Streptococcus pyogenes infection of tonsil explants is associated with a human ß-defensin 1 response from control but not recurrent acute tonsillitis patients.

Host defence peptides (HDP), including the defensins and hCAP-18, function as part of the innate immune defences, protecting the host epithelia from microbial attachment and invasion. Recurrent acute tonsillitis (RAT), in which patients suffer repeated symptomatic tonsil infections, is linked to Streptococcus pyogenes, a group A streptococcus, and may reflect the impaired expression of such peptides. To address this, the defensin and hCAP-18 messenger RNA expression profiles of 54 tonsils excised from control and RAT patients undergoing tonsillectomy were quantified and compared. Marked variation in expression was observed between individuals from the two groups, but statistically no significant differences were identified, suggesting that at the time of surgery the tonsil epithelial HDP barrier was not compromised in RAT subjects. Surgical removal of the tonsils occurs in a quiescent phase of disease, and so to assess the effects of an active bacterial infection, HaCaT cells an in vitro model of the tonsil epithelium, and explants of patient tonsils maintained in vitro were challenged with S. pyogenes. The HaCaT data supported the reduced expression of hCAP-18/LL-37, human ß-defensin 1 (HBD1;P < 0.01) and HBD2 (P < 0.05), consistent with decreased protection of the epithelial barrier. The tonsil explant data, although not as definitive, showed similar trends apart from HBD1 expression, which in the control tonsils but not the RAT patient tonsils was characterized by increased expression (P < 0.01). These data suggest that in vivo HBD1 may play a critical role in protecting the tonsil epithelia from S. pyogenes.

Cytokine expression in naïve and previously infected lambs after challenge with Teladorsagia circumcincta.

Infection of sheep with Teladorsagia circumcincta triggers an immune response with predominantly type-2 (Th2) characteristics, including local eosinophila, mastocytosis and increased mucus production. In order to better understand the protective immune responses elicited, we used RT-PCR assays to define the changes in expression levels of a range of cytokine transcripts in lymph nodes draining the ovine abomasum following a challenge infection with T. circumcincta. This study compared the changes in cytokine expression in the abomasal lymph node following challenge with T. circumcincta in naïve sheep (Group 2) and sheep immunised by a previous trickle infection (Group 3), in comparison to unchallenged naive sheep (Group 1). There was a significant up-regulation of interleukin-4 (IL-4), IL-5 and IL-13 in both the challenged groups compared to naïve individuals. There was also an up-regulation of IL-1beta, IL-6, IL-10, IL-18, transforming growth factor-beta1 (TGFbeta1) and tumour necrosis factor-alpha (TNFalpha) by day 5 after infection. IL-12p40 was found to be increased in the previously infected Group 3 animals by day 5 following challenge. By contrast, transcription of this cytokine was found to be reduced by day 10 following infection of Group 2 animals. Expression of IL-2 and Interferon-gamma (IFNgamma) did not significantly differ between the three groups.

Molecular characterization of a family of metalloendopeptidases from the intestinal brush border of Haemonchus contortus.

Substantial protection against the economically important parasitic nematode Haemonchus contortus has been achieved by immunizing sheep with a glycoprotein fraction isolated from the intestinal membranes of the worm (H-gal-GP). Previous studies showed that one of the major components of H-gal-GP is a family of at least 4 zinc metalloendopeptidases, designated MEPs 1-4. This paper describes aspects of the molecular architecture of this protease family, including the proteomic analysis of the MEP fraction of the H-gal-GP complex. These enzymes belong to the M13 zinc metalloendopeptidase family (EC 3.4.24.11), also known as neutral endopeptidases or neprilysins. The sequences of MEPs 1 and 3 suggested a typical Type II integral membrane protein structure, whilst MEPs 2 and 4 had putative cleavable signal peptides, typical of secreted proteins. Proteomic analysis of H-gal-GP indicated that the extracellular domain of all 4 MEPs had been cleaved close to the transmembrane region/signal peptide with additional cleavage sites mid-way along the polypeptide. MEP3 was present as a homo-dimer in H-gal-GP, whereas MEP1 or MEP2 formed hetero-dimers with MEP4. It was found that expression of MEP3 was confined to developing 4th-stage larvae and to adult worms, the stages of Haemonchus which feed on blood. MEP-like activity was detected in the H-gal-GP complex over a broad pH range (5-9). Since all 4 MEPs must share a similar microenvironment in the complex, this suggests that each might have a different substrate specificity.

