Chronic generalized fibrotic skin lesions from disseminated leishmaniasis caused by Leishmania martiniquensis in two patients from northern Thailand infected with HIV.

BACKGROUND: Leishmaniasis is a newly emerging infection in Thailand. Most of the previous human cases have presented with the clinical features of visceral leishmaniasis and were mainly found in southern Thailand. Here we report the first two patients from northern Thailand presenting with disseminated cutaneous leishmaniasis. OBJECTIVES: To determine the nature of the infection of leishmaniasis and to identify the species of parasite responsible. METHODS: Clinical investigations included the taking of biopsy samples and histology. Parasitological diagnosis was performed by establishment of Leishmania promastigote cultures, and identification was performed by DNA sequencing of four independent gene loci (ribosomal RNA internal transcribed spacer 1; large subunit of RNA polymerase II; heat shock protein 70; RPL23a intergenic sequence). RESULTS: Both patients were infected with HIV, and had multiple cutaneous lesions and accompanying visceral leishmaniasis. They had similar cutaneous manifestations characterized by chronic generalized fibrotic lesions, which were more prominent on traumatic areas. In both patients the parasite was identified as Leishmania martiniquensis. This is a recently described species that is distinct and only distantly related to the classical agents of cutaneous leishmaniasis in Asia (Leishmania major and Leishmania tropica) or of visceral leishmaniasis (Leishmania donovani and Leishmania infantum). Each patient responded well to therapy with intravenous amphotericin B followed by oral itraconazole. CONCLUSIONS: Leishmania martiniquensis is a cause of cutaneous leishmaniasis in Thailand.

Exsheathment and midgut invasion of nocturnally subperiodic Brugia malayi microfilariae in a refractory vector, Aedes aegypti (Thailand strain).

Exsheathment and midgut invasion of nocturnally subperiodic Brugia malayi microfilariae were analyzed using light and scanning electron microscopy in a refractory vector, Aedes aegypti (Thailand strain). Results showed that exsheathed microfilariae represented only approximately 1% of the total microfilaria midguts dissected at 5-min post-infected blood meal (PIBM). The percentage of exsheathed microfilariae found in midguts progressively increased to about 20, 60, 80, 90, and 100% at 1-, 2-5-, 6-12-, 18-36-, and 48-h PIBM, respectively. Importantly, all the microfilariae penetrating the mosquito midguts were exsheathed. Midgut invasion by the exsheathed microfilariae was observed between 2- and 48-h PIBM. SEM analysis revealed sheathed microfilariae surrounded by small particles and maceration of the microfilarial sheath in the midguts, suggesting that the midguts of the refractory mosquitoes might have protein(s) and/or enzyme(s) and/or factor(s) that induce and/or accelerate exsheathment. The microfilariae penetrated the internal face of the peritrophic matrix (PM) by their anterior part and then the midgut epithelium, before entering the hemocoel suggesting that PM was not a barrier against the microfilariae migrating towards the midgut. Melanized microfilariae were discovered in the hemocoel examined at 96-h PIBM suggesting that the refractory mosquitoes used melanization reactions against this parasite. This study provided evidence that A. aegypti (Thailand strain) has refractory mechanisms against B. malayi in both midgut and hemocoel.

The characterization of the Phlebotomus papatasi transcriptome.

As important vectors of human disease, phlebotomine sand flies are of global significance to human health, transmitting several emerging and re-emerging infectious diseases. The most devastating of the sand fly transmitted infections are the leishmaniases, causing significant mortality and morbidity in both the Old and New World. Here we present the first global transcriptome analysis of the Old World vector of cutaneous leishmaniasis, Phlebotomus papatasi (Scopoli) and compare this transcriptome to that of the New World vector of visceral leishmaniasis, Lutzomyia longipalpis. A normalized cDNA library was constructed using pooled mRNA from Phlebotomus papatasi larvae, pupae, adult males and females fed sugar, blood, or blood infected with Leishmania major. A total of 47 615 generated sequences was cleaned and assembled into 17 120 unique transcripts. Of the assembled sequences, 50% (8837 sequences) were classified using Gene Ontology (GO) terms. This collection of transcripts is comprehensive, as demonstrated by the high number of different GO categories. An in-depth analysis revealed 245 sequences with putative homology to proteins involved in blood and sugar digestion, immune response and peritrophic matrix formation. Twelve of the novel genes, including one trypsin, two peptidoglycan recognition proteins (PGRP) and nine chymotrypsins, have a higher expression level during larval stages. Two novel chymotrypsins and one novel PGRP are abundantly expressed upon blood feeding. This study will greatly improve the available genomic resources for P. papatasi and will provide essential information for annotation of the full genome.

