Cutaneous Leishmaniasis - Skin Barrier Properties And Drug Delivery Strategies

Cutaneous leishmaniasis (CL) is a parasitic disease caused by several species of the protozoan parasite Leishmania and affects approximately 10 million people worldwide. The drugs currently available such as miltefosine, amphotericin B and pentavalent antimonials, are limited by high cost, considerable side effects and restricted efficacy. CL could potentially be treated by a topical formulation. In its simplest form CL consists of a single nodule or papule, typically on exposed body parts, with the parasites residing in the lower epidermis and dermis. A topical treatment would minimise possible adverse effects by reducing the amount of drug taken up in the systemic circulation and would be easy to apply which is important for patient compliance. The overall aim of the work described in this thesis was to explore the use of different drugs in a topical formulation to cure CL. To be efficacious, a topical treatment requires the permeation of the active ingredient through the stratum corneum and into the deeper layers of the skin where the Leishmania parasites resides. Intact skin is a highly effective barrier against xenobiotics. However many skin diseases are known to affect the skin barrier making it more or less permeable to drugs. An understanding of the permeability of the skin hosting the Leishmania parasites is a prerequisite when trying to optimize drug delivery to the skin. Therefore the barrier function of Leishmania infected skin in early stages of CL was characterised with respect to histology, transepidermal water loss and drug permeation. Suitable anti-leishmanial drugs with the potential to permeate into the skin were identified through literature review, taking into account the information obtained from the skin barrier characterisation. The leishmanicidal activity of the drugs was evaluated in an intracellular amastigote-macrophage model and potent compounds were selected for further work. First, the re-formulation of miltefosine, currently used to treat leishmaniasis via oral administration, into a topical formulation was explored. The effects of different solvents on its permeation through and disposition in the skin was evaluated using in vitro Franz diffusion cell permeation studies. Secondly, a drug discovery approach was used to identify lead benzoxaboroles, a set of interesting anti-leishmanial compounds. In the early stages, in vitro ADME studies were conducted to establish basic pharmacokinetic parameters of a range of benzoxaboroles. This information together with previously unpublished data was used to select compounds for further testing such as stability and binding in skin homogenate and permeation evaluation. The three most promising compounds were tested in vivo in a CL model

Relation between Skin Pharmacokinetics and Efficacy in AmBisome Treatment of Murine Cutaneous Leishmaniasis

AmBisome® (LAmB), a liposomal formulation of amphotericin B (AmB), is a second-line treatment for the parasitic skin disease cutaneous leishmaniasis (CL). Little is known about its tissue distribution and pharmacodynamics to inform clinical use in CL. Here, we compared the skin pharmacokinetics of LAmB with Fungizone® (DAmB), the deoxycholate form of AmB, in murine models of Leishmania major CL. Drug levels at the target site (the localized lesion) 48 hours after single intravenous (IV) dosing of the individual AmB formulations (1 mg/kg of body weight) were similar, but were 3-fold higher for LAmB than for DAmB on day 10 after multiple administrations (1 mg/kg on days 0, 2, 4, 6 and 8). After single and multiple dosing, intralesional concentrations were respectively 5- and 20-fold higher compared to those in the healthy control skin of the same infected mice. We then evaluated how drug levels in the lesion after LAmB treatment relate to therapeutic outcomes. After five administrations of the drug at 0, 6.25 or 12.5 mg/kg (IV), there was a clear correlation between dose level, intralesional AmB concentration and relative reduction in parasite load and lesion size (R2 values > 0.9). This study confirms the improved efficacy of the liposomal over the deoxycholate AmB formulation in experimental CL, which is related to higher intralesional drug accumulation

A single dose of antibody-drug conjugate cures a stage 1 model of African trypanosomiasis.