Protection induced by vaccinating sheep with a thiol-binding extract of Haemonchus contortus membranes is associated with its protease components.

Previous work has shown that a protein extract enriched for cysteine protease activity (TSBP) prepared from adult Haemonchus contortus using thiol sepharose affinity chromatography confers substantial protection against a single challenge infection. TSBP comprised proteases and other proteins. Here, TSBP were further fractionated using anion-exchange chromatography and fractions pooled on the basis of containing (1) protease activity, (2) a prominent non-protease peptide and (3) material which did not bind to the column. A protection trial showed that only the protease-enriched material conferred protective immunity and this was comparable to that observed in a TSBP-immunized positive control group. Immunization stimulated a marked IgG response with the IgG2 isotype predominating.

Immunization against sheep scab: preliminary identification of fractions of Psoroptes ovis which confer protective effects.

In an attempt to enrich for potentially protective Psoroptes ovis antigens, three separate vaccine trials were conducted in which groups of sheep were immunized three times with various fractions of a soluble extract of P. ovis mites using QuilA as adjuvant. These groups, as well as controls that received adjuvant only, were challenged with P. ovis, and protective immunity was assessed by measuring lesion areas and conducting mite counts 4 and 6 weeks later. All fractions stimulated high titre serum antibodies. Most conferred some protection on sheep with active disease, although there was considerable variation between sheep in all groups, including the controls. Some fractions were more protective than the extract itself, suggesting that the protective components had been concentrated. Indeed the best fraction, obtained by ion exchange chromatography, followed by a gel filtration step, slowed lesion growth to less than a third by 6 weeks after challenge and reduced mite numbers by more than 13 times compared to control sheep vaccinated with QuilA only. However, as judged by polyacrylamide gels, the polypeptide profile of this fraction was still complex, indicating that further work is required to identify the protective components.

An ergonomic study of the optimum operating table height for laparoscopic surgery.

BACKGROUND: Laparoscopic surgery requires the use of longer instruments than open surgery, thus changing the relation between the height of the surgeon's hands and the desirable height of the operating room table. The optimum height of the operating room table for laparoscopic surgery is investigated in this study. METHODS: Twenty-one surgeons performed a two-handed, one-fourth circle cutting task using a laparoscopic video system and laparoscopic instruments positioned at five instrument handle heights relative to subjects' elbow height (-20, -10, 0, +10, and +20 cm) by adjusting the height of the trainer box. Subjects rated the difficulty and discomfort experienced during each task on a visual analog scale. Skin conductance (SC) was measured in Micromhos via paired surface electrodes placed near the ulnar edge of the palm of the right (cutting) hand. The mean electromyographic (EMG) signal from the right deltoid and trapezius muscles was measured. Arm orientation was measured in three dimensions using a magnetometer/accelerometer. Signals were acquired using analog circuitry and digitally sampled using a National Instruments DAQCard 700 connected to a Macintosh PowerBook 5300c running LabVIEW software. Statistical analysis was carried out by analysis of variance and post hoc testing. RESULTS: Statistically significant changes were found in the subjective rating of discomfort (p <0.002), deltoid EMG (p <0.0006), trapezius EMG (p <0.0001), and arm elevation (p <0.0001) between instrument handle heights. SC values and task times did not change significantly. Discomfort and difficulty ratings were lowest when instrument handles were positioned at elbow height. EMG values and arm elevation all decreased with lower instrument height. CONCLUSION: This study suggests that the optimum table height for laparoscopic surgery should position the laparoscopic instrument handles close to surgeons' elbow level to minimize discomfort and upper arm and shoulder muscle work. This corresponds to an approximate table height of 64 to 77 cm above floor level. A redesign of current operating room tables may be required to meet these ergonomic guidelines.

