Human dendritic cell sequestration onto the Necator americanus larval sheath during ex-sheathing: a possible mechanism for immune privilege.

Despite the profound health implications of Necator americanus infection in humans, many aspects of its interaction with the host immune system are poorly understood. Here we investigated the early events at the interface of N. americanus larvae (L3) and human dendritic cells (DCs). Our data show that co-culturing DCs and the larvae trigger ex-sheathing of hookworms rapidly where a majority of DCs are sequestered onto the larval sheath allowing the ex-sheathed larvae to migrate away unchallenged. Intriguingly, DCs show negligible interaction with the ex-sheathed larvae, alluding to differences between the surface chemistry of the larva and its sheath. Furthermore, blocking of two key C-type lectin receptors on DC surface (i.e. DC-SIGN and mannose receptor) resulted in inhibition of ex-sheathing process and DC sequestration, highlighting the importance of C-type lectins on DCs in the induction of the ex-sheathing. Analyses of DC phenotype and cytokine profile after co-culture with the N. americanus larvae showed an immature phenotype as evidenced by the low expression of the maturation markers and cytokines. These data provide new insights into early events at the interface of human DCs and N. americanus larvae and could explain how L3 evade immune recognition upon initial interaction with DCs.

Enhanced expression of Organic Cation Transporters in bronchial epithelial cell layers following insults associated with asthma - Impact on salbutamol transport.

Increasing evidence suggests Organic Cation Transporters (OCT) might facilitate the absorption of inhaled bronchodilators, including salbutamol, across the lung epithelium. This is essentially scarred and inflamed in asthma. Accordingly, the impact of epithelial insults relevant to asthma on OCT expression and salbutamol transport was evaluated in air-liquid interfaced layers of the human broncho-epithelial cell line Calu-3. These were physically injured and allowed to recover for 48h or exposed to the pro-inflammatory stimulant lipopolysaccharide (LPS) for 48h and the aeroallergen house dust mite (HDM) for 8h twice over 48h. Increases in transporter expression were measured following each treatment, with the protein levels of the OCTN2 subtype consistently raised by at least 50%. Interestingly, OCT upregulation upon LPS and HDM challenges were dependent on an inflammatory event occurring in the cell layers. Salbutamol permeability was higher in LPS exposed layers than in their untreated counterparts and in both cases, was sensitive to the OCT inhibitor tetraethylammonium. This study is the first to show epithelial injury, inflammation and allergen abuse upregulate OCT in bronchial epithelial cells, which might have an impact on the absorption of their substrates in diseased lungs.

Quality control of a medicinal larval (Lucilia sericata) debridement device based on released gelatinase activity.

Lucilia sericata Meigen (Diptera: Calliphoridae) larvae are manufactured worldwide for the treatment of chronic wounds. Published research has confirmed that the primary clinical effect of the product, debridement (the degradation of non-viable wound tissue), is accomplished by a range of enzymes released by larvae during feeding. The quality assessment of larval activity is currently achieved during production using meat-based assays, which monitor insect growth and/or the reduction in substrate mass. To support this, the present authors developed a complementary radial diffusion enzymatic assay to produce a visual and measureable indication of the activity of larval alimentary products (LAP) collected under standardized conditions, against a gelatin substrate. Using basic laboratory equipment and reagents, the assay is rapid and suited to high throughput. Assay reproducibility is high (standard deviation: 0.06-0.27; coefficient of variation: 0.75-4.31%) and the LAP collection procedure does not adversely affect larval survival (mortality: < 2%). Because it is both cost- and time-effective, this method is suited to both academic and industrial use and supports good manufacturing and laboratory practice as a quality control assay.

The impacts of larval density and protease inhibition on feeding in medicinal larvae of the greenbottle fly Lucilia sericata.

