Early immune suppression leads to uncontrolled mite proliferation and potent host inflammatory responses in a porcine model of crusted versus ordinary scabies.

Scabies is a neglected tropical disease of global significance. Our understanding of host-parasite interactions has been limited, particularly in crusted scabies (CS), a severe clinical manifestation involving hyper-infestation of Sarcoptes scabiei mites. Susceptibility to CS may be associated with immunosuppressive conditions but CS has also been seen in cases with no identifiable risk factor or immune deficit. Due to ethical and logistical difficulties with undertaking research on clinical patients with CS, we adopted a porcine model which parallels human clinical manifestations. Transcriptomic analysis using microarrays was used to explore scabies pathogenesis, and to identify early events differentiating pigs with ordinary (OS) and crusted scabies. Pigs with OS (n = 4), CS (n = 4) and non-infested controls (n = 4) were compared at pre-infestation, weeks 1, 2, 4 and 8 post-infestation. In CS relative to OS, there were numerous differentially expressed genes including pro-inflammatory cytokines (IL17A, IL8, IL19, IL20 and OSM) and chemokines involved in immune cell activation and recruitment (CCL20, CCL27 and CXCL6). The influence of genes associated with immune regulation (CD274/PD-L1 and IL27), immune signalling (TLR2, TLR8) and antigen presentation (RFX5, HLA-5 and HLA-DOB) were highlighted in the early host response to CS. We observed similarities with gene expression profiles associated with psoriasis and atopic dermatitis and confirmed previous observations of Th2/17 pronounced responses in CS. This is the first comprehensive study describing transcriptional changes associated with the development of CS and significantly, the distinction between OS and CS. This provides a basis for clinical follow-up studies, potentially identifying new control strategies for this severely debilitating disease.

Host-Defense Peptides Caerin 1.1 and 1.9 Stimulate TNF-Alpha-Dependent Apoptotic Signals in Human Cervical Cancer HeLa Cells.

Host defense caerin 1.1 and 1.9 peptides, isolated from the glandular secretion of Australian tree frogs, the genus Litoria, have been previously shown to have multiple biological activities, including the inhibition of human papillomavirus (HPV) 16 early protein E7 transformed murine as well as human cancerous cell proliferation both in vitro and in vivo. However, the mechanism underlying their anti-proliferative activities against HPV18+ cervical cancer HeLa cells remains unknown. This study comparatively investigated the anti-proliferation on HeLa cells by caerin 1.1, 1.9, and their mixture, followed by confocal microscopy examination to assess the cellular intake of the peptides. Tandem mass tag labeling proteomics was employed to reveal the proteins that were significantly regulated by the peptide treatment in cells and cell growth environment, to elucidate the signaling pathways that were modulated. Western blot was performed to confirm the modulation of the pathways. Both caerin 1.1 and 1.9 highly inhibited HeLa cell proliferation with a significant additive effect compared to untreated and control peptide. They entered the cells with different magnitudes. Intensive protein-protein interaction was detected among significantly upregulated proteins. Translation, folding and localization of proteins and RNA processing, apoptosis process was significantly enriched post the treatments. The apoptotic signaling was suggested as a result of tumor necrosis factor-α (TNF-α) pathway activation, indicated by the dose-dependent elevated levels of caspase 3 and caspase 9. The epidermal growth factor receptor and androgen receptor pathways appeared inhibited by the peptides. Moreover, the activation of T-cell receptor derived from the quantitation results further implies the likelihood of recruiting more T cells to the cell growth environment post the treatment and more sensitive to T cell mediated killing of HeLa cells. Our results indicate that caerin 1.1 and 1.9 mediate apoptotic signals of HeLa cells and may subsequently enhances adaptive T cell immune responses.

Topical application of temperature-sensitive caerin 1.1 and 1.9 gel inhibits TC-1 tumor growth in mice.