Performing laparoscopic surgery is significantly more stressful for the surgeon than open surgery.

BACKGROUND: The awkward visual and physical interface of video-endoscopic surgery (VES) has been shown to increase the physical workload of the surgeon, yet there is a lack of objective information on the mental effort and stress demanded by VES techniques. This study addresses the hypothesis that VES is more stressful than open surgery using an ergonomic analysis of surgical tasks in a laboratory setting. METHODS: A portable ergonomic work station was developed using a software Virtual Instrument (VI) interfaced with electronic hardware to compare the mental workload of 28 surgeons. The task was knot tying. The independent variable was work condition: rest, open technique, or VES technique. Dependent variables were tonic skin conductance level (SCL), electrooculogram (EOG), and subjective reports of concentration (CON) and stress (STR). Statistical analysis used nonparametric methods. RESULTS: Subjects tied fewer knots using the VES technique (p < 0.05). The SCL increased progressively from rest to the open task to the VES task (p < 0.05), correlating with the subjects' reported increase in mental stress level (p < 0.05). Eye blinks decreased from rest to the open task (p < 0.05), consistent with the subjects' reported increase in level of mental concentration. From the open to the VES task, eye blinks increased (p < 0.05), as would be expected given the greater demands of the VES task. Experienced subjects demonstrated less variability in SCL levels across tasks. CONCLUSIONS: VES technique requires greater concentration and places greater mental stress on surgeons than to open surgery. More experience with VES may decrease this effect. Studies are needed to improve the human-technology interface the stress.

Vaccination against gastrointestinal nematode parasites of ruminants using gut-expressed antigens.

To date, proteins isolated from the surface of the gut of gastrointestinal nematodes, particularly Haemonchus contortus, have generally proved to be useful protective antigens and several are being progressed towards recombinant protein-based vaccines. This paper describes the properties of some of the most promising antigens and summarises their performance in laboratory and field based trials. The antigens described include contortin, H11, H-gal-GP, GPI and cysteine proteinases. In addition, the discussion addresses the utility of selected antigens to protect against co-infecting nematode species such as Teladorsagia circumcincta and against related nematode infections such as Ostertagia ostertagi in cattle.

The effect of laparoscopic instrument working angle on surgeons' upper extremity workload.

BACKGROUND: Laparoscopic surgery may be kinder to the patient, but it is more demanding on the surgeon. Fixed trocar positions often require the surgeon to work with instruments at awkward angles to their body. We studied the effect of horizontal and vertical laparoscopic instrument working angle on the surgeon's thumb, forearm, and shoulder muscle work. METHODS: Electronyographic (EMG) signals were collected from the thenar compartment (TH), flexor digitorum superficialis (FDS), and deltoid (DEL) muscles of the dominant arm of eighth surgeons while they were closing a standard pistol-grip disposable laparoscopic grasper against a fixed resistance of 3 N. With the aid of a special testing bench, the instruments' position was randomly changed among 15 degrees, 45 degrees, and 75 degrees of horizontal angulation relative to the surgeons' sagital plane, and 15 degrees, 45 degrees, and 75 degrees degrees of vertical angulation relative to a horizontal plane. EMG signals were rectified and smoothed using analogue circuitry and digitally sampled at l0 Hz using a National Instruments DAQCard-700 connected to a Macintosh PowerBook 5300c running LabVIEW software. Statistical analysis was carried out by analysis of variance (ANOVA). RESULTS: The effects of vertical and horizontal working angles on the muscle effort were as follows: TH (horizontal, N.S.; vertical, N.S.), FDS (horizontal, p 45 degrees to the surgeon's sagital plane significantly increases the workload of the flexor digitorium superficialis and deltoid muscles. The deltoid muscle is also adversely affected by vertical angulation of the instrument. The instrument working angle has no effect on the thenar muscles. Whenever possible, laparoscopic surgeons should strive to place their instruments and trocars so as to minimize extreme horizontal or vertical displacement of their hands away from a resting position of comfort.

Dynamic ventilatory response to acute isocapnic hypoxia in septuagenarians.