Infections of humans and livestock with African trypanosomes are treated with drugs introduced decades ago that are not always fully effective and often have severe side effects. Here, the trypanosome haptoglobin-haemoglobin receptor (HpHbR) has been exploited as a route of uptake for an antibody-drug conjugate (ADC) that is completely effective against Trypanosoma brucei in the standard mouse model of infection. Recombinant human anti-HpHbR monoclonal antibodies were isolated and shown to be internalised in a receptor-dependent manner. Antibodies were conjugated to a pyrrolobenzodiazepine (PBD) toxin and killed T. brucei in vitro at picomolar concentrations. A single therapeutic dose (0.25 mg/kg) of a HpHbR antibody-PBD conjugate completely cured a T. brucei mouse infection within 2 days with no re-emergence of infection over a subsequent time course of 77 days. These experiments provide a demonstration of how ADCs can be exploited to treat protozoal diseases that desperately require new therapeutics

Hierarchy Theory of Evolution and the Extended Evolutionary Synthesis: Some Epistemic Bridges, Some Conceptual Rifts

Contemporary evolutionary biology comprises a plural landscape of multiple co-existent conceptual frameworks and strenuous voices that disagree on the nature and scope of evolutionary theory. Since the mid-eighties, some of these conceptual frameworks have denounced the ontologies of the Modern Synthesis and of the updated Standard Theory of Evolution as unfinished or even flawed. In this paper, we analyze and compare two of those conceptual frameworks, namely Niles Eldredge’s Hierarchy Theory of Evolution (with its extended ontology of evolutionary entities) and the Extended Evolutionary Synthesis (with its proposal of an extended ontology of evolutionary processes), in an attempt to map some epistemic bridges (e.g. compatible views of causation; niche construction) and some conceptual rifts (e.g. extra-genetic inheritance; different perspectives on macroevolution; contrasting standpoints held in the “externalism–internalism” debate) that exist between them. This paper seeks to encourage theoretical, philosophical and historiographical discussions about pluralism or the possible unification of contemporary evolutionary biology

Does size matter for horny beetles? A geometric morphometric analysis of interspecific and intersexual size and shape variation in Colophon haughtoni Barnard, 1929, and C. kawaii Mizukami, 1997 (Coleoptera: Lucanidae)

Colophon is an understudied, rare and endangered stag beetle genus with all species endemic to isolated mountain peaks in South Africa’s Western Cape. Geometric morphometrics was used to analyse intersexual and interspecific variation of size and shape in the mandibles, heads, pronota and elytra of two sympatric species: Colophon haughtoni and Colophon kawaii. All measured structures showed significant sexual dimorphism, which may result from male-male competition for females. Female mandibles were too small and featureless for analysis, but male Colophon beetles possess large, ornate mandibles for fighting. Males had significantly larger heads and pronota that demonstrated shape changes which may relate to resource diversion to the mandibles and their supporting structures. Females are indistinguishable across species, but males were accurately identified using mandibles, heads and pronota. Male C. kawaii were significantly larger than C. haughtoni for all structures. These results support the species status of C. kawaii, which is currently in doubt due to its hybridisation with C. haughtoni. We also demonstrate the value of geometric morphometrics as a tool which may aid Colophon conservation by providing biological and phylogenetic insights and enabling species identification

Speciation in little: the role of range and body size in the diversification of Malagasy mantellid frogs

<p>Abstract</p> <p>Background</p> <p>The rate and mode of lineage diversification might be shaped by clade-specific traits. In Madagascar, many groups of organisms are characterized by tiny distribution ranges and small body sizes, and this high degree of microendemism and miniaturization parallels a high species diversity in some of these groups. We here investigate the geographic patterns characterizing the radiation of the frog family Mantellidae that is virtually endemic to Madagascar. We integrate a newly reconstructed near-complete species-level timetree of the Mantellidae with georeferenced distribution records and maximum male body size data to infer the influence of these life-history traits on each other and on mantellid diversification.</p> <p>Results</p> <p>We reconstructed a molecular phylogeny based on nuclear and mitochondrial DNA for 257 species and candidate species of the mantellid frog radiation. Based on this phylogeny we identified 53 well-supported pairs of sister species that we used for phylogenetic comparative analyses, along with whole tree-based phylogenetic comparative methods. Sister species within the Mantellidae diverged at 0.2-14.4 million years ago and more recently diverged sister species had geographical range centroids more proximate to each other, independently of their current sympatric or allopatric occurrence. The largest number of sister species pairs had non-overlapping ranges, but several examples of young microendemic sister species occurring in full sympatry suggest the possibility of non-allopatric speciation. Range sizes of species included in the sister species comparisons increased with evolutionary age, as did range size differences between sister species, which rejects peripatric speciation. For the majority of mantellid sister species and the whole mantellid radiation, range and body sizes were associated with each other and small body sizes were linked to higher mitochondrial nucleotide substitution rates and higher clade diversity. In contrast, small range sizes were unexpectedly associated with a slow-down of mitochondrial substitution rates.</p> <p>Conclusions</p> <p>Based on these results we define a testable hypothesis under which small body sizes result in limited dispersal capabilities and low physiological tolerances, causing smaller and more strongly fragmented ranges. This can be thought to facilitate reproductive isolation and thus favor speciation. Contrary to the expectation of the faster speciation of such microendemic phenotype species, we only found small body sizes of mantellid frogs to be linked to higher diversification and substitution rates, but not small range sizes. A joint analysis of various species-rich regional anuran radiations might provide enough species with all combinations of range and body sizes for a more conclusive test of this hypothesis.</p