Genital warts, which are one of the most common sexually transmitted diseases (STDs), result from persistent infection with human papillomavirus (HPV), especially subtypes 6 or 11. Topical application of 5% imiquimod cream is currently recommended as a first-line treatment choice for genital warts, but the clearance and patient compliance rates remain less than sufficient. In the current study, we developed a temperature-sensitive gel that contains the host-defense peptides caerin 1.1 and 1.9, which were originally isolated from Australian tree frogs of the genus Litoria. Growth of HPV16 E6/E7-transformed TC-1 cells was inhibited in vitro and in vivo following injection of the tumor with the caerin gel in a TC-1 tumor mouse model. Furthermore, when the caerin gel was topically applied, the inhibitory effect remained, and T, NK cells were attracted to the tumor site. In addition, the gel maintained a similar level of bioactivity after incubation at room temperature for 30 days. Our results suggest that this caerin gel, following further optimization, may provide an alternative method for the management of genital warts.

Synthesized natural peptides from amphibian skin secretions increase the efficacy of a therapeutic vaccine by recruiting more T cells to the tumour site.

BACKGROUND: Therapeutic vaccines against cervical cancer remain ineffective. Previously, we demonstrated that blocking the signalling of a cytokine, interleukin 10, at the time of immunisation elicited significantly higher numbers of antigen specific T cells and inhibited tumour growth in mice. RESULTS: In the current paper, we demonstrate, in a HPV16 E6/E7 transformed TC-1 tumour mouse model, that despite increased antigen specific T cell numbers, blocking IL-10 signalling at the time of immunisation does not increase the survival time of the TC-1 tumour bearing mice compared to mice receiving the same immunisation with no IL-10 signalling blockade. Moreover, the function of tumour infiltrating T cells isolated 3 weeks post TC-1 transplantation is more suppressed than those isolated 2 weeks after tumour inoculation. We demonstrate that synthesized caerin peptides, derived from amphibian skin secretions, 1) were able to inhibit TC-1 tumour growth both in vitro and in vivo; 2) are environmentally stable; and 3) promote the secretion of pro-inflammatory interlukine-6 by TC-1 cells. Notably caerin peptides were able to increase the survival time of TC-1 tumour bearing mice after therapeutic vaccination with a HPV16E7 peptide-based vaccine containing IL-10 inhibitor, via recruiting increased levels of T cells to the tumour site. CONCLUSION: Caerin peptides increase the efficacy of a therapeutic vaccine by recruiting more T cells to the tumour site.

Comparative Proteomic Study of the Antiproliferative Activity of Frog Host-Defence Peptide Caerin 1.9 and Its Additive Effect with Caerin 1.1 on TC-1 Cells Transformed with HPV16 E6 and E7.

Caerin is a family of peptides isolated from the glandular secretion of Australian tree frogs, the genus Litoria, and has been previously shown to have anticancer activity against several cancer cells. In this work, we used two host-defence peptides, caerin 1.1 and caerin 1.9, to investigate their ability to inhibit a murine derived TC-1 cell transformed with human papillomavirus 16 E6 and E7 growth in vitro. Caerin 1.9 inhibits TC-1 cell proliferation, although inhibition is more pronounced when applied in conjunction with caerin 1.1. To gain further insights into the antiproliferative mechanisms of caerin 1.9 and its additive effect with caerin 1.1, we used a proteomics strategy to quantitatively examine (i) the changes in the protein profiles of TC-1 cells and (ii) the excretory-secretory products of TC-1 cells following caerin peptides treatment. Caerin 1.9 treatment significantly altered the abundance of several immune-related proteins and related pathways, such as the Tec kinase and ILK signalling pathways, as well as the levels of proinflammatory cytokines and chemokines. In conclusion, caerin peptides inhibit TC-1 cell proliferation, associated with modification in signalling pathways that would change the tumour microenvironment which is normally immune suppressive.

Treatment of scabies using a tea tree oil-based gel formulation in Australian Aboriginal children: protocol for a randomised controlled trial.

