genes in the RNAi pathway could indicate activation of the pathway in Caenorhabditis elegans and to develop an RNAi platform in T. circumcincta. In C. elegans, transcript levels of three candidate marker genes, Ce-dcr-1 (Dicer), Ce-ego-1 (Enhancer of Glp-One family member) and Ce-rsd-3 (RNAi Spreading Defective), were analysed and results indicated that activation of the pathway had no effect on transcript levels of these genes. In T. circumcincta, two vaccine candidate genes from the Activation-associated Secreted Protein (ASP) family were targets for knockdown. RNAi experiments showed successful silencing of both targets, although inconsistencies in efficacy were observed. After testing a number of parameters that might affect variability, it was found that the length of the storage period of the larvae plays an important role in the consistency of the RNAi results.

Elbow dislocation with complete triceps avulsion.

Radio-ulnar Fracture dislocation of the elbow is a high-energy trauma which can be associated with significant ligamentous injury in adults. We report an unusual triad of injury in a patient with avulsion injury of the triceps. This injury can be thought of as a variant of "terrible triad" with dislocation of radio-ulnar joint, radial head fracture, and medial collateral ligament injury with avulsion of the triceps. Elbow has to be stabilized with early repair of the ligaments for a successful outcome.

Amelioration of the periparturient relaxation of immunity to parasites through a reduction in mammalian reproductive effort.

The degree of periparturient relaxation of immunity to gastrointestinal parasites has a nutritional basis, as overcoming protein scarcity through increased protein supply improves lactational performance, enhances local immune responses and reduces worm burdens. Herein lactating rats, re-infected with Nippostrongylus brasiliensis, are used to test the hypothesis that a similar and rapid improvement of immunity can be achieved through reducing nutrient demand at times of dietary protein scarcity. Reducing litter size from 12 to three pups during lactation resulted, as expected, in cessation of maternal body weight loss and increased pup body weight gain compared with dams which continued to nurse 12 pups. This increase in performance concurred with a rapid decrease in parasitism; within 3 days post nutrient reduction, a 87% reduction in the number of worm eggs found in the colon and 83% reduction in worm burdens was observed, which concurred with increased local immune responses, i.e. 70% more mast cells and 44% more eosinophils in the small intestinal mucosa, to levels similar to those in dams nursing three pups throughout. However, there were no concurrent changes in goblet cell hyperplasia, serum anti-N. brasiliensis-specific antibody levels or mRNA expression of IL-4, IL-10 or IL-13 in the mesenteric lymph nodes. To our knowledge the current study is the first to employ a litter reduction strategy to assess the rate of immune improvement upon overcoming nutrient scarcity in a non-ruminant host. These data support the hypothesis that periparturient relaxation of immunity to gastrointestinal nematodes can be reduced by restoring nutrient adequacy and, importantly, that this improvement can occur very rapidly.

Glucocorticoid receptors and extinction retention deficits in the single prolonged stress model.

Single prolonged stress (SPS) is a rodent model of post traumatic stress disorder that is comprised of serial application of restraint (r), forced swim (fs), and ether (eth) followed by a 7-day quiescent period. SPS induces extinction retention deficits and it is believed that these deficits are caused by the combined stressful effect of serial exposure to r, fs, and eth. However, this hypothesis remains untested. Neurobiological mechanisms by which SPS induces extinction retention deficits are unknown, but SPS enhances glucocorticoid receptor (GR) expression in the hippocampus, which is critical for contextual modulation of extinction retrieval. Upregulation of GRs in extinction circuits may be a mechanism by which SPS induces extinction retention deficits, but this hypothesis has not been examined. In this study, we systematically altered the stressors that constitute SPS (i.e. r, fs, eth), generating a number of partial SPS (p-SPS) groups, and observed the effects SPS and p-SPSs had on extinction retention and GR levels in the hippocampus and prefrontal cortex (PFC). PFC GRs were assayed, because regions of the PFC are critical for maintaining extinction. We predicted that only exposure to full SPS would result in extinction retention deficits and enhance hippocampal and PFC GR levels. Only exposure to full SPS induced extinction retention deficits. Hippocampal and PFC GR expression was enhanced by SPS and most p-SPSs, however hippocampal GR expression was significantly larger following the full SPS exposure than all other conditions. Our findings suggest that the combined stressful effect of serial exposure to r, fs, and eth results in extinction retention deficits. The results also suggest that simple enhancements in GR expression in the hippocampus and PFC are insufficient to result in extinction retention deficits, but raise the possibility that a threshold-enhancement in hippocampal GR expression contributes to SPS-induced extinction retention deficits.