The sticking point: How integrins bind to their ligands.

The integrin adhesion receptors are alpha beta heterodimers that exist in different ligand-binding states. Because of their large size and conformational lability, it has been difficult to determine how they interact with their ligands. Ligand-binding sites have been identified in the beta subunit, and now more recently in the 'I' domain and EF-hand-like domains V and VI of the alpha subunit. We speculate here about how these various sites might operate together to bind ligand in a stable manner.

Second Blood Meal by Female Lutzomyia longipalpis: Enhancement by Oviposition and Its Effects on Digestion, Longevity, and Leishmania Infection.

Lutzomyia longipalpis is the main vector of visceral leishmaniasis (VL) in America. Physiological and molecular mechanisms of Leishmania infection in sand flies have been studied during the first gonotrophic cycle. There are few studies about these interactions during the second gonotrophic cycle mainly because of the difficulties maintaining sand flies through sequential feeds. Here we standardized conditions to perform the second blood feed efficiently, and our results show that oviposition is an essential factor for the success of multiple feeds. We evaluated the impact of the second blood meal on longevity, protein digestion, trypsin activity, and Leishmania mexicana development within L. longipalpis gut. Mortality of blood-fed females increases after second blood meal as compared to sugar-fed females. Trypsin activity was lower during the second gonotrophic cycle. However, no difference in protein intake was observed between blood meals. There was no difference in the population size of Leishmania in the gut after both blood meals. In this work, we presented an optimized protocol for obtaining sufficient numbers of sand fly females fed on a second blood meal, and we described some physiological and parasitological aspects of the second gonotrophic cycle which might influence the vectorial competence of sand flies.

Progress in protein structure prediction: assessment of CASP3.

The third comparative assessment of techniques of protein structure prediction (CASP3) was held during 1998. This is a blind trial in which structures are predicted prior to having knowledge of the coordinates, which are then revealed to enable the assessment. Three sections at the meeting evaluated different methodologies - comparative modelling, fold recognition and ab initio methods. For some, but not all of the target coordinates, high quality models were submitted in each of these sections. There have been improvements in prediction techniques since CASP2 in 1996, most notably for ab initio methods.

Species composition and population dynamics of phlebotomine sand flies in a Leishmania infected area of Chiang Mai, Thailand.

Phlebotomine sand flies are established vectors of leishmaniasis in humans. In Thailand, Leishmania martiniquensis and "Leishmania siamensis" have been described as causative agents of leishmaniasis. In this study, a survey of sand flies in the Leishmania infected area of Hang Dong district, Chiang Mai, Thailand was performed using CDC light traps for eight consecutive months, from January to August 2016. A total of 661 sand flies were collected, and of 280 female sand flies, four species of the genus Sergentomyia including Sergentomyia gemmea, S. barraudi, S. indica, and S. hivernus and one species of the genus Phlebotomus, Phlebotomus stantoni, were identified. S. gemmea and S. hivernus were found in Chiang Mai for the first time. The density of captured female sand flies was high in warm and humid periods from June to August, with temperatures of around 26°C and relative humidity about 74%. In addition, S. gemmea was the most predominant species in the area. Further studies as to whether or not these sand fly species could be a vector of Leishmaniasis in Thailand are required.

Didilia sp. infecting Phlebotomus stantoni in Thailand.

Nematode infection in wild caught Phlebotomine sand flies was investigated in Thailand. Light microscopy (LM) and scanning electron microscopy (SEM) were used to detect and morphologically characterize entomopathogenic nematodes that presented in the sand flies. Didilia sp. nematodes were found for the first time in the body cavity of wild caught male Phlebotomus stantoni sand flies. The Didilia sp. was identified based on the morphology of the adult nematodes, from their stylet and teeth at the anterior tip, body length, and egg shell sculpture. It was noted that every infected male sand fly had unrotated genitalia, which would not allow them to mate, thus leading to the loss of their offspring. This finding provided information that might lead to study on whether or not the Didilia sp. has the potential to control sand fly population.