Increased Inter-Colony Fusion Rates Are Associated with Reduced COI Haplotype Diversity in an Invasive Colonial Ascidian Didemnum vexillum

Considerable progress in our understanding of the population genetic changes associated with biological invasions has been made over the past decade. Using selectively neutral loci, it has been established that reductions in genetic diversity, reflecting founder effects, have occurred during the establishment of some invasive populations. However, some colonial organisms may actually gain an ecological advantage from reduced genetic diversity because of the associated reduction in inter-colony conflict. Here we report population genetic analyses, along with colony fusion experiments, for a highly invasive colonial ascidian, Didemnum vexillum. Analyses based on mitochondrial cytochrome oxidase I (COI) partial coding sequences revealed two distinct D. vexillum clades. One COI clade appears to be restricted to the probable native region (i.e., north-west Pacific Ocean), while the other clade is present in widely dispersed temperate coastal waters around the world. This clade structure was supported by 18S ribosomal DNA (rDNA) sequence data, which revealed a one base-pair difference between the two clades. Recently established populations of D. vexillum in New Zealand displayed greatly reduced COI genetic diversity when compared with D. vexillum in Japan. In association with this reduction in genetic diversity was a significantly higher inter-colony fusion rate between randomly paired New Zealand D. vexillum colonies (80%, standard deviation ±18%) when compared with colonies found in Japan (27%, standard deviation ±15%). The results of this study add to growing evidence that for colonial organisms reductions in population level genetic diversity may alter colony interaction dynamics and enhance the invasive potential of newly colonizing species

Integrative phylogenetic, phylogeographic and morphological characterisation of the Unio crassus species complex reveals cryptic diversity with important conservation implications

The global decline of freshwater mussels and their crucial ecological services highlight the need to understand their phylogeny, phylogeography and patterns of genetic diversity to guide conservation efforts. Such knowledge is urgently needed for Unio crassus, a highly imperilled species originally widespread throughout Europe and southwest Asia. Recent studies have resurrected several species from synonymy based on mitochondrial data, revealing U. crassus to be a complex of cryptic species. To address long-standing taxonomic uncertainties hindering effective conservation, we integrate morphometric, phylogenetic, and phylogeographic analyses to examine species diversity within the U. crassus complex across its entire range. Phylogenetic analyses were performed using cytochrome c oxidase subunit I (815 specimens from 182 populations) and, for selected specimens, whole mitogenome sequences and Anchored Hybrid Enrichment (AHE) data on ∼600 nuclear loci. Mito-nuclear discordance was detected, consistent with mitochondrial DNA gene flow between some species during the Pliocene and Pleistocene. Fossil-calibrated phylogenies based on AHE data support a Mediterranean origin for the U. crassus complex in the Early Miocene. The results of our integrative approach support 12 species in the group: the previously recognised Unio bruguierianus, Unio carneus, Unio crassus, Unio damascensis, Unio ionicus, Unio sesirmensis, and Unio tumidiformis, and the reinstatement of five nominal taxa: Unio desectus stat. rev., Unio gontierii stat. rev., Unio mardinensis stat. rev., Unio nanus stat. rev., and Unio vicarius stat. rev. Morphometric analyses of shell contours reveal important morphospace overlaps among these species, highlighting cryptic, but geographically structured, diversity. The distribution, taxonomy, phylogeography, and conservation of each species are succinctly described