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In this chapter, we describe the scientific, technical, clinical and regulatory aspects of establishing a controlled human hookworm infection (CHHI) model in non-endemic and endemic geographical regions, to facilitate a pathway towards accelerated vaccine development. The success achieved in establishing the CHHI platform specifically allows the Human Hookworm Vaccine Initiative (HHVI) to accelerate its progress by establishing a human hookworm vaccination/challenge model (HVCM) in a hookworm endemic area of Brazil. The HVCM will permit the rapid and robust determination of clinical efficacy in adults, allowing for early selection of the most efficacious human hookworm vaccine (HHV) candidate(s) to advance into later-stage pivotal paediatric clinical trials and reduce the overall number of participants required to assess efficacy (Diemert et al. 2018)., (© 2021. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Background: Helminth-associated changes in gut microbiota composition have been hypothesised to contribute to the immune-suppressive properties of parasitic worms. Multiple sclerosis is an immune-mediated autoimmune disease of the central nervous system whose pathophysiology has been linked to imbalances in gut microbial communities., Results: In the present study, we investigated, for the first time, qualitative and quantitative changes in the faecal bacterial composition of human volunteers with remitting multiple sclerosis (RMS) prior to and following experimental infection with the human hookworm, Necator americanus (N+), and following anthelmintic treatment, and compared the findings with data obtained from a cohort of RMS patients subjected to placebo treatment (PBO). Bacterial 16S rRNA high-throughput sequencing data revealed significantly decreased alpha diversity in the faecal microbiota of PBO compared to N+ subjects over the course of the trial; additionally, we observed significant differences in the abundances of several bacterial taxa with putative immune-modulatory functions between study cohorts. Parabacteroides were significantly expanded in the faecal microbiota of N+ individuals for which no clinical and/or radiological relapses were recorded at the end of the trial., Conclusions: Overall, our data lend support to the hypothesis of a contributory role of parasite-associated alterations in gut microbial composition to the immune-modulatory properties of hookworm parasites.

The allergic phenotype manifests itself in a spectrum of troublesome to life-threatening diseases, from seasonal hay fever, through the food allergies, atopic eczema, asthma, to anaphylaxis. Allergy, that is an overreaction to allergen in hypersensitive individuals, results from the production of IgE, mast cell and basophil sensitisation and degranulation, requiring a range of medications to manage the conditions. Yet it is highly likely that allergy evolved for a purpose and that allergic diseases are accidental consequences of an insufficiently regulated immune response. This article presents a viewpoint from which to restore the immunological reputation of the allergic phenotype. We consider the evolutionary origins of potential allergens, toxins and parasites, and how they might have influenced early-mammal species in existence when IgE first developed. We conclude that the allergic phenotype has likely saved the lives of many more mammals than have ever died from allergy, so justifying the positive role of IgE in our evolution., (© 2020 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Importance: Studies suggest gut worms induce immune responses that can protect against multiple sclerosis (MS). To our knowledge, there are no controlled treatment trials with helminth in MS., Objective: To determine whether hookworm treatment has effects on magnetic resonance imaging (MRI) activity and T regulatory cells in relapsing MS., Design, Setting, and Participants: This 9-month double-blind, randomized, placebo-controlled trial was conducted between September 2012 and March 2016 in a modified intention-to-treat population (the data were analyzed June 2018) at the University of Nottingham, Queen's Medical Centre, a single tertiary referral center. Patients aged 18 to 61 years with relapsing MS without disease-modifying treatment were recruited from the MS clinic. Seventy-three patients were screened; of these, 71 were recruited (2 ineligible/declined)., Interventions: Patients were randomized (1:1) to receive either 25 Necator americanus larvae transcutaneously or placebo. The MRI scans were performed monthly during months 3 to 9 and 3 months posttreatment., Main Outcomes and Measures: The primary end point was the cumulative number of new/enlarging T2/new enhancing T1 lesions at month 9. The secondary end point was the percentage of cluster of differentiation (CD) 4+CD25highCD127negT regulatory cells in peripheral blood., Results: Patients (mean [SD] age, 45 [9.5] years; 50 women [71%]) were randomized to receive hookworm (35 [49.3%]) or placebo (36 [50.7%]). Sixty-six patients (93.0%) completed the trial. The median cumulative numbers of new/enlarging/enhancing lesions were not significantly different between the groups by preplanned Mann-Whitney U tests, which lose power with tied data (high number of zeroactivity MRIs in the hookworm group, 18/35 [51.4%] vs 10/36 [27.8%] in the placebo group). The percentage of CD4+CD25highCD127negT cells increased at month 9 in the hookworm group (hookworm, 32 [4.4%]; placebo, 34 [3.9%]; P = .01). No patients withdrew because of adverse effects. There were no differences in adverse events between groups except more application-site skin discomfort in the hookworm group (82% vs 28%). There were 5 relapses (14.3%) in the hookworm group vs 11 (30.6%) receiving placebo., Conclusions and Relevance: Treatment with hookworm was safe and well tolerated. The primary outcome did not reach significance, likely because of a low level of disease activity. Hookworm infection increased T regulatory cells, suggesting an immunobiological effect of hookworm. It appears that a living organism can precipitate immunoregulatory changes that may affect MS disease activity., Trial Registration: ClinicalTrials.gov Identifier: NCT01470521.

Background: Epstein-Barr virus (EBV) infection is strongly associated with multiple sclerosis (MS). Helminth infection can downregulate antiviral immune responses, potentially protecting against MS, but with a theoretical risk for reactivating latent EBV infection. Objective: To investigate parameters of EBV infection and their relationship with disease activity in people with MS (PwMS) therapeutically vaccinated with Necator americanus (hookworm). Methods: Sequential serum samples from 51 PwMS; 26 therapeutically infected (25 larvae) with N. americanus and 25 controls were tested for EBV virus capsid antigen (VCA) IgG and IgM, EBV nuclear antigen-1 (EBNA-1) IgG, and EBV early antigen (EA) IgG. Disease activity was assessed by periodic MRI. Significance was set at p < 0.05. Results: All PwMS were EBV VCA IgG and EBNA-1 IgG positive, and 35.2% were EBV EA IgG positive. EBV antibody levels were generally stable, and EBV reactivation in PwMS was not demonstrated by significant increases in IgG titre over 12 months. Disease activity was most frequent in PwMS possessing high levels of EBV VCA IgG (>600 units/mL) or EBNA-1 IgG (>150 units/mL); however, there was no association with hookworm treatment. Interpretation: Therapeutic hookworm vaccination was not associated with EBV reactivation. Multiple sclerosis disease activity was associated with high levels of EBV VCA IgG or EBNA-1 IgG.