Subcutaneous anthrax in three intravenous drug users: a new clinical diagnosis.

Anthrax is extremely rare in the western world but is endemic to areas of south and central Asia. In early 2010 an outbreak was identified in heroin-injecting intravenous drug users in the United Kingdom and Europe. Afghanistan is currently the principal source of heroin which reaches the United Kingdom. When anthrax occurs, cutaneous disease accounts for over 95% of cases. At least 47 cases with 13 deaths have been confirmed so far. We present three cases presenting during this time with marked swelling, one resulting in compartment syndrome but all with an absence of the expected cutaneous appearances. We suggest that rather than cutaneous anthrax, these patients represent a new subcutaneous presentation of anthrax.

Dissecting the impact of protein versus energy host nutrition on the expression of immunity to gastrointestinal parasites during lactation.

Many mammals exhibit a periparturient relaxation of previously established immune responses (PPRI) to gastrointestinal nematodes culminating in increased worm burdens. It has been suggested that the extent of PPRI may have a nutritional basis as it is considerably augmented when protein supply is scarce. Subsequent studies have shown that increased dietary protein intake can ameliorate this phenomenon. However, this effect is often confounded with increased food intake and thus increased energy levels. Herein, we aimed to dissect the effects of protein and energy nutrition on the immune status and resistance to re-infection with gastrointestinal nematodes in the periparturient host. The lactating, Nippostrongylus brasiliensis re-infected rat was utilised as an established model for mammalian PPRI. Experimental animals were assigned to restricted feeding regimens designed to achieve four pre-determined levels of crude protein (CP) at one of two levels of metabolisable energy (ME) and parasitological and immunological measurements taken at either day 6 or day 9 post re-infection. We clearly show that increased supply of dietary CP, but not increased dietary ME, significantly reduced worm burdens. The increased magnitude of worm expulsion with increased dietary CP supply strongly correlated with mucosal mast cell accumulation in the small intestine. In addition, increased CP and not ME supply increased mucosal eosinophil numbers. Furthermore, increased CP led to higher levels of total IgG at high ME only and there were interactive effects of CP and ME on serum levels of IgG1 and IgG2a. Perhaps surprisingly, CP nutrition did not affect expression of either Th1 (IFN-γ) or Th2 (IL-4, IL-13) cytokines in the mesenteric lymph nodes. These data emphasise the role of immunonutrition, and particularly dietary protein, in combating infectious disease such as gastrointestinal parasitism.

Microsatellite standardization and evaluation of genotyping error in a large multi-partner research programme for conservation of Atlantic salmon (Salmo salar L.).

Microsatellite genotyping is a common DNA characterization technique in population, ecological and evolutionary genetics research. Since different alleles are sized relative to internal size-standards, different laboratories must calibrate and standardize allelic designations when exchanging data. This interchange of microsatellite data can often prove problematic. Here, 16 microsatellite loci were calibrated and standardized for the Atlantic salmon, Salmo salar, across 12 laboratories. Although inconsistencies were observed, particularly due to differences between migration of DNA fragments and actual allelic size ('size shifts'), inter-laboratory calibration was successful. Standardization also allowed an assessment of the degree and partitioning of genotyping error. Notably, the global allelic error rate was reduced from 0.05 ± 0.01 prior to calibration to 0.01 ± 0.002 post-calibration. Most errors were found to occur during analysis (i.e. when size-calling alleles; the mean proportion of all errors that were analytical errors across loci was 0.58 after calibration). No evidence was found of an association between the degree of error and allelic size range of a locus, number of alleles, nor repeat type, nor was there evidence that genotyping errors were more prevalent when a laboratory analyzed samples outside of the usual geographic area they encounter. The microsatellite calibration between laboratories presented here will be especially important for genetic assignment of marine-caught Atlantic salmon, enabling analysis of marine mortality, a major factor in the observed declines of this highly valued species.

A calcium-activated apyrase from Teladorsagia circumcincta: an excretory/secretory antigen capable of modulating host immune responses?