This study compared the ventilatory response to 20 min of acute isocapnic hypoxia (end-tidal P(O(2)), 50 mmHg) using the technique of dynamic end-tidal forcing in young (Y) and old (O) men. Two groups of non-smoking male subjects (mean +/- s.d. age: Y, 29.8 +/- 6.9 years; O, 73.4 +/- 2.8 years) with similar body size, normal age-predicted spirometry, and normal moderate levels of physical activity were studied. Compared with baseline ventilation in euoxia (10.79 +/- 1.99 and 11.88 +/- 0.91 l min-1) both groups responded to the abrupt onset of isocapnic hypoxia with peak ventilatory responses of 22.58 +/- 2.60 and 24.56 +/- 2.54 l min-1 for Y and O, respectively (not significant, n.s.). Both groups demonstrated a significant increment in neuromuscular drive (i.e. tidal volume (V(T))/inspiratory time (T(I)); 0.46 +/- 0.06 to 0.91 +/- 0.15 and 0.48 +/- 0.06 to 0.91 +/- 0.12 l s-1 for Y and O, respectively) with a small (but also significant) change in central timing (T(I)/total ventilation time (T(tot)); 0.38 +/- 0.02 to 0.41 +/- 0.02 and 0.42 +/- 0.02 to 0.45 +/- 0.02 for Y and O, respectively). Oxygen sensitivity was assessed using Weil's equation, and gave a hyperbolic factor (A) of 282 +/- 75 and 317 +/- 72, and using the linear equation: change in expiratory minute volume (DeltaV.(E))/change in arterial O(2) saturation (DeltaS(a,O(2))) which gave -1.17 +/- 0.57 and -1.17 +/- 0.42 l min-1 %-1 (n.s.) for Y and O, respectively. After 20 min of sustained isocapnic hypoxia, ventilation declined to 14.29 +/- 1.92 and 16.85 +/- 2.34 l min-1 for Y and O, respectively (n.s.). The acute response to hypoxia was characterised by similar time constants (16.0 +/- 5.4 and 18.5 +/- 6.7 s) and time delays (4.8 +/- 2.1 and 4.6 +/- 1.9 s) for Y and O, respectively. Thus, the dynamic ventilatory response to acute isocapnic hypoxia is maintained into the eighth decade in a group of habitually active elderly men. Experimental Physiology (2001) 86.1, 117-126.

Cloning and expression of cystatin, a potent cysteine protease inhibitor from the gut of Haemonchus contortus.

A cDNA encoding a cysteine protease inhibitor (cystatin) was identified by immunoscreening a Haemonchus contortus cDNA library with antisera from lambs vaccinated with a protective membrane protein complex (H-gal-GP) derived from the gut of the parasite. The cDNA sequence, designated Cys-1, showed significant levels of similarity with cystatins from several species of nematode as well as with human cystatin. Recombinant H. contortus cystatin was expressed in Escherichia coli in a soluble and functionally active form, which proved to be a potent inhibitor of both mammalian cathepsin B and native H. contortus cysteine proteases. Immunolocalization studies using antisera raised against recombinant H. contortus cystatin showed that the inhibitor was predominantly expressed in the cytoplasm of intestinal cells. To determine whether H. contortus had any protective capacity against infection, lambs were vaccinated with the recombinant molecule and subsequently given a single challenge infection. Although vaccination did not confer any protection against infection with H. contortus, as judged by faecal egg output or worm counts, cystatin will be a valuable tool in the analysis of the function of the cysteine proteases which are the subject of on-going study as potential vaccine components.

Cross-protection studies with gut membrane glycoprotein antigens from Haemonchus contortus and Teladorsagia circumcincta.

Gel profiles of the peanut and ConA lectin binding integral membrane glycoproteins of Teladorsagia circumcincta and Haemonchus contortus were compared and found to be considerably different. However, some of the Teladorsagia polypeptides were recognized by antisera specific for Haemonchus amino-, metallo- or aspartyl peptidases, enzymes which are known to be protective antigens for that parasite. As expected, an experimental vaccine containing these Haemonchus proteases was extremely effective against homologous challenge, reducing egg and worm counts by more than 99% and 92%, respectively, but it did not provide any useful cross-protection against either T. circumcincta, Trichostrongylus axei or Cooperiaoncophora. A reciprocal experiment, where sheep were immunized with the equivalent glycoproteins from T. circumcincta, showed that, while they were not protected against homologous challenge, there was some cross-protection against Haemonchus as measured by a significant reduction in worm egg output.