Larval therapy, the therapeutic use of blowfly larvae to treat chronic wounds, is primarily used in debridement. There are, however, gaps in current knowledge of the optimal clinical application of the therapy and mechanisms of action in the debridement process. Using an artificial assay, two studies were undertaken to investigate these aspects of larval debridement by Lucilia sericata Meigen (Diptera: Calliphoridae); the first studied the effects of the density of larvae on tissue digestion and larval mass, and the second considered the effects on the same parameters of incorporating protease inhibitors into the feeding substrate. The total mass of tissue digested increased with larval density until saturation was observed at 5.0-7.5 larvae/cm(2) . This range was considered optimal as lower doses resulted in the removal of less tissue and higher doses offered no additional tissue removal and appeared to exacerbate competition for feeding. In the second study, increased protease inhibitor concentration led to significant decreases in tissue digestion and larval mass, suggesting that serine proteases, particularly trypsin, may play major roles in larval digestion. Such information is important in elucidating the main constituents that make up larval digestive products and may be significant in the development of new therapies.

Maximising the secondary beneficial effects of larval debridement therapy.

Laboratory-based clinical investigations have shown that maggots and their secretions promote, among other activities, fibroblast motogenesis and angiogenesis. These events would contribute to re-granulation if translated to the wound environment. Maggot secretions also have ascribed antibacterial actions and may exhibit anti-inflammatory effects. Many of these biological events would be lost in the presence of necrotic tissue, making debridement a prerequisite for the release of larval-secreted secondary beneficial effects on the wound. We argue that Larval Debridement Therapy (LDT) should be considered as a primary and secondary treatment in wound management, with the primary application designed to debride the wound, and with subsequent applications to the debrided wound targeted to cellular events that promote healing. This review lends support to a re-evaluation of larval application protocols, in order to optimally harness the potential secondary beneficial clinical effects of larval therapy.

Digital pattern recognition-based image analysis quantifies immune infiltrates in distinct tissue regions of colorectal cancer and identifies a metastatic phenotype.

BACKGROUND: Several studies in colorectal cancer (CRC) indicate a relationship between tumour immune infiltrates and clinical outcome. We tested the utility of a digital pattern recognition-based image analysis (DPRIA) system to segregate tissue regions and facilitate automated quantification of immune infiltrates in CRC. METHODS: Primary CRC with matched hepatic metastatic (n=7), primary CRC alone (n=18) and primary CRC with matched normal (n=40) tissue were analysed immunohistochemically. Genie pattern recognition software was used to segregate distinct tissue regions in combination with image analysis algorithms to quantify immune cells. RESULTS: Immune infiltrates were observed predominately at the invasive margin. Quantitative image analysis revealed a significant increase in the prevalence of Foxp3 (P<0.0001), CD8 (P<0.0001), CD68 (<0.0001) and CD31 (<0.0001) positive cells in the stroma of primary and metastatic CRC, compared with tumour cell mass. A direct comparison between non-metastatic primary CRC (MET-) and primary CRC that resulted in metastasis (MET+) showed an immunosuppressive phenotype, with elevated Foxp3 (P<0.05) and reduced numbers of CD8 (P<0.05) cells in the stroma of MET+ compared with MET- samples. CONCLUSION: By combining immunohistochemistry with DPRIA, we demonstrate a potential metastatic phenotype in CRC. Our study accelerates wider acceptance and use of automated systems as an adjunct to traditional histopathological techniques.

Polyethylene glycol-drug ester conjugates for prolonged retention of small inhaled drugs in the lung.

Typically, inhaled drugs are rapidly absorbed into the bloodstream, which results in systemic side effects and a brief residence time in the lungs. PEGylation was evaluated as a novel strategy for prolonging the retention of small inhaled molecules in the pulmonary tissue. Hydrolysable ester conjugates of PEG1000, PEG2000, 2000, PEG3400 and prednisolone, a model drug cleared from the lungs within a few minutes, were synthesised and thoroughly characterised. The conjugates were stable in buffers with hydrolysis half-lives ranging from 1h to 70 h, depending on the pH and level of substitution. With the exception of PEG3400-prednisolone, conjugates did not induce a significant lactate dehydrogenase (LDH) release from Calu-3 cells after a 20 h exposure. Following nebulisation to isolated perfused rat lungs (IPRL), the PEG2000 and mPEG2000 conjugates reduced the maximum prednisolone concentration in the perfusate (Cmax) by 3.0 and 2.2 fold, respectively. Moreover, while prednisolone was undetectable in the perfusion solution beyond 20 min when the free drug was administered, prednisolone concentrations were still quantifiable after 40 min following delivery of the conjugates. This study is the first to demonstrate hydrolysable PEG drug ester conjugates are a promising approach for optimising the pharmacokinetic profile of small drugs delivered by inhalation.