Caenorhabditis elegans is a free-living nematode that resides in soil and typically feeds on bacteria. We postulate that haematophagic C. elegans could provide a model to evaluate vaccine responses to intestinal proteins from hematophagous nematode parasites, such as Necator americanus. Human erythrocytes, fluorescently labelled with tetramethylrhodamine succinimidyl ester, demonstrated a stable bright emission and facilitated visualization of feeding events with fluorescent microscopy. C. elegans were observed feeding on erythrocytes and were shown to rupture red blood cells upon capture to release and ingest their contents. In addition, C. elegans survived equally on a diet of erythrocytes. There was no statistically significant difference in survival when compared with a diet of Escherichia coli OP50. The enzymes responsible for the digestion and detoxification of haem and haemoglobin, which are key components of the hookworm vaccine, were found in the C. elegans intestine. These findings support our postulate that free-living nematodes could provide a model for the assessment of neutralizing antibodies to current and future hematophagous parasite vaccine candidates.

The effective use of larvae of the greenbottle fly, Lucilia sericata, in wound debridement requires a working knowledge of how feeding changes over time. Using a laboratory assay and bagged larval dressings, the effect of incubation time on larval feeding rates and body mass was investigated for up to 120 hours at 32°C. The mass of tissue digested increased significantly in incremental 24-hour periods up to 72 hours, with no significant consumption occurring afterwards. Larval mass increased only up to 48 hours. A further test comparing the efficacy of a single 96-hour application of larvae against two consecutive 48-hour applications found that the mass of tissue digested in the latter was 14.3% higher than the former, a difference that was statistically significant. Current clinical guidance suggests a 4-day application period for bagged larvae. Based on these results, an incubation time of 72 hours (3 days) for bagged larvae would be the most effective at the study temperature. However, it is acknowledged that wound temperature can vary, whereby feeding rates would likely differ. In view of this, we conclude that a period of 3 to 4 days is optimum for the application of larvae, and current guidelines should be adhered to., (© 2018 Medicalhelplines.com Inc and John Wiley & Sons Ltd.)

Background: Controlled human hookworm infection (CHHI) is a central component of a proposed hookworm vaccination-challenge model (HVCM) to test the efficacy of candidate vaccines. Critical to CHHI is the manufacture of Necator americanus infective larvae ( Na L3) according to current Good Manufacturing Practice (cGMP) and the determination of an inoculum of Na L3 that is safe and reliably induces patent infection., Methods: cGMP-grade Na L3 were produced for a phase 1 trial in 20 healthy, hookworm-naïve adults in the United States, who received either 25 or 50 Na L3. Participants were monitored for 12-18 weeks postinfection for safety, tolerability, and patency of N. americanus infection., Results: Both Na L3 doses were well tolerated. Early manifestations of infection included pruritus, pain, and papulovesicular rash at the application site. Gastrointestinal symptoms and eosinophilia appeared after week 4 postinfection. The 50 Na L3 inoculum induced patent N. americanus infection in 90% of this dose group., Conclusions: The inoculum of 50 Na L3 was well tolerated and consistently induced patent N. americanus infection suitable for future HVCM trials., Clinical Trials Registration: NCT01940757.

Necator americanus, a haematophagous hookworm parasite, infects ~10% of the world's population and is considered to be a significant public health risk. Its lifecycle has distinct stages, permitting its successful transit from the skin via the lungs (L3) to the intestinal tract (L4 maturing to adult). It has been hypothesised that the L3 larval sheath, which is shed during percutaneous infection (exsheathment), diverts the immune system to allow successful infection and reinfection in endemic areas. However, the physicochemical properties of the L3 larval cuticle and sheath, which are in direct contact with the skin and its immune defences, are unknown. In the present study, we controlled exsheathment, to characterise the sheath and underlying cuticle surfaces in situ, using atomic force microscopy (AFM) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). AFM revealed previously unseen surface area enhancing nano-annuli exclusive to the sheath surface and confirmed greater adhesion forces exist between cationic surfaces and the sheath, when compared to the emergent L3 cuticle. Furthermore, ToF-SIMS elucidated different chemistries between the surfaces of the cuticle and sheath which could be of biological significance. For example, the phosphatidylglycerol rich cuticle surface may support the onward migration of a lubricated infective stage, while the anionic and potentially immunologically active heparan sulphate rich deposited sheath could result in the diversion of immune defences to an inanimate antigenic nidus. We propose that our initial studies into the surface analysis of this hookworm provides a timely insight into the physicochemical properties of a globally important human pathogen at its infective stage and anticipate that the development and application of this analytical methodology will support translation of these findings into a biological context.

Alimentary products of medicinal Lucilia sericata larvae are studied to determine their mechanisms of action, particularly in the contexts of wound debridement and disinfection. Furthermore, the larvae can be applied to patients in contained medical devices (such as the BioBag; BioMonde). Here, we tested the materials and larval content of the most commonly used debridement device (the "BB-50") to explore the possibility that endotoxins may be contributing to the bio-activity of the product, given that endotoxins are potent stimulants of cellular activation. Using standardised protocols to collect larval alimentary products (LAP), we proceeded to determine residual endotoxin levels in LAP derived from the device, before and after the neutralisation of interfering enzymatic activity. The debridement device and its associated larval content was not a significant source of lipopolysaccharide (LPS) activity. However, it is clear from these experiments that a failure to remove the confounding serine proteinase activity would have resulted in spuriously high and erroneous results. The residual LPS levels detected are unlikely to be active in wound healing assays, following cross-referencing to publications where LPS at much higher levels has been shown to have positive and negative effects on processes associated with wound repair and tissue regeneration., (© 2017 by the Wound Healing Society.)