Conformational analysis of the first observed non-proline cis-peptide bond occurring within the complementarity determining region (CDR) of an antibody.

An analysis has been performed on the first example of a non-proline cis- peptide bond found within a complementarity determining region (CDR) of an antibody. The bond is located in CDR 3 of the heavy chain (H3) and makes substantial interactions to a peptide from a breast tumour-associated antigen. The antibody-peptide complex is compared, both in H3 length (six residues) and peptide conformation, to a number of other such complexes in the Brookhaven Data Bank (PDB). There is only one other H3 loop of the same length. Analysis of loop searches of the PDB, taken over the H3 framework of SM3, suggest that there is a limited repertoire of conformations for loops of length 6 compared to loops of length 5 and 7. It is argued that the cis-peptide bond is present because of the limited number of loop conformations of length 6, plus, the requirement of the H3 loop to contact the bound peptide. Modelling suggests that an all-trans-peptide loop conformation can replace the H3 loop and this raises the question of whether there is a trans- to cis-peptide bond isomerization upon peptide binding.

An automated classification of the structure of protein loops.

Conformational clusters and consensus sequences for protein loops have been derived by computational analysis of their structures in a non-redundant set of 233 proteins with less than 25% sequence homology (X-ray resolution better than 2.5 A). Loops have been classified into five types (alpha-alpha, beta-beta links, beta-beta hairpins, alpha-beta and beta-alpha) according to the secondary structures they embrace. Four variables have been used to describe the loop geometry, three angles and one distance between the secondary structure elements embracing the loop. Ramachandran angles (phi, psi) are used to define the loop conformations within each brace geometry. All loops from the non-redundant set have been clustered by means of these geometric features. A total of 56 classes (9 alpha-alpha, 11 beta-beta links, 14 beta-beta hairpins, 13 alpha-beta and 9 beta-alpha) were identified with consensus Ramachandran angles in the loops. These classes were divided into subclasses based on the brace geometry. This clustering procedure captures most of the clusters analysed by predominantly visual inspection methods and finds other clusters that have hitherto not been described. Consensus sequence patterns were identified for the subclasses. An extensive characterisation of loop conformations has therefore been achieved and the computational approach is readily open to the incorporation of information from newly determined structures. These clusters should also enhance model building by comparison studies.

Recognition of analogous and homologous protein folds: analysis of sequence and structure conservation.

An analysis was performed on 335 pairs of structurally aligned proteins derived from the structural classification of proteins (SCOP http://scop.mrc-lmb.cam.ac.uk/scop/) database. These similarities were divided into analogues, defined as proteins with similar three-dimensional structures (same SCOP fold classification) but generally with different functions and little evidence of a common ancestor (different SCOP superfamily classification). Homologues were defined as pairs of similar structures likely to be the result of evolutionary divergence (same superfamily) and were divided into remote, medium and close sub-divisions based on the percentage sequence identity. Particular attention was paid to the differences between analogues and remote homologues, since both types of similarities are generally undetectable by sequence comparison and their detection is the aim of fold recognition methods. Distributions of sequence identities and substitution matrices suggest a higher degree of sequence similarity in remote homologues than in analogues. Matrices for remote homologues show similarity to existing mutation matrices, providing some validity for their use in previously described fold recognition methods. In contrast, matrices derived from analogous proteins show little conservation of amino acid properties beyond broad conservation of hydrophobic or polar character. Secondary structure and accessibility were more conserved on average in remote homologues than in analogues, though there was no apparent difference in the root-mean-square deviation between these two types of similarities. Alignments of remote homologues and analogues show a similar number of gaps, openings (one or more sequential gaps) and inserted/deleted secondary structure elements, and both generally contain more gaps/openings/deleted secondary structure elements than medium and close homologues. These results suggest that gap parameters for fold recognition should be more lenient than those used in sequence comparison. Parameters were derived from the analogue and remote homologue datasets for potential used in fold recognition methods. Implications for protein fold recognition and evolution are discussed.

Automated classification of antibody complementarity determining region 3 of the heavy chain (H3) loops into canonical forms and its application to protein structure prediction.