In-cell Western™ detection of organic cation transporters in bronchial epithelial cell layers cultured at an air-liquid interface on Transwell(®) inserts.

INTRODUCTION: Organic cation transporters (OCT) have been shown to mediate the transport of inhaled drugs in bronchial epithelial cells and might have important physiological functions in the airway epithelium. However, a quantitative method to evaluate OCT protein expression in physiologically relevant airway epithelial cell culture models is currently lacking. In-cell Western™ (ICW) techniques might fill that gap but to date, have only been performed on cells grown on 96 or 384-well microplates. METHODS: An ICW assay was designed for measuring levels of the different OCT subtypes in intact layers of the human bronchial epithelial Calu-3 cell line cultured at an air-liquid interface on Transwell(®) inserts. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal standard for normalisation of cell number between the layers. The protocol was subsequently validated by exposing cell layers to compounds known to cause variations in OCT expression. RESULTS: Antibody signals above the background fluorescence were detected for OCT1, OCT3, OCTN1 and OCTN2 but not for OCT2 in 21day old Calu-3 layers, in agreement with previous studies which had reported OCT2 was absent in the Calu-3 cell line. Furthermore, increases in the fluorescence signal associated with OCT1, OCTN1 and OCTN2 were obtained following treatment of the layers with, respectively, the nitric oxide inducer sodium nitroprusside, the peroxisome proliferator activated receptor α (PPARα) agonist fenofibrate or the PPARγ agonist rosiglitazone, confirming the reliability of the ICW method developed. However, a suitable positive control for OCT3 could not be identified. DISCUSSION: This novel ICW assay can be exploited to quantify basal OCT protein expression as well as changes in transporter levels following external stimuli in various in vitro models. It can also be easily adapted to probe any protein in epithelial layers maintained on permeable filters.

Blow fly Lucilia sericata nuclease digests DNA associated with wound slough/eschar and with Pseudomonas aeruginosa biofilm.

In chronic wounds, it may be clinically important to remove extracellular bacterial and patient DNA as its presence may impede wound healing and promote bacterial survival in biofilm, in which extracellular DNA forms part of the biofilm architecture. As medicinal maggots, larvae of Lucilia sericata Meigen (Diptera: Calliphoridae) have been shown to efficiently debride wounds it became of interest to investigate their excretions/secretions (ES) for the presence of a deoxyribonuclease (DNAse) activity. Excretions/secretions products were shown to contain a DNAse, with magnesium, sodium and calcium metal ion dependency, and a native molecular mass following affinity purification of approximately 45 kDa. The affinity purified DNAse degraded genomic bacterial DNA per se, DNA from the slough/eschar of a venous leg ulcer, and extracellular bacterial DNA in biofilms pre-formed from a clinical isolate of Pseudomonas aeruginosa. The latter finding highlights an important attribute of the DNAse, given the frequency of P. aeruginosa infection in non-healing wounds and the fact that P. aeruginosa virulence factors can be toxic to maggots. Maggot DNAse is thus a competent enzyme derived from a rational source, with the potential to assist in clinical wound debridement by removing extracellular DNA from tissue and biofilm, and promoting tissue viability, while liberating proteinaceous slough/eschar for debridement by the suite of proteinases secreted by L. sericata.

Evaluation of layers of the rat airway epithelial cell line RL-65 for permeability screening of inhaled drug candidates.