Wound bed preparation (WBP) is an integral part of the care programme for chronic wounds. The acronym TIME is used in the context of WBP and describes four barriers to healing in chronic wounds; namely, dead Tissue, Infection and inflammation, Moisture imbalance and a non-migrating Edge. Larval debridement therapy (LDT) stems from observations that larvae of the blowfly Lucilia sericata clean wounds of debris. Subsequent clinical studies have proven debriding efficacy, which is likely to occur as a result of enzymatically active alimentary products released by the insect. The antimicrobial, anti-inflammatory and wound healing activities of LDT have also been investigated, predominantly in a pre-clinical context. This review summarises the findings of investigations into the molecular mechanisms of LDT and places these in context with the clinical concept of WBP and TIME. It is clear from these findings that biotherapy with L. sericata conforms with TIME, through the enzymatic removal of dead tissue and its associated biofilm, coupled with the secretion of defined antimicrobial peptides. This biotherapeutic impact on the wound serves to reduce inflammation, with an associated capacity for an indirect effect on moisture imbalance. Furthermore, larval serine proteinases have the capacity to alter fibroblast behaviour in a manner conducive to the formation of granulation tissue., (© 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd.)

Venous leg ulcer slough is unpleasant to the patient and difficult to manage clinically. It harbours infection, also preventing wound management materials and dressings from supporting the underlying viable tissues. In other words, slough has significant nuisance value in the tissue viability clinic. In this study, we have sought to increase our knowledge of slough by building upon a previous but limited analysis of this necrotic tissue. In particular, slough has been probed using Western blotting for the presence of proteins with the capacity to engage microbial surface components recognising adhesive matrix macromolecules. Although the samples were difficult to resolve, we detected fibrinogen, fibronectin, IgG, collagen, human serum albumin and matrix metalloproteinase-9. Furthermore, the effect of a maggot-derived debridement enzyme, chymotrypsin 1 on macromolecules in slough was confirmed across seven patient samples. The effect of chymotrypsin 1 on slough confirms our thesis that this potential debridement enzyme could be effective in removing slough along with its associated bacteria, given its observed resistance to intrinsic gelatinase activity. In summary, we believe that the data provide scientists and clinicians with further insights into the potential molecular interactions between bacteria, wound tissue and Lucilia sericata in a clinically problematic yet scientifically interesting wound ecosystem., (© 2013 The Authors. International Wound Journal © 2013 Medicalhelplines.com Inc and John Wiley & Sons Ltd.)

The impact of P-glycoprotein (MDR1, ABCB1) on drug disposition in the lungs as well as its presence and activity in in vitro respiratory drug absorption models remain controversial to date. Hence, we characterised MDR1 expression and the bidirectional transport of the common MDR1 probe (3)H-digoxin in air-liquid interfaced (ALI) layers of normal human bronchial epithelial (NHBE) cells and of the Calu-3 bronchial epithelial cell line at different passage numbers. Madin-Darby Canine Kidney (MDCKII) cells transfected with the human MDR1 were used as positive controls. (3)H-digoxin efflux ratio (ER) was low and highly variable in NHBE layers. In contrast, ER=11.4 or 3.0 were measured in Calu-3 layers at a low or high passage number, respectively. These were, however, in contradiction with increased MDR1 protein levels observed upon passaging. Furthermore, ATP depletion and the two MDR1 inhibitory antibodies MRK16 and UIC2 had no or only a marginal impact on (3)H-digoxin net secretory transport in the cell line. Our data do not support an exclusive role of MDR1 in (3)H-digoxin apparent efflux in ALI Calu-3 layers and suggest the participation of an ATP-independent carrier. Identification of this transporter might provide a better understanding of drug distribution in the lungs., (Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.)

In Europe ≈15,000 patients receive larval therapy for wound treatment annually. Over the past few years, clinical studies have demonstrated the success of larvae of Lucilia sericata as debridement agents. This is based on a combination of physical and biochemical actions. Laboratory investigations have advanced our understanding of the biochemical mechanisms underlying the beneficial effects of larval secretions, including removal of dead tissue, reduction of the bacterial burden, and promotion of tissue regeneration. The present article summarizes our current understanding of the microbiological, immunological, and wound healing actions of larval therapy, and the molecules involved in these beneficial effects. Future studies will focus on the isolation, identification, and (pre)clinical testing of the effective molecules of L. sericata larvae. These molecules may be candidates for the development of new agents for the treatment of several infectious and inflammatory diseases, including chronic wounds., (© 2013 WILEY Periodicals, Inc.)

Bacteria deploy a range of chemistries to regulate their behaviour and respond to their environment. Quorum sensing is one method by which bacteria use chemical reactions to modulate pre-infection behaviour such as surface attachment. Polymers that can interfere with bacterial adhesion or the chemical reactions used for quorum sensing are therefore a potential means to control bacterial population responses. Here, we report how polymeric 'bacteria sequestrants', designed to bind to bacteria through electrostatic interactions and therefore inhibit bacterial adhesion to surfaces, induce the expression of quorum-sensing-controlled phenotypes as a consequence of cell clustering. A combination of polymer and analytical chemistry, biological assays and computational modelling has been used to characterize the feedback between bacteria clustering and quorum sensing signalling. We have also derived design principles and chemical strategies for controlling bacterial behaviour at the population level.

Extended dynamic range pH-sensitive ratiometric nanosensors, capable of accurately mapping the full physiological pH range, have been developed and used to characterize the pH of the pharyngeal and intestinal lumen of Caenorhabditis elegans in real-time. Nanosensors, 40 nm in diameter, were prepared by conjugating pH-sensitive fluorophores, carboxyfluorescein (FAM) and Oregon Green (OG) in a 1:1 ratio, and a reference fluorophore, 5-(and-6)-carboxytetramethylrhodamine (TAMRA) to an inert polyacrylamide matrix. Accurate ratiometric pH measurements were calculated through determination of the fluorescence ratio between the pH-sensitive and reference fluorophores. Nanosensors were calibrated with an automated image analysis system and validated to demonstrate a pH measurement resolution of ±0.17 pH units. The motility of C. elegans populations, as an indicator for viability, showed nematodes treated with nanosensors, for concentrations ranging from 50.00 to 3.13 mg/mL, were not statistically different to nematodes not challenged with nanosensors up to a period of 4 days (p < 0.05). The nanosensors were also found to remain in the C. elegans lumen >24 h after nanosensor challenge was removed. The pH of viable C. elegans lumen was found to range from 5.96 ± 0.31 in the anterior pharynx to 3.59 ± 0.09 in the posterior intestine. The pharyngeal pumping rate, which dictates the transfer of ingested material from the pharynx to the intestine, was found to be temperature dependent. Imaging C. elegans at 4 °C reduced the pharyngeal pumping rate to 7 contractions/min and enabled the reconstruction of rhythmic pH oscillations in the intestinal lumen in real-time with fluorescence microscopy.