A cDNA representing the gene Teladorsagia circumcincta apyrase-1 (Tci-apy-1) was isolated, by PCR, from a T. circumcincta fourth-stage larval (L4) cDNA library. The closest orthologue of this gene is a Ca(2+)-dependent apyrase from Ostertagia ostertagi, with 92% amino acid identity across all 339 residues. Tci-apy-1 is transcribed in a stage-specific manner, the transcript being predominant in L4, detectable in the adult cDNA, but absent from eggs and infective third-stage larvae (L3). The protein, Tci-APY-1, was detected by immunoblotting in extracts of L4 nematodes and was present in excretory/secretory products from the same developmental stage. A recombinant version of Tci-APY-1 was expressed in bacteria as an active enzyme that hydrolysed nucleoside triphosphate substrates with a preference of ATP over other nucleoside triphosphates. Recombinant Tci-APY-1 hydrolysed ATP and ADP but not AMP. Apyrase activity was divalent cation-dependent, with no hydrolysis in the presence of Mg(2+), but activation in the presence of Ca(2+). Recombinant Tci-APY-1 was bound by IgG present in serum and both IgG and IgA present in abomasal mucus from trickle-infected, immune sheep but not in material derived from lambs exposed to a single infection. The potential immunomodulatory roles of this Tci-APY-1 are discussed in relation to purinergic signalling.

Using lectins to identify hidden antigens in Fasciola hepatica.

Fasciola hepatica is the causative agent of fascioliasis, one of the most economically important helminth diseases of livestock worldwide. Traditionally, fascioliasis has been controlled by the strategic use of fasciolicidal drugs, but the emergence of resistant parasites has spurred an interest in developing vaccines as an alternative means of control. Most vaccine studies to date have evaluated conventional antigens, which are exposed to the host's immune system during the course of a natural infection. 'Hidden' antigens have proven to be effective vaccine candidates in other parasite species, most notably the blood-feeding nematode parasite, Haemonchus contortus, and tend to be expressed in the intestine or gut of the parasite. Fasciola hepatica is known to ingest large quantities of blood and may be vulnerable to this approach. Most, if not all, of the candidate antigens identified thus far have been membrane-bound glycoproteins which were solubilized by detergents. Here, we have attempted to employ lectins to select gut-associated glycoproteins from complex mixtures of somatic extracts of adult F. hepatica. We have conducted a comprehensive lectin-binding screen on adult histological sections with a panel of 16 fluorescently labelled lectins. Seven of the lectins bound to molecules within the gastrodermis but also bound to a range of other tissues. Within the gut tissues, jacalin and peanut lectins bound selectively to the gut lamellae and gastrodermal cells, respectively. These lectins were then used to isolate proteins from the integral membrane protein component of the adult fluke. Both lectins showed selectivity for relatively simple subsets of proteins compared to the original crude extracts.

A macrophage migration inhibitory factor-like tautomerase from Teladorsagia circumcincta (Nematoda: Strongylida).

A macrophage migration inhibitory factor (MIF)-like molecule, Tci-MIF-1, was isolated from Teladorsagia circumcincta and subjected to detailed characterization. A cDNA representing Tci-mif-1 was isolated following its identification in third-stage larvae (L3)-enriched cDNA population. Sequencing of the cDNA indicated a 348-bp open reading frame (ORF) with the closest orthologue being a MIF derived from the human hookworm Ancylostoma ceylanicum. Messenger RNA (mRNA) representing the Tci-MIF-1 transcript was detected in eggs, L3 and adult stages of T. circumcincta. The transcript was also present, but to a lesser extent in fourth-stage larvae (L4). Detection of Tci-MIF-1 protein in T. circumcincta developmental stages reflected the transcript levels identified by reverse transcriptase-PCR. Using immunohistochemistry, the Tci-MIF-1 protein was shown to have a diffuse distribution in L3 tissue, and in L4 and adult stages, the protein was localized to the nematode gut. A recombinant version of Tci-MIF-1 was produced, and enzymic assays indicated that this recombinant protein and a somatic extract of L3 possessed dopachrome tautomerase activity as has been observed previously in other MIF-like molecules. Neither native, purified Tci-MIF nor recombinant Tci-MIF-1 dramatically influenced the in vitro migration of sheep monocytes.

daf-7-related TGF-beta homologues from Trichostrongyloid nematodes show contrasting life-cycle expression patterns.