A rat respiratory epithelial cell culture system for in vitro prediction of drug pulmonary absorption is currently lacking. Such a model may however enhance the understanding of interspecies differences in inhaled drug pharmacokinetics by filling the gap between human in vitro and rat in/ex vivo drug permeability screens. The rat airway epithelial cell line RL-65 was cultured on Transwell inserts for up to 21 days at an air-liquid (AL) interface and cell layers were evaluated for their suitability as a drug permeability measurement tool. These layers were found to be morphologically representative of the bronchial/bronchiolar epithelium when cultured for 8 days in a defined serum-free medium. In addition, RL-65 layers developed epithelial barrier properties with a transepithelial electrical resistance (TEER) >300 Ω cm(2) and apparent (14)C-mannitol permeability (P(app)) values between 0.5-3.0 × 10(-6)cm/s; i.e., in the same range as established in vitro human bronchial epithelial absorption models. Expression of P-glycoprotein was confirmed by gene analysis and immunohistochemistry. Nevertheless, no vectorial transport of the established substrates (3)H-digoxin and Rhodamine123 was observed across the layers. Although preliminary, this study shows RL-65 cell layers have the potential to become a useful in vitro screening tool in the pre-clinical development of inhaled drug candidates.

Wound debridement potential of glycosidases of the wound-healing maggot, Lucilia sericata.

The wound-healing maggot, Lucilia sericata Meigen (Diptera: Calliphoridae), degrades extracellular matrix components by releasing enzymes. The purpose of this study was to investigate the glycosylation profiles of wound slough/eschar from chronic venous leg ulcers and the complementary presence of glycosidase activities in first-instar excretions/secretions (ES1) and to define their specificities. The predominant carbohydrate moieties present in wound slough/eschar were determined by probing one-dimensional Western blots with conjugated lectins of known specificities. The presence of specific glycosidase activities in ES1 was determined using chromogenic and fluorogenic substrates. The removal of carbohydrate moieties from slough/eschar proteins by glycosidases in ES1 was determined by two-dimensional electrophoresis and Emerald 300 glycoprotein staining. α-D-glucosyl, α-D-mannosyl and N-acetylglucosamine residues were detected on slough/eschar-derived proteins. Furthermore, it was demonstrated that the treatment of slough/eschar with ES1 significantly reduced uptake of the carbohydrate-specific stain. Subsequently, α-D-glucosidase, α-D-mannosidase and N-acetylglucosaminidase activities were identified in ES1. Specific chromogenic and fluorogenic substrates and gel filtration chromatography showed that these activities result from distinct enzymes. These activities were mirrored in the removal of α-D-glucosyl, α-D-mannosyl and N-acetylglucosamine residues from proteins of slough/eschar from maggot-treated wounds. These data suggest that maggot glycosidases remove sugars from slough/eschar proteins. This may contribute to debridement, which is ultimately accomplished by a suite of biochemically distinct enzymes present in ES1.

Evaluation of air-interfaced Calu-3 cell layers for investigation of inhaled drug interactions with organic cation transporters in vitro.

A physiologically pertinent in vitro model is urgently needed for probing interactions between inhaled drugs and the organic cation transporters (OCT) in the bronchial epithelium. This study evaluated OCT expression, functionality, inhibition by common inhaled drugs and impact on formoterol transepithelial transport in layers of human bronchial epithelial Calu-3 cells grown at an air-liquid interface. 21 day old Calu-3 layers expressed OCT1, OCT3, OCTN1 and OCTN2 whereas OCT2 could not be detected. Quantification of the cellular uptake of the OCT substrate ASP(+) in presence of inhibitors suggested several OCT were functional at the apical side of the cell layers. ASP(+) uptake was reduced by the bronchodilators formoterol, salbutamol (albuterol), ipratropium and the glucocorticoid budesonide. However, the OCT inhibitory properties of the two ß(2)-mimetics were suppressed at therapeutically relevant concentrations. The absorptive permeability of formoterol across the cell layers was enhanced at a high drug concentration shown to decrease ASP(+) uptake by ∼50% as well as in presence of the OCT inhibitor tetraethylammonium (TEA). Secretory transport was unaffected by the drug concentration but was reduced by TEA. Our data indicate air-interfaced Calu-3 layers offer a low-cost in vitro model suitable for assessing inhaled drug-OCT interactions in the bronchial epithelium.