INTRODUCTION: In remote Aboriginal communities in Australia, scabies affects 7 out of 10 children before their first birthday. This is more than six times the rate seen in the rest of the developed world. Scabies infestation is frequently complicated by bacterial infection, leading to the development of skin sores and other more serious consequences, such as septicaemia and chronic heart and kidney diseases. Tea tree oil (TTO) has been used as an antimicrobial agent for several decades with proven clinical efficacy. Preclinical investigations have demonstrated superior scabicidal properties of TTO compared with widely used scabicidal agents, such as permethrin 5% cream and ivermectin. However, current data are insufficient to warrant a broad recommendation for its use for the management of scabies because previous studies were small or limited to in vitro observations. METHODS AND ANALYSIS: A pragmatic first trial will examine the clinical efficacy of a simple and low-cost TTO treatment against paediatric scabies and the prevention of associated secondary bacterial infections, with 1:1 randomisation of 200 participants (Aboriginal children, aged 5-16 years and living in remote Australia) into active control (permethrin 5% cream) and treatment (5% TTO gel) groups. The primary outcome for the study is clinical cure (complete resolution). Secondary outcome measures will include relief of symptoms, recurrence rate, adverse effects, adherence to treatment regimen and patient acceptability. ETHICS AND DISSEMINATION: The project has received approvals from the University of Canberra Human Research Ethics Committee (HREC 16-133), Wurli-Wurlinjang Health Service Indigenous subcommittee and the Aboriginal Medical Services Alliance Northern Territory reference group. The results of this study will be published in core scientific publications, with extensive knowledge exchange activities with non-academic audiences throughout the duration of the project. TRIAL REGISTRATION: ACTRN12617000902392; Pre-results.

Host immune responses to the itch mite, Sarcoptes scabiei, in humans.

Scabies is a parasitic disease due to infestation of skin by the burrowing mite Sarcoptes scabiei. Scabies is a major public health problem and endemic in resource poor communities worldwide affecting over 100 million people. Associated bacterial infections cause substantial morbidity, and in severe cases can lead to renal and cardiac diseases. Mite infestation of the skin causes localised cutaneous inflammation, pruritus, skin lesions, and allergic and inflammatory responses are mounted by the host against the mite and its products. Our current understanding of the immune and inflammatory responses associated with the clinical manifestations in scabies is far outweighed by the significant global impact of the disease. This review aims to provide a better understanding of human immune responses to S. scabiei in ordinary and crusted scabies phenotypes.

Characterization of Sarcoptes scabiei Tropomyosin and Paramyosin: Immunoreactive Allergens in Scabies.

Scabies is a human skin disease due to the burrowing ectoparasite Sarcoptes scabiei var. hominis resulting in intense itching and inflammation and manifesting as a skin allergy. Because of insufficient mite material and lack of in vitro propagation system for antigen preparation, scabies is a challenging disease to develop serological diagnostics. For allergen characterization, full-length S. scabiei tropomyosin (Sar s 10) was cloned, expressed in pET-15b, and assessed for reactivity with IgE antibodies from human sera. IgE binding was observed to Sar s 10 with sera collected from subjects with ordinary scabies, house dust mite (HDM)-positive and naive subjects and a diagnostic sensitivity of < 30% was observed. S. scabiei paramyosin (Sar s 11) was cloned, and expressed in pET-28a in three overlapping fragments designated Sspara1, Sspara2, and Sspara3. IgE and IgG binding was observed to Sspara2 and Sspara3 antigens with sera collected from ordinary scabies, and HDM-positive subjects, but no binding was observed with sera collected from naive subjects. Sspara2 displayed excellent diagnostic potential with 98% sensitivity and 90% specificity observed for IgE binding and 70% sensitivity for IgG. In contrast, the diagnostic sensitivity of Sspara3 was 84% for IgE binding and 40% for IgG binding. In combination, Sspara2 and Sspara3 provided an IgE sensitivity of 94%. This study shows that IgE binding to Sspara2 and Sspara3 is a highly sensitive method for diagnosis of scabies infestation in clinical practice. The developed enzyme-linked immunosorbent assay helps direct future development of a specific diagnostic tool for scabies.

In Vitro Efficacy of Moxidectin versus Ivermectin against Sarcoptes scabiei.

Moxidectin is under consideration for development as a treatment for human scabies. As some arthropods show decreased sensitivity to moxidectin relative to ivermectin, it was important to assess this for Sarcoptes scabieiIn vitro assays showed that the concentration of moxidectin required to kill 50% of mites was lower than that of ivermectin (0.5 µM versus 1.8 µM at 24 h; P < 0.0001). This finding provides further support for moxidectin as a candidate for the treatment of human scabies.