A computer-based algorithm was used to cluster the loops forming the complementarity determining region (CDR) 3 of the heavy chain (H3) into canonical classes. Previous analyses of the three-dimensional structures of CDR loops (also known as the hypervariable regions) within antibody immunoglobulin variable domains have shown that for five of the six CDRs there are only a few main-chain conformations (known as canonical forms) that show clear relationships between sequence and structure. However, the larger variation in length and conformation of loops within H3 has limited the classification of these loops into canonical forms. The clustering procedure presented here is based on aligning the Ramachandran-coded main-chain conformation of the residues using a dynamic algorithm that allows the insertion of gaps to obtain an optimum alignment. A total of 41 H3 loops out of 62 non-identical loops, extracted from the Brookhaven Protein Data Bank, have been automatically grouped into 22 clusters. Inspection of the clusters for consensus sequences or intra-loop interactions or invariant conformation led to the proposal of 13 canonical forms representing 31 loops. These canonical forms include a consideration of the geometry of both the take-off region adjacent to the bracing beta-strands and the remaining loop apex. Subsequently a new set of 15 H3 loops not included in the initial analysis was considered. The clustering procedure was repeated and nine of these 15 loops could be assigned to original clusters, including seven to canonical forms. A sequence profile was generated for each canonical form from the original set of loops and matched against the sequences of the new H3 loops. For five out of the seven new H3 loops that were in a canonical form, the correct form was identified at first rank by this predictive scheme.

Crystal structure at 1.95 A resolution of the breast tumour-specific antibody SM3 complexed with its peptide epitope reveals novel hypervariable loop recognition.

The anti-breast tumour antibody SM3 has a high selectivity in reacting specifically with carcinoma-associated mucin. SM3 recognises the core repeating motif (Pro-Asp-Thr-Arg-Pro) of aberrantly glycosylated epithelial mucin MUC1, and has potential as a therapeutic and diagnostic tool. Here we report the crystal structure of the Fab fragment of SM3 in complex with a 13-residue MUC1 peptide antigen (Thr1P-Ser2P-Ala3P-Pro4P-Asp5P-Thr6P -Arg7P-Pro8P-Ala9P-Pro10P-Gly11P- Ser12P-Thr13P). The SM3-MUC1 peptide structure was solved by molecular replacement, and the current model is refined at 1.95 A resolution with an R-factor of 21.3% and R-free 28.3%. The MUC1 peptide is bound both by non-polar interactions and hydrogen bonds in an elongated groove in the antibody-combining site through interactions with Complimentarity Determining Regions (CDRs), three of the light chain (L1, L2, L3) and two of the heavy chain (H1 and H3). The conformation of the peptide is mainly extended with no discernable standard secondary structure. There is a single non-proline cis-peptide bond in H3 (Val95H-Gly96H-Gln97H-Phe98H-Ala101H-Ty r102H) between Gly96H and Gln97H, which appears to play a role in SM3-peptide antigen interactions, and represents the first such example within an antibody hypervariable loop. The SM3-MUC1 peptide structure has implications for rational therapeutic and diagnostic antibody engineering.

New insights into the developmental biology and transmission mechanisms of Leishmania.

Leishmania alternates between two main morphological forms in its life cycle: intracellular amastigotes in the mammalian host and motile promastigotes in the sandfly vector. Several different forms of promastigote can be recognised in sandfly infections. The first promastigote forms, which are found in the sandfly in the bloodmeal phase, are multiplicative procyclic promastigotes. These differentiate into nectomonad promastigotes, which are a non-dividing migratory stage moving from the posterior to the anterior midgut. When nectomonad promastigotes arrive at the anterior midgut they differentiate into leptomonad forms, a newly named life cycle stage, which resume replication. Leptomonad promastigotes, which are found in the anterior midgut, are the developmental precursors of the metacyclic promastigotes, the mammal-infective stages. Leptomonad forms also produce promastigote secretory gel, a substance that plays a key role in transmission by forming a physical obstruction in the gut, forcing the sandfly to regurgitate metacyclic promastigotes during bloodfeeding.

SETD2 loss-of-function promotes renal cancer branched evolution through replication stress and impaired DNA repair.