Staphylococcus aureus and Staphylococcus epidermidis biofilms cause chronic infections due to their ability to form biofilms. The excretions/secretions of Lucilia sericata larvae (maggots) have effective activity for debridement and disruption of bacterial biofilms. In this paper, we demonstrate how chymotrypsin derived from maggot excretions/secretions disrupts protein-dependent bacterial biofilm formation mechanisms.

Parasite immunologists have contributed significantly to our understanding of the human immune system, to the extent that live parasites are being tested as investigational medicinal products (IMPs) and their secretions are being analysed for potentially novel and effective immune regulatory molecules (IRMs). This article expresses an opinion on the current status of research, and suggests that parasite immunologists and the pharmaceutical industry combine to source non-immunogenic IRMs from parasites selected for their immune modulatory potential. The article also suggests that parasite immunologists should be perhaps more rigorous in their choice of infection and disease models in rodents.

Previously, we demonstrated the effectiveness of a research grade recombinant chymotrypsin, derived from the larvae of Lucilia sericata, in "debriding" slough/eschar from venous leg ulcers ex vivo. Furthermore, we were able to formulate this enzyme for successful delivery to in vitro wound healing assays, from a prototype hydrogel wound dressing, and showed that enzyme delivered in this way could degrade wound tissue ex vivo. Recently, to progress biotechnological development of the enzyme as a potential therapeutic product, we explored expression using current good manufacturing practice (cGMP) guidelines, and now report that a recombinant chymotrypsin I zymogen from L. sericata can be expressed in the cGMP acceptable strain of Escherichia coli (BLR-DE3). In addition, the conditions required for purification, refolding and activation of the chymotrypsinogen have been determined. The activated enzyme was stable, and effective in digesting wound slough/eschar tissue. To summarise, we have successfully initiated the production and characterisation of a novel cGMP compatible product for use in future clinical trials., (Copyright © 2012 American Institute of Chemical Engineers (AIChE).)

Some infectious diseases have been shown to halt the onset of autoimmune disease in animal models and have been suggested to also influence autoimmune pathology in humans. The isolation and study of small molecules and proteins from the infectious agents responsible for the protective effect will enable a mechanistic understanding of how these components may prevent or delay the onset of autoimmunity. In this study we confirm that the quorum-sensing signal molecule OdDHL from Pseudomonas aeruginosa can delay the onset of type 1 diabetes in the NOD mouse model. Furthermore, using an antigen-presenting cell-free system, we find not only that OdDHL inhibits the proliferation of naïve T cells but also that it directly inhibits the differentiation of T cell subsets. OdDHL was shown to have no effect on the inhibition of primed and committed differentiated T cell responses, suggesting that that immune mechanism mediated by this molecule may be more restricted to initial stages of infection.

The Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)-l-homoserine lactone (1) is involved not only in bacterial activation but also in subversion of the host immune system, and this compound might thus be used as a template to design immunosuppressive agents, provided derivatives devoid of quorum-sensing activity could be discovered. By use of a leukocyte proliferation assay and a newly developed bioluminescent P. aeruginosa reporter assay, systematic modification of 1 allowed us to delineate the bacterial LasR-induction and host immunosuppressive activities. The main determinant is replacement of the methylene group proximal to the β-ketoamide in the acyl chain of 1 with functions containing heteroatoms, especially an NH group. This modification can be combined with replacement of the homoserine lactone system in 1 with stable cyclic groups. For example, we found the simple compound N(1)-(5-chloro-2-hydroxyphenyl)-N(3)-octylmalonamide (25d) to be over twice as potent as 1 as an immune suppressor while displaying LasR-induction antagonist activity.

Several studies have linked tumor-infiltration by regulatory T cells with poor patient outcome. Targeting the mechanisms by which regulatory T cells traffic to and persist in the tumor may circumvent tumor immune-escape by de-restricting T cell-mediated cytotoxicity. In this review, we describe the principle axes that govern regulatory T cell migration and the mechanisms that underpin their immunosuppressive activity in cancer. Inhibiting either the migration or function of regulatory T cells may enhance host-anti-cancer immune responses and as such are attractive and tractable targets for therapeutic intervention.

Knowledge of the molecular mechanisms used by Necator americanus larvae to penetrate the human skin and the vasculature would aid the development of effective vaccines against this important pathogen. In this work, the impact of N. americanus exsheathing fluid (EF) and excretory/secretory products (ES) on the endothelial barrier was examined using human umbilical vein endothelial cells (HUVEC). Cellular responses were assessed by investigating molecular changes at cell-cell junctions and by determining levels of secreted IL-6, IL-8, and vascular endothelial growth factor (VEGF) in the culture medium. It would appear that a repertoire of larval proteases caused a dose-related increase in endothelial permeability as characterized by a decrease in monolayer resistance with increased permeation of tracer-albumin. These barrier changes were associated with disruption of junctional vascular endothelial cadherin (VE-cadherin) and F-actin and an increase in endothelial secretion of IL-6 and IL-8. Our data suggest that larval proteases play an important role in negotiating the endothelium.

Background: Parasitic worms induce a strong, polarized T(H)2-type immune response. The kinetics of gastrointestinal nematode-induced T(H)2-type responses, especially in the context of primary infection, have been extensively studied in experimental infection models but not in human subjects., Objective: We sought to determine the kinetics of basophil sensitization in subjects infected with Necator americanus during the first 12 weeks after infection., Methods: Thirty nonasthmatic subjects with allergic rhinoconjunctivitis were randomized in a double-blind manner to cutaneous administration of either 10 hookworm infective larvae or histamine placebo. Blood samples were taken at regular intervals for 12 weeks, and basophil activation was determined in whole blood by measuring CD63 and CD203c levels on stimulation with N americanus excretions/secretions. Parasite-specific immunoglobulin responses were assessed by means of ELISA and Western blotting., Results: Median values reflecting basophil activation (CD203c/CD63 double-positive cells) in the excretion/secretion-stimulated infected group steadily increased after week 4, consistently achieving statistical significance compared with the placebo group between 6 and 12 weeks after infection. Only parasite-specific IgM levels increased significantly during this period, whereas total and parasite-specific IgE levels did not differ between groups., Conclusion: Basophils are sensitized early in the context of a low-dose primary infection with N americanus in the absence of measurable total and specific IgE serum level increase.