Therapeutic Potential of Tea Tree Oil for Scabies.

Globally, scabies affects more than 130 million people at any time. In the developed world, outbreaks in health institutions and vulnerable communities result in a significant economic burden. A review of the literature demonstrates the emergence of resistance toward classical scabicidal treatments and the lack of effectiveness of currently available scabicides in reducing the inflammatory skin reactions and pyodermal progression that occurs in predisposed patient cohorts. Tea tree oil (TTO) has demonstrated promising acaricidal effects against scabies mites in vitro and has also been successfully used as an adjuvant topical medication for the treatment of crusted scabies, including cases that did not respond to standard treatments. Emerging acaricide resistance threatens the future usefulness of currently used gold standard treatments (oral ivermectin and topical permethrin) for scabies. The imminent development of new chemical entities is doubtful. The cumulative acaricidal, antibacterial, antipruritic, anti-inflammatory, and wound healing effects of TTO may have the potential to successfully reduce the burden of scabies infection and the associated bacterial complications. This review summarizes current knowledge on the use of TTO for the treatment of scabies. On the strength of existing data for TTO, larger scale, randomized controlled clinical trials are warranted.

Scabies: an ancient global disease with a need for new therapies.

BACKGROUND: Scabies is an ancient disease (documented as far back as 2500 years ago). It affects about 300 million people annually worldwide, and the prevalence is as high as about 60% in Indigenous and Torres Strait Islander children in Australia. This is more than six times the rate seen in the rest of the developed world. Scabies is frequently complicated by bacterial infection leading to the development of skin sores and other more serious consequences such as septicaemia and chronic heart and kidney diseases. This causes a substantial social and economic burden especially in resource poor communities around the world. DISCUSSION: Very few treatment options are currently available for the management of scabies infection. In this manuscript we briefly discuss the clinical consequences of scabies and the problems found (studies conducted in Australia) with the currently used topical and oral treatments. Current scabies treatment options are fairly ineffective in preventing treatment relapse, inflammatory skin reactions and associated bacterial skin infections. None have ovicidal, antibacterial, anti-inflammatory and/or anti-pruritic properties. Treatments which are currently available for scabies can be problematic with adverse effects and perhaps of greater concern the risk of treatment failure. The development of new chemical entities is doubtful in the near future. Though there may be potential for immunological control, the development of a vaccine or other immunotherapy modalities may be decades away. The emergence of resistance among scabies mites to classical scabicides and ineffectiveness of current treatments (in reducing inflammatory skin reactions and secondary bacterial infections associated with scabies), raise serious concerns regarding current therapy. Treatment adherence difficulties, and safety and efficacy uncertainties in the young and elderly, all signal the need to identify new treatments for scabies.

Prospective study in a porcine model of sarcoptes scabiei indicates the association of Th2 and Th17 pathways with the clinical severity of scabies.

BACKGROUND: Understanding of scabies immunopathology has been hampered by the inability to undertake longitudinal studies in humans. Pigs are a useful animal model for scabies, and show clinical and immunologic changes similar to those in humans. Crusted scabies can be readily established in pigs by treatment with the glucocorticoid dexamethasone (Dex). METHODOLOGY/ PRINCIPAL FINDINGS: Prospective study of 24 pigs in four groups: a) Scabies+/Dex+, b) Scabies+/Dex-, c) Scabies-/Dex+ and d) Scabies-/Dex-. Clinical symptoms were monitored. Histological profiling and transcriptional analysis of skin biopsies was undertaken to compare changes in cell infiltrates and representative cytokines. A range of clinical responses to Sarcoptes scabiei were observed in Dex treated and non-immunosuppressed pigs. An association was confirmed between disease severity and transcription of the Th2 cytokines IL-4 and IL-13, and up-regulation of the Th17 cytokines IL-17 and IL-23 in pigs with crusted scabies. Immunohistochemistry revealed marked infiltration of lymphocytes and mast cells, and strong staining for IL-17. CONCLUSIONS/ SIGNIFICANCE: While an allergic Th2 type response to scabies has been previously described, these results suggest that IL-17 related pathways may also contribute to immunopathology of crusted scabies. This may lead to new strategies to protect vulnerable subjects from contracting recurrent crusted scabies.