Parasitic worm therapy for allergy: is this incongruous or avant-garde medicine?

Helminth (worm) therapy has already been used in clinical trials associated with allergy. These were generally small scale, safety orientated trials of short duration, justified by epidemiological and experimental data indicating potentially beneficial immune modulation by some parasites. However, parasites by definition are disadvantageous to their hosts, and helminth infection in particular almost invariably induces an allergic phenotype, rendering this somewhat paradoxical therapeutic approach for allergy open to scrutiny. Is parasitic worm therapy for allergy incongruous medicine, or avant-garde medicine? In the present article, we assess the strength of evidence supporting the use of helminth therapy for allergy and critically appraise the trials already completed. Then, should this approach prove successful, we suggest strategies to improve the delivery of helminth therapy, and ways to discover immune response modifiers derived from worms.

Maggot chymotrypsin I from Lucilia sericata is resistant to endogenous wound protease inhibitors.

BACKGROUND: A chymotrypsin found in the secretions of Lucilia sericata and manufactured as a recombinant enzyme degrades chronic wound eschar ex vivo. OBJECTIVES: To characterize the inhibition profile of the L. sericata recombinant chymotrypsin I. METHODS: Activity of recombinant chymotrypsin I and its sensitivity to endogenous inhibitors were determined enzymatically using the fluorogenic substrate succinyl-alanyl-alanyl-prolyl-phenylalanyl-aminomethyl coumarin. RESULTS: We report the presence of high concentrations of two endogenous inhibitors, α1-antichymotrypsin and α1-antitrypsin, in wound eschar and a trace of a third, α2-macroglobulin, with the potential to inhibit this debridement process. However, the addition of a soluble and inhibitor-containing extract of chronic wound eschar to chymotrypsin I did not affect activity of the enzyme, neither did the addition of purified native α1-antichymotrypsin or α1-antitrypsin, although chymotrypsin I was inhibited by α2-macroglobulin. Conversely, the mammalian equivalent, α-chymotrypsin, was inhibited by the purified native α1-antichymotrypsin, α1-antitrypsin and α2-macroglobulin and by the soluble extract of wound eschar. CONCLUSIONS: The data suggest that the maggot-derived chymotrypsin I is biochemically distinct from human α-chymotrypsin and the lack of inhibition by wound eschar suggests a means by which chymotrypsin I activity survives within the wound to contribute towards debridement during maggot biotherapy.

Degradation of eschar from venous leg ulcers using a recombinant chymotrypsin from Lucilia sericata.

BACKGROUND: Larvae of the greenbottle Lucilia sericata are used to debride nonhealing wounds and stimulate the production of fresh granulation tissue. Previous publications have shown that secretions from L. sericata contain a number of proteolytic activities including a chymotrypsin that degrades a number of extracellular matrix components such as fibronectin, laminin and collagen. OBJECTIVES: To produce a recombinant L. sericata chymotrypsin (chymotrypsin I) and determine its effects on the degradation of patient wound eschar. METHODS: An active recombinant chymotrypsin I from L. sericata was cloned and expressed in Sf9 cells and its subsequent effects ex vivo on eschar from venous leg ulcers were determined by two-dimensional electrophoresis. RESULTS: The recombinant enzyme had the attributes of a chymotrypsin, possessing sequence homology with other chymotrypsins and demonstrating attributes of the native enzyme including cleavage of the chymotrypsin substrate succinyl-alanyl-alanyl-prolyl-phenylalanyl-7-amino-4-methyl coumarin, inhibition by phenylmethylsulphonyl fluoride and lack of inhibition by amidinophenylmethylsulphonyl fluoride. Importantly, the recombinant chymotrypsin cleaved the majority of proteins from slough/eschar from venous leg ulcers in a superior manner to chymotrypsins from human and bovine sources. CONCLUSIONS: The ex vivo degradation of eschar from venous leg ulcers indicates the potential value of recombinant chymotrypsin I as a novel, stand-alone debridement agent.