An ergonomic evaluation of surgeons' axial skeletal and upper extremity movements during laparoscopic and open surgery.

BACKGROUND: Many surgeons have complained of fatigue and musculoskeletal pain after laparoscopic surgery. We evaluated differences in surgeons' axial skeletal and upper extremity movements during laparoscopic and open operations. METHODS: Five surgeons were videotaped performing 16 operations (8 laparoscopic and 8 open) to record their neck, trunk, shoulder, elbow, and wrist movements during the first hour of surgery. We also compared postprocedural complaints of pain, stiffness, or numbness between the two groups. RESULTS: Compared with surgeons performing open surgery, surgeons performing laparoscopic surgery exhibited less lateral neck flexion; less trunk flexion; more internal rotation of the shoulders; more elbow flexion; more wrist supination and wrist ulnar and radial deviation. There was a trend of more shoulder stiffness after laparoscopic operations than after open operations. CONCLUSIONS: Laparoscopic surgery involves a more static posture of the neck and trunk, but more frequent awkward movements of the upper extremities than open surgery. Ergonomic changes in the operating room environment and instrument design could ease the physical stress imposed on surgeons during laparoscopic operations.

A virtual instrument ergonomics workstation for measuring the mental workload of performing video-endoscopic surgery.

The visual and physical interface imposed on the surgeon by video-endoscopic surgery (VES) increases the surgeon's mental workload. Ergonomic studies are needed to develop ways to reduce this workload. We used virtual instrumentation to devise a portable ergonomic workstation to compare the surgeon's mental workloads during simulated open surgery and VES. The system measures palmar tonic skin conductance level (SCL) and electrooculogram (EOG) and frontalis electrical activity to monitor mental stress and concentration levels. We used the system at a national surgery conference on volunteer subjects during a rest period and as they performed simulated surgery, consisting of typing knots using open and VES techniques. The subjects were asked to self-rate their levels of mental concentration and stress during these activities and reported that both progressively increased from rest to the open surgery task to the VES task. The subjects tied fewer knots during the VES than the open task, consistent with the increased demands of the VES task. The SCL progressively increased from rest to the open task to the VES task, correlating with the subjects' reported increase in mental stress level. Eye blinks and low frequency EOG activity decreased from rest to the open task, consistent with the subjects' reported increase in mental concentration level. From the open to the VES task, eye blinks and EOG activity increased, as expected given the greater demands of the VES task. High frequency frontalis activity merits further study as another indicator of the subjects' levels of mental concentration and stress.

Molecular cloning and characterization of gut-derived cysteine proteinases associated with a host protective extract from Haemonchus contortus.

Cysteine proteinases have been implicated in the protection conferred by vaccination with detergent-soluble extracts of Haemonchus contortus. In the present study, antisera from sheep refractory to Haemonchus challenge following vaccination with a 'proteinase-enriched' Haemonchus gut membrane extract, were employed to screen a cDNA expression library of the adult parasite. This resulted in the isolation of 3 cDNAs (designated hmcp1, 4 and 6) encoding cathepsin B-like cysteine proteinases. Immunocytochemical studies specifically localized the products of these genes to the microvillar surface of the parasite's gut and RT-PCR experiments revealed that these were developmentally regulated, being expressed exclusively during the blood-feeding parasitic stages. In addition, a generic PCR approach was adopted in order to identify the predominant cysteine proteinases in a UK strain of Haemonchus. A panel of 5 cDNAs, including hmcp1 and 4, was amplified in this way. Genomic Southern blot analysis indicated that some of these enzymes were encoded by single-copy genes, whereas others were encoded by multi-copy genes. Subsequent sequence analysis revealed that the proteases identified in this study were distinct from those previously reported in USA strains of the parasite.

A virtual instrument ergonomics workstation to measure surgeons' physical stress.