The transforming growth factor-beta (TGF-beta) gene family regulates critical processes in animal development, and plays a crucial role in regulating the mammalian immune response. We aimed to identify TGF-beta homologues from 2 laboratory model nematodes (Heligmosomoides polygyrus and Nippostrongylus brasiliensis) and 2 major parasites of ruminant livestock (Haemonchus contortus and Teladorsagia circumcincta). Parasite cDNA was used as a template for gene-specific PCR and RACE. Homologues of the TGH-2 subfamily were isolated, and found to differ in length (301, 152, 349 and 305 amino acids respectively), with variably truncated N-terminal pre-proteins. All contained conserved C-terminal active domains (>85% identical over 115 amino acids) containing 9 cysteine residues, as in C. elegans DAF-7, Brugia malayi TGH-2 and mammalian TGF-beta. Surprisingly, only the H. contortus homologue retained a conventional signal sequence, absent from shorter proteins of other species. RT-PCR assays of transcription showed that in H. contortus and N. brasiliensis expression was maximal in the infective larval stage, and very low in adult worms. In contrast, in H. polygyrus and T. circumcincta, tgh-2 transcription is higher in adults than infective larvae. The molecular evolution of this gene family in parasitic nematodes has diversified the pre-protein and life-cycle expression patterns of TGF-beta homologues while conserving the structure of the active domain.

Immunomodulatory effects of dietary protein during Nippostrongylus brasiliensis re-infection in lactating rats.

Periparturient relaxation of immunity (PPRI) to secondary infection with nematodes is believed to have a nutritional basis due to differential partitioning of scarce nutrient resources, particularly protein, to reproductive rather than immune functions. At times of protein scarcity, an increase in protein supply has been reported to assuage this phenomenon. The Nippostrongylus brasiliensis reinfected lactating rat model is now being utilized to investigate the immune reactions underlying the modifying role of dietary protein on PPRI. Herein, we demonstrate that lactating rats reinfected with N. brasiliensis under high protein (HP) dietary conditions exhibit decreased worm burdens and reduced colon egg counts compared to their low protein (LP) counterparts. These reductions correlated with increased mastocytosis and greater goblet cell hyperplasia. Additionally, the local antibody profile revealed that HP reinfected lactating rats developed a stronger antigen specific IgG2b response earlier in infection in comparison with their LP counterparts. Our study provides evidence that increased dietary protein content reduces the PPRI to N. brasiliensis re-infection in the lactating rat through improved mucosal immune responses.

Molecular characterization, expression and localization of a peroxiredoxin from the sheep scab mite, Psoroptes ovis.

The sheep scab mite, Psoroptes ovis, induces an intensely pruritic exudative dermatitis which is responsible for restlessness, loss of appetite and weight loss. Within the first 24 h of infection, there is a rapid inflammatory influx of eosinophils and apoptosis of the keratinocytes at the site of infection. The former cell type is capable of a sustained respiratory burst, toxic products of which may directly damage the mite and also contribute to lesion formation. Analysis of a P. ovis expressed sequence tag (EST) database identified a number of antioxidant enzyme-encoding sequences, including peroxiredoxin (thioredoxin peroxidase EC 1.11.1.15), all of which may help the mite endure the potentially toxic skin environment. A full length sequence encoding Po-TPx, a protein of 206 amino acids which showed high homology to a peroxiredoxin from the salivary gland of the tick Ixodes scapularis, was amplified from P. ovis cDNA. Recombinant Po-TPx was expressed in bacteria and antiserum to this protein was used to localize native Po-TPx in mite sections. Peroxiredoxin was localized, amongst other sites, to a subpharyngeal region in mite sections. The recombinant protein was recognized by sera from sheep infested with the mite suggesting that it may be secreted or excreted by the mite and interact with the host immune response.

Immune recognition of the surface associated antigen, Tc-SAA-1, from infective larvae of Teladorsagia circumcincta.