Reduced helminth burden increases allergen skin sensitization but not clinical allergy: a randomized, double-blind, placebo-controlled trial in Vietnam.

BACKGROUND: Observational evidence suggests that infection with helminths protects against allergic disease and allergen skin sensitization. It is postulated that such effects are mediated by helminth-induced cytokine responses, in particular IL-10. OBJECTIVE: We tested this hypothesis in a rural area of central Vietnam where hookworm infection is endemic. METHODS: One thousand five hundred and sixty-six schoolchildren aged 6-17 were randomly allocated to receive either anti-helminthic therapy or a placebo at 0, 3, 6, and 9 months. We compared changes in the prevalence of exercise-induced bronchoconstriction, allergen skin sensitization, flexural eczema on skin examination, questionnaire-reported allergic disease (wheeze and rhinitis symptoms), and immunological parameters (hookworm-induced IFN-gamma, IL-5, IL-10) between 0 and 12 months. RESULTS: One thousand four hundred and eighty-seven children (95% of these randomized) completed the study. The most common helminth infections were hookworm (65%) and Ascaris lumbricoides (7%). There was no effect of the therapy on the primary outcome, exercise-induced bronchoconstriction (within-participant mean percent fall in peak flow from baseline after anti-helminthic treatment 2.25 (SD 7.3) vs. placebo 2.19 (SD 7.8, P=0.9), or on the prevalence of questionnaire-reported wheeze [adjusted odds ratio (OR)=1.16, 95% confidence interval (CI) 0.35-3.82, P=0.8] and rhinitis (adjusted OR=1.39, 0.89-2.15, P=0.1), or flexural dermatitis on skin examination (adjusted OR=1.15, 0.39-3.45, P=0.8). However, anti-helminthic therapy was associated with a significantly higher allergen skin sensitization risk (adjusted OR=1.31, 1.02-1.67, P=0.03). This effect was particularly strong for children infected with A. lumbricoides at baseline (adjusted OR=4.90, 1.48-16.19, P=0.009). Allergen skin sensitization was inversely related to hookworm-specific IL-10 at baseline (adjusted OR=0.76, 0.59-0.99, P=0.04). No cytokine tested, including IL-10, changed significantly after the anti-helminthic therapy compared with the placebo. CONCLUSION: A significant reduction in worm burden over a 12-month period in helminth-infected children increases the risk of allergen skin sensitization but not of clinical allergic disease. The effect on skin sensitization could not be fully explained by any of the immunological parameters tested.

Experimental hookworm infection: a randomized placebo-controlled trial in asthma.

BACKGROUND: Epidemiological studies suggest that hookworm infection protects against asthma, and therefore that hookworm infection may have a direct or an indirect therapeutic potential in this disease. We now report the first clinical trial of experimental hookworm infection in people with allergic asthma. OBJECTIVES: To determine the effects of experimental hookworm infection in asthma. METHODS: Thirty-two individuals with asthma and measurable airway responsiveness to adenosine monophosphate (AMP) were randomized and double blinded to cutaneous administration of either ten Necator americanus larvae, or histamine solution (placebo), and followed for 16 weeks. The primary outcome was the change in provocation dose of inhaled AMP required to reduce forced expiratory volume in 1 s by 20% (PD(20)AMP) from baseline to week 16. Secondary outcomes included change in several measures of asthma control and allergen skin sensitivity and the occurrence of adverse effects. RESULTS: Mean PD(20)AMP improved in both groups, more in the hookworm [1.49 doubling doses (DD)] than the placebo group (0.98 DD), but the difference between groups was not significant (0.51 DD; 95% confidence interval: -1.79 to 2.80; P=0.65). There were no significant differences between the two groups for other measures of asthma control or allergen skin sensitization. Infection was generally well tolerated. CONCLUSIONS: Experimental infection with ten hookworm larvae in asthma did not result in significant improvement in bronchial responsiveness or other measures of asthma control in this study. However, infection was well tolerated and resulted in a non-significant improvement in airway responsiveness, indicating that further studies that mimic more closely natural infection are feasible and should be undertaken.