We have previously shown a reduction in anaemia and wasting malnutrition in infants <3 years old in Pemba Island, Zanzibar, following repeated anthelminthic treatment for the endemic gastrointestinal (GI) nematodes Ascaris lumbricoides, hookworm and Trichuris trichiura. In view of the low intensity of worm infections in this age group, this was unexpected, and it was proposed that immune responses to the worms rather than their direct effects may play a significant role in morbidity in infants and that anthelminthic treatment may alleviate such effects. Therefore, the primary aims of this study were to characterise the immune response to initial/early GI nematode infections in infants and the effects of anthelminthic treatment on such immune responses. The frequency and levels of Th1/Th2 cytokines (IL-5, IL-13, IFN-gamma and IL-10) induced by the worms were evaluated in 666 infants aged 6-24 months using the Whole Blood Assay. Ascaris and hookworm antigens induced predominantly Th2 cytokine responses, and levels of IL-5 and IL-13 were significantly correlated. The frequencies and levels of responses were higher for both Ascaris positive and hookworm positive infants compared with worm negative individuals, but very few infants made Trichuris-specific cytokine responses. Infants treated every 3 months with mebendazole showed a significantly lower prevalence of infection compared with placebo-treated controls at one year following baseline. At follow-up, cytokine responses to Ascaris and hookworm antigens, which remained Th2 biased, were increased compared with baseline but were not significantly affected by treatment. However, blood eosinophil levels, which were elevated in worm-infected children, were significantly lower in treated children. Thus the effect of deworming in this age group on anaemia and wasting malnutrition, which were replicated in this study, could not be explained by modification of cytokine responses but may be related to eosinophil function.

Patent and pathologic infections of the human hookworm Necator americanus were established in the common marmoset (Callithrix jacchus). In a pilot study, a laboratory strain of N. americanus was compared with a fresh field isolate. Pathology was more severe in animals infected with a fresh isolate. In all animals, infection was associated with increased total plasma IgE and production of IgG specific to adult worm excretory/secretory (ES) products. Histamine was released by basophils in response to IgE, ES products, and a recombinant hookworm allergen, calreticulin. The pilot study indicated the potential of this animal model of hookworm infection and led us to investigate the consequences of infecting a further cohort with the fresh field isolate. This second study confirmed our initial findings, that it is possible to investigate the human hookworm N. americanus in a model exhibiting many of the characteristics of the immunology of hookworm infection in its definitive host.

The Pseudomonas aeruginosa quorum-sensing signal molecule N-3-oxododecanoyl)-L-homoserine lactone (OdDHL) has been reported to affect the function of a wide range of mammalian cell types, including cells of the immune system. In T cells, it has been reported to inhibit the production of most cytokines, and it has been reported to inhibit the function of antigen-presenting cells. The intracellular target of OdDHL in these cells remains to be identified, although the lipophilic nature of the molecule suggested that the target could be membrane associated. We explored the association of radiolabelled OdDHL with the membrane and cytoplasm of Jurkat T-cell lines and of primary murine T cells and dendritic cells. We found that not only did 3H-OdDHL enter the cytoplasm of Jurkat cells without disproportionate association with the cell membrane, it also reached maximum levels in the cytoplasm very quickly, and that the intracellular concentration was proportional to the extracellular concentration. Similar results were obtained when 3H-OdDHL was incubated with primary murine T cells or cultured dendritic cells. In addition, we show that the cellular distribution of OdDHL does not significantly alter after stimulation of Jurkat cells or primary murine CD4 T cells with immobilized anti-CD3, with little activity being associated with nuclear fractions. Together, these data strongly suggest that OdDHL enters mammalian cells by passive mechanisms, and that it does not preferentially associate with the membrane or nucleus upon T-cell receptor ligation.

A popular hypothesis to explain parasite survival in the presence of a pronounced T helper 2 phenotype in helminth-parasitized populations has been Fc epsilonRI blockade by parasite-induced polyclonal IgE. To begin to test the hypothesis that Fc epsilonRI-bearing cells would be refractory to activation in parasitized populations, we investigated basophil function in 43 individuals from a hookworm endemic area. Study individuals had high levels of total IgE and eosinophilia and a mean hookworm burden of 2,257 epg. Basophils from all members of this parasitized population were shown to release histamine to a number of agonists, including anti IgE and a hookworm allergen, calreticulin. These data would indicate that Fc epsilonRI blockade at the level of the basophil did not occur in this parasitized population despite the presence of possible immunologic blocking agents. This would suggest that this effector arm of the T helper 2 phenotype remains operative in infected populations.

This methodological study was carried out in preparation for a major long term study, also reported in this volume, which was designed to investigate whether the combination of vaccines and pyridostigmine bromide (PB) could have been responsible for adverse signs and symptoms reported by a number of veterans of the 1990/1991 Gulf conflict. In this context, the marmoset has been used to model aspects of the human immune system. The purposes of this methodological study were to select appropriate immunochemical reagents to measure humoral responses induced in marmosets in response to selected health and hygiene and biological warfare vaccines and to initially assess the effects of PB on the responses recorded. Vaccines were administered at 1/5th of a human dose, and also investigated in combination with the nerve agent pretreatment compound PB. PB dosing was selected to induce an inhibition of erythrocyte acetylcholinesterase by 30%. In order to assess the functionality of the immune system, antibody responses to a neo-antigen (keyhole limpet haemocyanin--KLH), administered some 2 months following the completion of the vaccination schedule, were measured. The present study identified appropriate isotyping reporter reagents which cross-reacted with equivalent marmoset immunoglobulins. Robust antibody responses were identified against anthrax protective antigen (PA), whole cell pertussis vaccine and KLH, while weaker responses were measured against cholera and typhoid vaccines. The killed whole cell plague vaccine induced a response which was at the limit of detection of the assay. Coadministered PB had no discernable effect on immunological responses in this study.

