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.

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.

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.

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.

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.

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.

Molecular characterisation of a pH-gated chloride channel from Sarcoptes scabiei.

Reports of ivermectin resistance in scabies mites raise concerns regarding the sustainability of mass intervention programs for scabies worldwide and for the treatment of crusted scabies. Ligand gated ion channels (LGICs) are the primary targets of ivermectin in invertebrates. We report the molecular characterisation of SsCl--a novel LGIC from Sarcoptes scabiei var. hominis. While SsCl shows sequence similarity to other LGICs, phylogenetic analysis does not suggest strong homology to conventional glutamate, histamine or GABA gated channels. Instead, it is most similar to Drosophila pH-sensitive and group 1 clades. When expressed in Xenopus oocytes, SsCl forms a homomeric, pH-gated chloride channel that is irreversibly activated by ivermectin. These results provide the first confirmation that this group of LGIC exists in arachnids, and suggest that SsCl may be an in vivo target of ivermectin in S. scabiei.

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.

Scabies mite inactivated serine protease paralogues are present both internally in the mite gut and externally in feces.

The scabies mite, Sarcoptes scabiei, is the causative agent of scabies, a disease that is common among disadvantaged populations and facilitates streptococcal infections with serious sequelae. Previously, we encountered large families of genes encoding paralogues of house dust mite protease allergens with their catalytic sites inactivated by mutation (scabies mite inactivated protease paralogues [SMIPPs]). We postulated that SMIPPs have evolved as an adaptation to the parasitic lifestyle of the scabies mite, functioning as competitive inhibitors of proteases involved in the host-parasite interaction. To propose testable hypotheses for their functions, it is essential to know their locations in the mite. Here we show by immunohistochemistry that SMIPPs exist in two compartments: 1) internal to the mite in the gut and 2) external to the mite after excretion from the gut in scybala (fecal pellets). SMIPPs may well function in both of these compartments to evade host proteases.

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.

Identification and characterization of Sarcoptes scabiei and Dermatophagoides pteronyssinus glutathione S-transferases: implication as a potential major allergen in crusted scabies.

The astigmatid mite Sarcoptes scabiei is the causative agent of scabies, a highly infectious parasitic disease of the skin. Although the mite causes marked hypersensitivity reactions, particularly in crusted (severe) scabies, little is known about the specific scabies mite molecules involved in such immunologic responses. We have identified six genes encoding scabies mite homologues of mu and delta-like glutathione S-transferases (GSTs) as well as novel house dust mite GSTs. A mu class S. scabiei GST was subcloned into a prokaryotic expression system. The purified recombinant protein rSsGST01 reacted strongly with IgE and IgG4 in sera from crusted scabies patients. This response was not observed with control antigens or with ordinary scabies and uninfested patient sera. In addition, the specific IgE response to rSsGST01 did not correlate with the total IgE level of the patient. These results suggest that GST may play a role in the pathophysiology associated with crusted 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.

Mechanisms for a novel immune evasion strategy in the scabies mite sarcoptes scabiei: a multigene family of inactivated serine proteases.

Parasitic infestation of the skin by the mite Sarcoptes scabiei is a significant problem worldwide, particularly in socially disadvantaged communities. A multigene family of at least 24 homologs of a serine protease allergen have been identified in S. scabiei. Surprisingly, the products of all but one of these genes are predicted to be catalytically inactive, due to mutations at a critical triad of amino acids at the active site. We discuss the possibility that these genes for inactivated proteases have been conserved because they mediate a novel host defense evasion strategy that the mite has evolved as an adaptation to parasitism of the epidermis. The identification of this family, and elucidation of its value to the parasite, may present an unanticipated approach to protective vaccination.

First documentation of in vivo and in vitro ivermectin resistance in Sarcoptes scabiei.

Ivermectin is increasingly being used to treat scabies, especially crusted (Norwegian) scabies. However, treatment failures, recrudescence, and reinfection can occur, even after multiple doses. Ivermectin resistance has been documented for some intestinal helminths in animals with intensive ivermectin exposure. Ivermectin resistance has also been induced in arthropods in laboratory experiments but, to date, has not been documented among arthropods in nature. We report clinical and in vitro evidence of ivermectin resistance in 2 patients with multiple recurrences of crusted scabies who had previously received 30 and 58 doses of ivermectin over 4 and 4.5 years, respectively. As predicted, ivermectin resistance in scabies mites can develop after intensive ivermectin use.

Scabies: new future for a neglected disease.

Scabies is a disease of global proportions in both human and animal populations, resulting from infestation of the skin with the "itch" mite Sarcoptes scabiei. Despite the availability of effective chemotherapy the intensely itching lesions engender significant morbidity primarily due to secondary sepsis and post-infective complications. Some patients experience an extreme form of the disease, crusted scabies, in which many hundreds of mites may infest the skin causin severe crusting and hyperkeratosis. Overcrowded living conditions and poverty have been identified as significant confounding factors in transmission of the mite in humans. Control is hindered by difficulties with diagnosis, the cost of treatment, evidence for emerging resistance and lack of effective vaccines. Historically research on scabies has been extremely limited because of the difficulty in obtaining sufficient quantities of the organism. Recent molecular approaches have enabled considerable advances in the study of population genetics and transmission dynamics of S. scabiei. However, the most exciting and promising development is the potential exploitation of newly available data from S. scabiei cDNA libraries and EST projects. Ultimately this knowledge may aid early identification of disease, novel forms of chemotherapy, vaccine development and new treatment possibilities for this important but neglected parasite.

Generation and characterization of cDNA clones from Sarcoptes scabiei var. hominis for an expressed sequence tag library: identification of homologues of house dust mite allergens.

Molecular studies on scabies, a disease of considerable human and veterinary significance, have been limited because of the difficulty of obtaining the causative organism Sarcoptes scabiei, the "itch mite." We have used skin from the bedding of crusted scabies patients as a source of mites for the construction of libraries of cDNAs from S. scabiei var. hominis in the bacteriophage lambda vector lambdaZAP express. Sequences of 145 clones established that the libraries predominantly contain sequences from S. scabiei, enabling a major sequencing program to begin. Among those sequenced to date, cDNAs encoding S. scabiei homologues of 3 house dust mite allergens-the M-177 apolipoprotein, glutathione S-transferase, and paramyosin--were identified. The availability of cDNA libraries from S. scabiei var. hominis and S. scabiei var. vulpes and the emerging public sequence databases from both opens up new possibilities in scabies research.