Safety of hookworm infection in individuals with measurable airway responsiveness: a randomized placebo-controlled feasibility study.

BACKGROUND: Epidemiological evidence suggests that hookworm infection protects against asthma. However, for ethical and safety reasons, before testing this hypothesis in a clinical trial in asthma it is necessary to establish whether experimental hookworm infection might exacerbate airway responsiveness during larval lung migration. OBJECTIVE: To determine whether hookworm larval migration through the lungs increases airway responsiveness in allergic individuals with measurable airway responsiveness but not clinical asthma, and investigate the general tolerability of infection and effect on allergic symptoms. METHODS: Thirty individuals with allergic rhinoconjunctivitis and measurable airway responsiveness to adenosine monophosphate (AMP) but not clinically diagnosed asthma were randomized, double-blind to cutaneous administration of either 10 hookworm larvae or histamine placebo, and followed for 12 weeks. The primary outcome was the maximum fall from baseline in provocative dose of inhaled AMP required to reduce 1-s forced expiratory volume by 10% (PD(10)AMP) measured at any time over the 4 weeks after active or placebo infection. Secondary outcomes included peak flow variability in the 4 weeks after infection, rhinoconjunctivitis symptom severity and adverse effect diary scores over the 12-week study period, and change in allergen skin test responses between baseline and 12 weeks. RESULTS: Mean maximum change in PD(10)AMP from baseline was slightly but not significantly greater in the hookworm than the placebo group (-1.67 and -1.16 doubling doses; mean difference -0.51, 95% confidence interval -1.80 to 0.78, P=0.42). Symptom scores of potential adverse effects were more commonly reported in the hookworm group, but infection was generally well tolerated. There were no significant differences in peak-flow variability, rhinoconjunctivitis symptoms or skin test responses between groups. CONCLUSION: Hookworm infection did not cause clinically significant exacerbation of airway responsiveness and was well tolerated. Suitably powered trials are now indicated to determine the clinical effectiveness of hookworm infection in allergic rhinoconjunctivitis and asthma.

T-cell motility in the early stages of the immune response modeled as a random walk amongst targets.

The transport process by which a T cell makes high-frequency encounters with antigen-presenting cells following infection is an important element of adaptive immunity. Recent experimental work has allowed in vivo cell motility to be characterized in detail. On the basis of experimental data we develop a quantitative model for encounters between T cells and antigen-presenting cells. We model this as a transport-limited chemical reaction with the dynamics dependent on physical contact between randomly moving reactants. We use asymptotic methods to calculate a time distribution which characterizes the delay before a T cell is activated and use Monte Carlo simulations to verify the analysis. We find that the density of antigen-primed dendritic cells within the lymph node paracortex must be greater than 35 cells/mm3 for a T cell to have a more than 50% chance of encountering a dendritic cell within 24 h. This density is much larger than existing estimates based on calculations which neglect the transport process. We also use simulations to compare a T cell which re-orients isotropically with a T cell which turns according to an experimentally observed distribution and find that the effects of anisotropy on the solution are small.

Alleviation of insulitis and moderation of diabetes in NOD mice following treatment with a synthetic Pseudomonas aeruginosa signal molecule, N-(3-oxododecanoyl)-L-homoserine lactone.

Quorum sensing signal molecules (QSSMs) from the bacterium Pseudomonas aeruginosa control bacterial population density and the expression of virulence determinants. Coincidentally, and possibly to allow this pathogen to gain a foothold in the human body, certain signal molecules also downregulate immunological responses in an apparently T-helper 1-selective manner, which would suggest their application as therapeutics to some autoimmune diseases. In the present paper, experiments are described that indicate that one particular signal molecule, a synthetic N-(3-oxododecanoyl)-L-homoserine lactone, can be used to alleviate insulitis and diabetes in non-obese diabetic (NOD) mice, suggesting that bacterial signal molecules may represent a novel source of immune modulatory compounds for the treatment of type 1 diabetes, which afflicts more than 2 million individuals in Europe and North America.