A cDNA encoding a surface-associated antigen was amplified by reverse transcriptase polymerase chain reaction (PCR) from RNA extracted from Teladorsagia circumcincta exsheathed third stage larvae (xL3). The protein encoded by this cDNA, Tc-SAA-1, displays 77% identity over 162 amino acid residues to a surface associated antigen from Ancylostoma caninum (Ac-SAA-1). Antiserum raised against a bacterially-expressed recombinant form of Tc-SAA-1 reacted with a native protein in somatic and surface extracts of xL3 but not with L4 or adult parasites. Limited binding of anti-Tc-SAA-1 antibody was observed on the cuticular surface of xL3 s, however, regions of localization underlying the cuticle were observed. Incubation of xL3 T. circumcincta with anti-SAA rabbit serum failed to significantly inhibit penetration of the abomasal mucosa in vitro. IgA in abomasal mucus derived from sheep that had received a trickle infection of T. circumcincta bound recombinant Tc-SAA-1.

Analysis of the transthyretin-like (TTL) gene family in Ostertagia ostertagi--comparison with other strongylid nematodes and Caenorhabditis elegans.

The transthyretin-like (ttl) gene family is one of the largest conserved nematode-specific gene families, coding for a group of proteins with significant sequence similarity to transthyretins (TTR) and transthyretin-related proteins (TRP). In the present study, we investigated the ttl family in Ostertagia ostertagi (a nematode of the abomasum of cattle). Mining of expressed sequence tag (EST) databases revealed the presence of at least 18 ttl genes in O. ostertagi (Oo-ttl), most of which are constitutively transcribed from the free-living, third larval stage onwards. The full-length cDNA of one of these genes (Oo-ttl-1) was amplified and cloned for recombinant expression. Western blot analysis using a specific antiserum showed that the native protein Oo-TTL-1 was highly present in the excretory-secretory (ES) products of adults of O. ostertagi. The protein was immunolocalized to the pseudocoelomic fluid of adult worms. A phylogenetic-bioinformatic analysis of all amino acid sequence data for TTL proteins from a range of strongylid nematodes showed that they could be divided into at least five different classes. This classification was based on conserved amino acids in the first TTL signature domain and the number and location of cysteine residues. The biological role(s) of the TTLs in nematode biology is still unclear. A theoretical three-dimensional model of Oo-TTL-1 indicated that it had a similar structure to TTRs (i.e., containing ß-sheets, arranged in a ß-sandwich). In contrast to TTRs, competitive binding studies using recombinant Oo-TTL-1 indicated that the protein was devoid of any hydrophobic ligand- or thyroid hormone-binding properties. Finally, combinatorial analysis by double-stranded RNA interference of five ttl genes in the free-living nematode Caenorhabditis elegans did not reveal any visible phenotypes. More information on the transcription profile and tissue distribution of TTLs in nematodes is needed to provide new insights into the biological role of this gene family.

Control of parasitic disease using vaccines: an answer to drug resistance?

Antiparasitic drugs have been used successfully to control parasitic diseases in animals for many years, as they are safe, cheap and effective against a broad spectrum of parasites. One drawback of this success appears to be the emergence of drug resistance in many target parasites. Moreover, issues of residues in the food chain and environment have arisen, which threaten their sustained use. Control methods in which vaccines would have a central role provide attractive alternatives. However, while attenuated parasite vaccines have been successful, sub-unit vaccines are still rare. The advent of new techniques in molecular biology allows the elucidation of entire parasite genomes and the identification of individual genes. It is envisaged that a further understanding of parasite genes and the role of their products in parasite biology may lead to the identification of useful antigens, which could then be produced in recombinant systems. However, for this aim to be realised, continued investment in basic research on the complex interplay between parasite and host will be necessary.

Proteinase inhibitors and helminth parasite infection.

The concept that parasites may utilize proteinase inhibitors to survive within the host has been with us for 100 years. Given that we now know that proteinases are involved in key areas of the host anti-parasite immune response including antigen presentation, effector cell function and tissue dissolution and remodelling, it is somewhat surprising that the proteinase inhibitors of parasite origin have not generally been the subject of intense research effort. There is now substantial evidence to show that nematode parasites utilize these inhibitors to protect themselves from degradation by host proteinases, to facilitate feeding and to manipulate the host response to the parasite. The diversity of the parasite-derived inhibitors is also being revealed and they target the four major proteinase classes, namely serine, cysteine, aspartic and metallo-proteinases. This review summarizes the information available on nematode-derived proteinase inhibitors and what is known of their putative functions. Their potential as targets for immunological control is also addressed.