An aspartic protease of the scabies mite Sarcoptes scabiei is involved in the digestion of host skin and blood macromolecules.

BACKGROUND: Scabies is a disease of worldwide significance, causing considerable morbidity in both humans and other animals. The scabies mite Sarcoptes scabiei burrows into the skin of its host, obtaining nutrition from host skin and blood. Aspartic proteases mediate a range of diverse and essential physiological functions such as tissue invasion and migration, digestion, moulting and reproduction in a number of parasitic organisms. We investigated whether aspartic proteases may play role in scabies mite digestive processes. METHODOLOGY/PRINCIPLE FINDINGS: We demonstrated the presence of aspartic protease activity in whole scabies mite extract. We then identified a scabies mite aspartic protease gene sequence and produced recombinant active enzyme. The recombinant scabies mite aspartic protease was capable of digesting human haemoglobin, serum albumin, fibrinogen and fibronectin, but not collagen III or laminin. This is consistent with the location of the scabies mites in the upper epidermis of human skin. CONCLUSIONS/SIGNIFICANCE: The development of novel therapeutics for scabies is of increasing importance given the evidence of emerging resistance to current treatments. We have shown that a scabies mite aspartic protease plays a role in the digestion of host skin and serum molecules, raising the possibility that interference with the function of the enzyme may impact on mite survival.

A novel clinical grading scale to guide the management of crusted scabies.

BACKGROUND: Crusted scabies, or hyperinfestation with Sarcoptes scabiei, occurs in people with an inadequate immune response to the mite. In recent decades, data have emerged suggesting that treatment of crusted scabies with oral ivermectin combined with topical agents leads to lower mortality, but there are no generally accepted tools for describing disease severity. Here, we describe a clinical grading scale for crusted scabies and its utility in real world practice. METHODOLOGY/PRINCIPAL FINDINGS: In 2002, Royal Darwin Hospital (RDH), a hospital in tropical Australia developed and began using a clinical grading scale to guide the treatment of crusted scabies. We conducted a retrospective observational study including all episodes of admission to RDH for crusted scabies during the period October 2002-December 2010 inclusive. Patients who were managed according to the grading scale were compared with those in whom the scale was not used at the time of admission but was calculated retrospectively. There were 49 admissions in 30 patients during the study period, of which 49 (100%) were in Indigenous Australians, 29 (59%) were male and the median age was 44.1 years. According to the grading scale, 8 (16%) episodes were mild, 24 (49%) were moderate, and 17 (35%) were severe. Readmission within the study period was significantly more likely with increasing disease severity, with an odds ratio (95% CI) of 12.8 (1.3-130) for severe disease compared with mild. The patients managed according to the grading scale (29 episodes) did not differ from those who were not (20 episodes), but they received fewer doses of ivermectin and had a shorter length of stay (11 vs. 16 days, p = 0.02). Despite this the outcomes were no different, with no deaths in either group and a similar readmission rate. CONCLUSIONS/SIGNIFICANCE: Our grading scale is a useful tool for the assessment and management of crusted scabies.

Antibody responses to Sarcoptes scabiei apolipoprotein in a porcine model: relevance to immunodiagnosis of recent infection.

No commercial immunodiagnostic tests for human scabies are currently available, and existing animal tests are not sufficiently sensitive. The recombinant Sarcoptes scabiei apolipoprotein antigen Sar s 14.3 is a promising immunodiagnostic, eliciting high levels of IgE and IgG in infected people. Limited data are available regarding the temporal development of antibodies to Sar s 14.3, an issue of relevance in terms of immunodiagnosis. We utilised a porcine model to prospectively compare specific antibody responses to a primary infestation by ELISA, to Sar s 14.3 and to S. scabiei whole mite antigen extract (WMA). Differences in the antibody profile between antigens were apparent, with Sar s 14.3 responses detected earlier, and declining significantly after peak infestation compared to WMA. Both antigens resulted in >90% diagnostic sensitivity from weeks 8-16 post infestation. These data provide important information on the temporal development of humoral immune responses in scabies and further supports the development of recombinant antigen based immunodiagnostic tests for recent scabies infestations.