Videoendoscopic (VES) instruments have poor force transmission properties and often require surgeons to employ awkward hand and arm positions. In order to compare the physical workload of laparoscopic surgery to open surgery, we collected long-duration EMG records from the thumb (thenar compartment) of six surgeons performing suturing and knot tying in a training box using both open and VES techniques. EMG signals were acquired using a LabVIEW Virtual Instrument and analyzed using a Modified Exposure Variation Analysis (MEVA) algorithm. Standard EMG indices and the MEVA analysis demonstrated significantly greater amplitude and duration of EMG signals using VES technique compared to open technique. Our results suggest that the use VES techniques requires a greater intensity of physical effort than open surgery techniques.

Further immunization and biochemical studies with a protective antigen complex from the microvillar membrane of the intestine of Haemonchus contortus.

Immunization of sheep with a protective antigen complex from the intestinal cells of Haemonchus contortus in Freund's adjuvant stimulated individually variable antibody responses but still conferred significant protection against parasite infection. Correlation between antibody concentration and degree of protection was suggestive of antibody being the effector mechanism. The antigen is known as Haemonchus galactose-containing glycoprotein complex (H-gal-GP) because it binds to lectins with a specificity for N-acetyl-galactosamine. Polypeptide composition analysis by polyacrylamide gel electrophoresis indicated an apparent molecular weight of about 1000 kDa and SDS gels revealed four major polypeptides, containing between 2 and 5 disulphide linked subunits, nearly all being glycosylated. N-terminal amino acid sequence was obtained from 12 subunits, ten showing homologies with cDNAs from Haemonchus encoding either pepsin, metalloprotease or cysteine protease-like enzymes. pH optima, inhibitor and various substrate studies confirmed that the native complex possessed proteolytic activities in agreement with the sequence data. Although the cDNAs predicted water soluble enzymes, little of the complex was solubilized from worm membranes without the use of a detergent, such as Triton X-100. It is hypothesized that H-gal-GP is a gut membrane associated multiprotease complex which is involved in the digestion of the blood meal and which can be neutralized by specific antibodies with drastic consequences for the parasite.

Immunization with an affinity purified protein extract from the adult parasite protects lambs against infection with Haemonchus contortus.

Young lambs were immunized with membrane protein extracts from adult Haemonchus contortus which had been enriched for cysteine protease activity using Thiol-Sepharose chromatography. The resulting extract comprised several peptides including cysteine as well as serine and metallo-protease activities although the former predominated. A limited number of the peptide components were glycosylated. Following a single challenge infection with the same parasite, vaccinated lambs had substantially reduced faecal egg outputs and final worm burdens (overall means of 77% and 47%, respectively, from three trials) compared with challenge controls. Antibody in sera from vaccinated lambs was almost exclusively bound to the surface of the parasite gut indicating that protection may be mediated by the inhibition of parasite digestion.

Ergonomic problems associated with laparoscopic surgery.

BACKGROUND: The Society of American Gastrointestinal Endoscopic Surgeons (SAGES) Task Force on Ergonomics conducted a subjective and objective assessment of ergonomic problems associated with laparoscopic instrument use. The goal was to assess the prevalence, causes, and consequences of operational difficulties associated with the use of laparoscopic instruments. METHODS: A questionnaire was distributed asking respondents to rate the frequency with which they experienced pain, stiffness, or numbness in several body areas after laparoscopic operations. An ergonomics station was assembled to quantify forearm and thumb muscle workload. Processed electromyogram (EMG) signals were acquired from 27 volunteer surgeon subjects while they completed simulated surgical tasks using a hemostat and an Ethicon laparoscopic grasper, with the aid of an endoscopic trainer and video monitoring system. RESULTS: Of 149 surgeons responding to the questionnaire, 8% to 12% reported frequent pain in the neck and upper extremities associated with laparoscopic surgery. The ergonomics station demonstrated that the peak and total muscle effort of forearm and thumb muscles were significantly greater (p < 0.01) when the grasping task was performed using the laparoscopic instrument rather than the hemostat. CONCLUSION: These findings indicate that laparoscopic surgical technique is more taxing on the surgeon.