The resurgence of larval biotherapy as a debridement tool in wound management has been accompanied by several clinical reports highlighting concomitant tissue regeneration. Studies employing in vitro cell motility assays have found that purified excretory/secretory (ES) products from Greenbottle larvae (blowfly, Lucilia sericata) are motogenic for human dermal fibroblasts when used as a supplement in culture media. The objective of the present study was to determine whether ES delivered using a prototype hydrogel wound dressing induced similar motogenic effects on fibroblastic (3T3) and epithelial cells (HaCaTs) comprising a scratched-monolayer wound model. Quantitative analysis by MTT assay failed to detect significant mitogenic effects of ES on either cell type. Quantitative image analysis revealed that ES exposure markedly accelerated wound closure through a motogenic effect on both fibroblasts and keratinocytes. Quantitative histochemical analysis detected significantly higher phosphotyrosine (pTyr) expression in ES-exposed cell cultures than in controls; moreover immunocytochemistry revealed conspicuously raised levels of pTyr expression in cells located at the wound margin. By attenuation with a panel of enzyme inhibitors these effects were attributed to the protease components of ES. The present results suggest that controlled delivery of ES as a follow-up to maggot debridement therapy may be an effective therapeutic option for stimulation of tissue regeneration in wound management.

Lucilia sericata larvae, or green bottle fly maggots are applied to chronic wounds to aid healing. Previously, our laboratory has characterized the enzymatic activities present within maggot excretions/secretions (ES). Since then, we have related these to the degradation of extracellular matrix components, alteration of human, dermal fibroblast adhesion to surfaces and the stimulation of fibroblast migration within a two-dimensional in vitro assay. In this study, we developed a novel three-dimensional in vitro assay in which to observe fibroblast migration and morphology in response to maggot ES. Here, primary human foreskin fibroblasts were embedded within collagen gels containing fibronectin. Phase contrast and confocal microscopy were used in conjunction with image analysis software to examine and quantify aspects of fibroblast behavior. Our results showed that maggot ES stimulated fibroblast migration through the matrix and induced altered cell morphologies. Remodelling of the extracellular matrix located between individual fibroblasts was also induced, providing a mechanism by which cells may detect each other's presence over considerable distances. Thus, mechanisms by which maggots enhance tissue formation within wounds may be via the promotion of fibroblast motility, acceleration of extracellular matrix remodelling and coordination of cellular responses.

In order to assess age-related changes in the immune status of Labrador retriever dogs, leukocyte phenotypes, lymphocyte proliferative capacity, and serum antibody levels were measured in four cohorts of dogs, ranging from 2 to 10 years of age. Absolute numbers of white blood cells, lymphocytes, monocytes, granulocytes, and CD3+, CD4+, CD8+ and CD21+ lymphocytes significantly decreased with increasing age. Relative percentages of lymphocytes and CD4 cells were significantly decreased, and relative percentages of granulocytes and CD8 cells significantly increased, with age. The CD4:CD8 ratio showed a significant age-related decrease. Proliferative responses of T-cells to mitogens in whole-blood cultures either increased (Concanavalin A) or remained the same (phytohemagglutinin) with age when data was normalised to allow for differences in responding cell number. Similarly, normalised data of proliferative response to anti-CD3 stimulation together with phorbol 12-myristate 13-acetate showed an age-related increase. Serum levels of total IgA significantly increased with age whereas total IgG levels remained unchanged. These observations illustrate a significant change to a number of immune parameters with age. However, further work is required to determine whether the differences reported here are sufficient to cause overt or functional immune senescence in Labrador retriever dogs.

Lucilia sericata larvae, or greenbottle fly maggots, placed within chronic wounds have been observed to remove necrotic tissue and infection. They are also believed to actively promote granulation tissue formation. Interactions between fibroblasts and the surrounding extracellular matrix play a crucial role in tissue formation, influencing fibroblast proliferation, migration, and tissue remodeling. For example, the strength of cell adhesion to surfaces coated with extracellular matrix influences cell motility. L. sericata larval excretory/secretory products having previously been shown to modify fibroblast adhesion to collagen and particularly fibronectin, it was hypothesized that these products would alter fibroblast migration. This was investigated using a two-dimensional in vitro wound assay, time-lapse digital photography, enzyme class-specific substrates and inhibitors, and gel electrophoresis. Results showed that L. sericata excretory/secretory products promoted fibroblast migration upon a fibronectin-coated surface. This was related to the degradation of fibronectin by serine proteinases within maggot excretion/secretions. The presence of a metalloproteinase activity may also have played a role. Thus, a possible mechanism by which maggots enhance tissue formation within wounds may be via the promotion of fibroblast motility, providing for a wider distribution of viable fibroblasts.

Asthma has reached epidemic proportions globally. This has been attributed by many to improved hygiene. The frequent failure of conventional pharmaceuticals to manage the disease has led to the introduction of parasites as a potential alternative therapy for asthma and other immunological diseases. In this article, we briefly review the immunological rationale underpinning therapeutic parasitic infection, describe recently initiated trials, and highlight potential risks and benefits of introducing parasites into patient cohorts.

Immune regulation, either via the autonomic nervous system or by a proposed "non-neuronal" cholinergic system, suggests that the immune system may be susceptible to perturbation by compounds affecting cholinergic function. Here, the current UK and US nerve agent pre-treatment, pyridostigmine bromide (PB) and the related anti-acetylcholinesterase (AChE) compounds physostigmine (PHY) and BW284c51 were tested for their ability to affect mouse splenocyte function in vitro. In addition, PB, at a dose equivalent to that received during pre-treatment for nerve agent poisoning, was tested for its effect on a T-cell-dependent humoral response to antigen in vivo in the mouse. None of the anti-AChEs tested affected concanavalin A (Con A)-, anti-CD3- or lipopolysaccharide LPS-driven splenocyte proliferation, in vitro, at concentrations expected to give effective nerve agent pre-treatment. However, higher concentrations (>100 microM) particularly of PHY caused some inhibition of the proliferative responses. In vivo, PB or saline was administered via 28-day mini-osmotic pumps to give a 25-40% inhibition of whole blood AChE in the PB-treated animals. During PB or saline administration, primary and secondary doses (i.p.) of sheep red blood cells (SRBC) were given and the humoral response determined by monitoring anti-SRBC IgM and IgG levels. Splenocytes isolated from the experimental animals were also examined for their proliferative and cytokine responses to stimulation. No remarkable effects of PB were seen during the period of AChE inhibition on the humoral immune response. However, a modest elevation in IL-2 and IFN(gamma) in Con A-stimulated lymphocytes was seen in PB-treated animals following pump removal. Overall these data suggest that, in vivo, the SRBC stimulated T-cell-dependent immune response is unaffected by the administration of PB at pre-treatment doses.