Immunology of scabies and translational outcomes: identifying the missing links.

PURPOSE OF REVIEW: We propose that a major gap in the control, prevention, diagnosis and treatment of scabies exists because of lack of key translational understandings related to the immunopathology of scabies and the associated severe form of the disease, crusted scabies. Understanding the complex network of innate and adaptive immune responses, including the long lag period from infection to clinical symptoms, is fundamental to developing early interventions and decreasing transmission. Such interventions must be driven by clinical need and include user-friendly translational outcomes for improved control in endemic and resource-poor settings. RECENT FINDINGS: In the last few years, we have seen an increase in the molecular characterization of scabies mite proteins. However, owing to limited capacity in scabies immunology-related research, little is still known regarding the immunological effects of the mite or mite products on disease progression or protection. SUMMARY: Detailing the skin immunopathogenesis in relation to scabies, including the role of macrophages, mast cells and eosinophils, as well as the immunomodulatory effects of parasite evasion mechanisms are essential for the rational design of future therapeutic, diagnostic and preventative tools. Resolving this knowledge gap could ultimately lead to significant improvements in clinical and public health interventions. This article proposes a conceptual model for capacity building to inform future research activities in the field.

Scratching the itch: new tools to advance understanding of scabies.

Scabies remains a significant public health problem worldwide. Research into aspects of Sarcoptes scabiei biology and host-parasite interactions has been impeded by an inability to maintain mites in vitro and by limited access to parasite material and infected subjects. The generation of comprehensive expressed sequence tag libraries has enabled the initial characterisation of molecules of interest to diagnostics, vaccines, and drug resistance. The recent development and utilisation of animal models, combined with next-generation technologies, is anticipated to lead to new strategies to prevent, diagnose, and treat scabies, ultimately improving skin health in both human and veterinary settings. This article will summarise recent molecular and immunologic advances on scabies, and will address priorities for the exciting 'next chapter' of scabies research.

The interplay between diet and emerging allergy: what can we learn from Indigenous Australians?

The pathophysiology of atopic diseases, including asthma and allergy, is the result of complex gene-environment interactions. Since European colonization the Indigenous population of Australia has undergone significant changes with respect to their lifestyle as hunter-gatherers. These changes have had a detrimental effect on Aboriginal health, in part due to immunological modification. This review provides a comparative look at both the traditional Aboriginal/Indigenous diet and modern Western diets, examines some common allergies increasingly reported in contemporary Indigenous populations, and reviews concepts such the effect of vitamin deficiencies and changes in gut microbiota on immune function.

A diagnostic test for scabies: IgE specificity for a recombinant allergen of Sarcoptes scabiei.

Scabies infestations are difficult to diagnose clinically and current serologic tests have less than 50% accuracy. To develop more reliable diagnosis of scabies, specific IgE antibodies to a major scabies antigen recombinant Sar s 14.3 (rSar s 14.3) were measured in 140 plasma samples from scabies-infested and control subject groups using dissociation-enhanced lanthanide fluorescent immunoassays (DELFIA). Levels of rSar s 14.3-specific IgE were quantified, and cross-reactivity with its house dust mite homologue, Der p 14, was assessed. The rSar s 14.3 DELFIA showed excellent diagnostic capability, with 100% sensitivity and 93.75% specificity for distinguishing subjects with current scabies infestation from control, uninfested subjects. Recombinant Der p 14 preparation was ineffective at inhibiting IgE binding to rSar s 14.3. This study shows that quantification of levels of IgE antibody to rSar s 14.3 is a highly sensitive method for diagnosis of scabies infestation in clinical practice.

The evolution of infectious agents in relation to sex in animals and humans: brief discussions of some individual organisms.

The following series of concise summaries addresses the evolution of infectious agents in relation to sex in animals and humans from the perspective of three specific questions: (1) what have we learned about the likely origin and phylogeny, up to the establishment of the infectious agent in the genital econiche, including the relative frequency of its sexual transmission; (2) what further research is needed to provide additional knowledge on some of these evolutionary aspects; and (3) what evolutionary considerations might aid in providing novel approaches to the more practical clinical and public health issues facing us currently and in the future?