Pseudomonas aeruginosa releases a spectrum of well-regulated virulence factors, controlled by intercellular communication (quorum sensing) and mediated through the production of small diffusible quorum-sensing signal molecules (QSSM). We hypothesize that QSSM may in fact serve a dual purpose, also allowing bacterial colonization via their intrinsic immune-modulatory capacity. One class of signal molecule, the N-acylhomoserine lactones, has pleiotropic effects on eukaryotic cells, particularly those involved in host immunity. In the present study, we have determined the comparative effects of two chemically distinct and endobronchially detectable QSSM, N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and 2-heptyl-3-hydroxy-4 (1H)-quinolone or the Pseudomonas quinolone signal (PQS), on human leukocytes exposed to a series of stimuli designed to detect differential immunological activity in vitro. 3-Oxo-C12-HSL and PQS displayed differential effects on the release of interleukin-2 (IL-2) when human T cells were activated via the T-cell receptor and CD28 (a costimulatory molecule). 3-Oxo-C12-HSL inhibited cell proliferation and IL-2 release; PQS inhibited cell proliferation without affecting IL-2 release. Both molecules inhibited cell proliferation and the release of IL-2 following mitogen stimulation. Furthermore, in the presence of Escherichia coli lipopolysaccharide, 3-oxo-C12-HSL inhibited tumor necrosis factor alpha release from human monocytes, as reported previously (K. Tateda et al., Infect. Immun. 64:37-43, 1996), whereas PQS did not inhibit in this assay. These data highlight the presence of two differentially active immune modulatory QSSM from P. aeruginosa, which are detectable endobronchially and may be active at the host/pathogen interface during infection with P. aeruginosa, should the bronchial airway lymphoid tissues prove to be accessible to QSSM.

The current pretreatment against nerve agent poisoning deployed by the UK and US armed forces is the acetylcholinesterase (EC 3.1.1.7) inhibitor pyridostigmine bromide (PB). At higher doses, PB is also used to treat the autoimmune disease myasthenia gravis. In both cases, the therapeutic effect is mediated by inhibition of acetylcholinesterase (AChE) at cholinergic synapses. However, the location of AChE is not restricted to these sites. AChE, acetylcholine (ACh) receptors and choline acetyltransferase have been reported to be expressed by T cells, suggesting that cholinergic signalling may exert some modulatory influence on T-cell function and consequently on the immune system. The aim of this study was to investigate the role of the T-cell cholinergic system in the immunological activation process and to examine whether inhibitors of AChE such as PB affect immune function. To investigate this, human peripheral blood mononuclear cells (PBMC) were stimulated using either mitogen, cross-linking of the T-cell receptor and co-receptors with antibodies (anti-CD3/CD28) or by antigen presentation in the presence of various AChE inhibitors and ACh receptor agonists or antagonist. Several indices were used to assess T-cell activation, including the secretion of IL-2, cell proliferation and expression of CD69. Treatment with PB had no significant effect on the immunological assays selected. Physostigmine (PHY), a carbamate compound similar to PB, consistently showed inhibition of T-cell activation, but only at concentrations in excess of those required to inhibit AChE. No evidence was found to support previously published findings showing muscarinic enhancement of cell proliferation or IL-2 secretion.

Cytokine and proliferative responses to Necator americanus infection were measured in a treatment-reinfection study of infected subjects from an area of Papua New Guinea where N. americanus is highly endemic. Before treatment, most subjects produced detectable interleukin (IL)-4 (97%), IL-5 (86%), and interferon (IFN)- gamma (64%) in response to adult N. americanus antigen. Pretreatment IFN- gamma responses were negatively associated with hookworm burden, decreasing by 18 pg/mL for each increase of 1000 eggs/gram (epg) (n=75; P<.01). Mean IFN- gamma responses increased significantly after anthelmintic treatment, from 166 to 322 pg/mL (n=42; P<.01). The intensity of reinfection was significantly negatively correlated with pretreatment IL-5 responses, decreasing by 551 epg for each 100 pg/mL increase in production of IL-5 (n=51; P<.01). These data indicate that there is a mixed cytokine response in necatoriasis, with worm burden-associated suppression of IFN- gamma responses to adult N. americanus antigen. Resistance to reinfection is associated with the parasite-specific IL-5 response.

Helminth parasites are well known to induce an immune response in their hosts characterised by elevated IgE, peripheral blood or local tissue eosinophilia, and in some cases, intestinal mastocytosis. This immunological response has a strong T-helper 2 (Th2) cytokine bias and is reminiscent of the immunological constellation found in allergic diseases. However, the molecular forces driving the Th2 response to helminth parasites are still not understood. By using the human hookworm parasite Necator americanus as an example, the authors of the current article propose that in the course of its life cycle, this parasite becomes innately allergenic through the secretion of a molecular array designed to promote tissue migration and homing, feeding and survival against immunological attack. This complex array comprises proteases, lectins and other classes of molecules. Subsequent immunological and physiological events seemingly protect the host from both the allergic sequelae of exposure to environmental allergens and, moreover, from the parasite itself.

Hookworms are voracious blood-feeders. The cloning and functional expression of an aspartic protease, Na-APR-2, from the human hookworm Necator americanus are described here. Na-APR-2 is more similar to a family of nematode-specific, aspartic proteases than it is to cathepsin D or pepsin, and the term "nemepsins" for members of this family of nematode-specific hydrolases is proposed. Na-apr-2 mRNA was detected in blood-feeding, developmental stages only of N. americanus, and the protease was expressed in the intestinal lumen, amphids, and excretory glands. Recombinant Na-APR-2 cleaved human hemoglobin (Hb) and serum proteins almost twice as efficiently as the orthologous substrates from the nonpermissive dog host. Moreover, only 25% of the Na-APR-2 cleavage sites within human Hb were shared with those generated by the related N. americanus cathepsin D, Na-APR-1. Antiserum against Na-APR-2 inhibited migration of 50% of third-stage N. americanus larvae through skin, which suggests that aspartic proteases might be effective vaccines against human hookworm disease.

Comparative immune modulatory activity for a range of synthetic analogues of a Pseudomonas aeruginosa signal molecule, N-(3-oxododecanoyl)-l-homoserine lactone (3O, C(12)-HSL), is described. Twenty-four single or combination systematic alterations of the structural components of 3O, C(12)-HSL were introduced as described. Given the already defined immunological profile of the parent compound, 3O, C(12)-HSL, these compounds were assayed for their ability to inhibit murine and human leucocyte proliferation and TNF-alpha secretion by lipopolysaccharide (LPS) stimulated human leucocytes in order to provide an initial structure-activity profile. From IC(50) values obtained with a murine splenocyte proliferation assay, it is apparent that acylated l-homoserine lactones with an 11-13 C side chain containing either a 3-oxo or a 3-hydroxy group are optimal structures for immune suppressive activity. These derivatives of 3O, C(12)-HSL with monounsaturation and/or a terminal nonpolar substituent on the side chain were also potent immune suppressive agents. However, structures lacking the homoserine lactone ring, structures lacking the l-configuration at the chiral center, and those with polar substituents were essentially devoid of activity. The ability of compounds selected from the optimal activity range to modulate mitogen-driven human peripheral blood mononuclear cell proliferation and LPS-induced TNF-alpha secretion indicates the suitability of these compounds for further investigation in relation to their molecular mechanisms of action in TNF-alpha driven immunological diseases, particularly autoimmune diseases such as psoriasis, rheumatoid arthritis, and type 1 (autoimmune) diabetes.

The proteolytic activities frequently associated with sources of allergens and parasite secretions have been suggested as important immunomodulators. We have investigated whether the protease activity of the house dust mite allergen Der p1 and the secreted proteases of the hookworm Necator americanus are able to directly induce type 2 cytokine production by basophils. Der p1 and the secretions of N. americanus induced interleukin (IL)-4, IL-5, and IL-13 but not interferon-gamma mRNA in KU812 basophils. Enzyme-linked immunosorbent assay confirmed that IL-4 and IL-13 were secreted. A nonproteolytic antigen failed to induce cytokine expression, and preincubation of Der p1 or N. americanus secretions with protease inhibitors inhibited cytokine expression. Data were confirmed using basophils purified from human peripheral blood. We speculate that this innate mechanism may contribute to the development of a cytokine milieu that could promote immunoglobulin E synthesis, eosinophil recruitment, and the development of type 2 T cells.

Hookworms routinely reach the gut of nonpermissive hosts but fail to successfully feed, develop, and reproduce. To investigate the effects of host-parasite coevolution on the ability of hookworms to feed in nonpermissive hosts, we cloned and expressed aspartic proteases from canine and human hookworms. We show here that a cathepsin D-like protease from the canine hookworm Ancylosotoma caninum (Ac-APR-1) and the orthologous protease from the human hookworm Necator americanus (Na-APR-1) are expressed in the gut and probably exert their proteolytic activity extracellularly. Both proteases were detected immunologically and enzymatically in somatic extracts of adult worms. The two proteases were expressed in baculovirus, and both cleaved human and dog hemoglobin (Hb) in vitro. Each protease digested Hb from its permissive host between twofold (whole molecule) and sixfold (synthetic peptides) more efficiently than Hb from the nonpermissive host, despite the two proteases' having identical residues lining their active site clefts. Furthermore, both proteases cleaved Hb at numerous distinct sites and showed different substrate preferences. The findings suggest that the paradigm of matching the molecular structure of the food source within a host to the molecular structure of the catabolic proteases of the parasite is an important contributing factor for host-parasite compatibility and host species range.

Passage of helminth larvae through the lungs can cause pulmonary eosinophilia that may have evolved as a means of parasite attrition. If allergic responses represent a misdirected activation of this arm of the immune system, then mechanisms governing eosinophil recruitment during infection would be expected to be closely related to those seen in allergy. We studied primary Necator americanus infection and compared this to multiply-infected or vaccinated mice. The arrival of larvae in the lungs triggered rapid eosinophil recruitment, which was greatly enhanced in previously sensitized mice. Interestingly, the presence of larvae in the lung was sufficient to trigger eosinophil chemoattractant production, including the chemokines eotaxin and MIP-1alpha, and was not enhanced by prior exposure to the parasites. Infection stimulated IL-5 production in all groups; however, this and IgE production were greatly enhanced in sensitized animals. Elevated IL-5 increased bone marrow production of eosinophils, and eosinophilia was abrogated by treatment with anti-IL-5 antibody. Therefore, trapping of larvae in the pulmonary vasculature is sufficient to trigger eosinophil recruitment, by induction of chemokines and IL-5. Primed cognate Th2 immunity does not increase local chemokine production, but does increase IL-5 production, which greatly enhances the availability of eosinophils for recruitment to the lung.

C-type lectins (C-TLs) are carbohydrate-binding proteins central to diverse physiological processes including immunity, venom-induced haemostasis and wound repair. Here we describe the cloning of Na-ctl-2, a cDNA encoding a secreted C-TL from the human hookworm Necator americanus. The transcript was detected in mRNA from adult worms but not infective larvae. The cDNA encoded an N-terminal secretory signal peptide followed by a long-form C-TL domain with sequence similarity to C-TL-like proteins from Caenorhabditis elegans and mammalian antigen presenting cell receptors, suggesting that hookworms might utilise this class of lectin to interrupt anti-parasite immune responses or interfere with host clotting mechanisms. This is the first report of a full-length cDNA encoding a lectin from hookworms. The unusually skewed representation of this protein family within different nematode genera and its subsequent impact on the evolution of nematode parasitism is discussed.