Defining mechanisms that generate intratumour heterogeneity and branched evolution may inspire novel therapeutic approaches to limit tumour diversity and adaptation. SETD2 (Su(var), Enhancer of zeste, Trithorax-domain containing 2) trimethylates histone-3 lysine-36 (H3K36me3) at sites of active transcription and is mutated in diverse tumour types, including clear cell renal carcinomas (ccRCCs). Distinct SETD2 mutations have been identified in spatially separated regions in ccRCC, indicative of intratumour heterogeneity. In this study, we have addressed the consequences of SETD2 loss-of-function through an integrated bioinformatics and functional genomics approach. We find that bi-allelic SETD2 aberrations are not associated with microsatellite instability in ccRCC. SETD2 depletion in ccRCC cells revealed aberrant and reduced nucleosome compaction and chromatin association of the key replication proteins minichromosome maintenance complex component (MCM7) and DNA polymerase δ hindering replication fork progression, and failure to load lens epithelium-derived growth factor and the Rad51 homologous recombination repair factor at DNA breaks. Consistent with these data, we observe chromosomal breakpoint locations are biased away from H3K36me3 sites in SETD2 wild-type ccRCCs relative to tumours with bi-allelic SETD2 aberrations and that H3K36me3-negative ccRCCs display elevated DNA damage in vivo. These data suggest a role for SETD2 in maintaining genome integrity through nucleosome stabilization, suppression of replication stress and the coordination of DNA repair.

Filamentous proteophosphoglycan secreted by Leishmania promastigotes forms gel-like three-dimensional networks that obstruct the digestive tract of infected sandfly vectors.

Development of Leishmania parasites in the digestive tract of their sandfly vectors involves several morphological transformations from the intracellular mammalian amastigote via a succession of free and gut wall-attached promastigote stages to the infective metacyclic promastigotes. At the foregut midgut transition of Leishmania-infected sandflies a gel-like plug of unknown origin and composition is formed, which contains high numbers of parasites, that occludes the gut lumen and which may be responsible for the often observed inability of infected sandflies to draw blood. This "blocked fly" phenotype has been linked to efficient transmission of infectious metacyclic promastigotes from the vector to the mammalian host. We show by immunofluorescence and immunoelectron microscopy on two Leishmania/sandfly vector combinations (Leishmania mexicana/Lutzomyia longipalpis and L. major/Phlebotomus papatasi) that the gel-like mass is formed mainly by a parasite-derived mucin-like filamentous proteophosphoglycan (fPPG) whereas the Leishmania polymeric secreted acid phosphatase (SAP) is not a major component of this plug. fPPG forms a dense three-dimensional network of filaments which engulf the promastigote cell bodies in a gel-like mass. We propose that the continuous secretion of fPPG by promastigotes in the sandfly gut, that causes plug formation, is an important factor for the efficient transmission to the mammalian host.

Genetic analysis of integrin function in man: LAD-1 and other syndromes.

The integrins are cell membrane receptors composed of alpha and beta subunits which orchestrate adhesive events in all tissues of the body (Hynes, R.O., 1992. Integrins: versatility, modulation, and signalling in cell adhesion. Cell 69, 11-25; and Hynes, R.O., 1999. Cell adhesion: old and new questions. Trends Cell Biol. 9, M33-37). At present 18 alpha subunits and 8 beta subunits have been identified which are loosely organised into families. There are three inherited autosomal recessive diseases in man which involve germline mutations in genes coding for integrins. Leukocyte adhesion deficiency-1 (LAD-1) is the result of mutations in the beta2 subunit of the CD11/CD18 integrins, LFA-1, Mac-1, p150,95 and alphadbeta2. The bleeding disorder Glanzmann thrombasthenia is caused by mutations in either the alpha or beta subunit of the platelet integrin, alphaIIbbeta3. Thirdly, it is now recognised than one of the variants of the usually lethal skin blistering disorder, epidermolysis bullosa (JEB-PA), is caused by mutation in either the alpha or beta subunit of the epithelial hemidesmosome integrin, alpha6beta4. Many of the mutations cause defective alphabeta heterodimer formation. The majority of the beta subunit mutations are in the conserved N-terminal region known as the betaI domain. It is suggested that this region participates in alphabeta heterodimer formation.

A predicted three-dimensional structure for the carcinoembryonic antigen (CEA).

A three-dimensional model for the carcinoembryonic antigen (CEA) has been constructed by knowledge-based computer modelling. Each of the seven extracellular domains of CEA are expected to have immunoglobulin folds. The N-terminal domain of CEA was modelled using the first domain of the recently solved NMR structure of rat CD2, as well as the first domain of the X-ray crystal structure of human CD4 and an immunoglobulin variable domain REI as templates. The remaining domains were modelled from the first and second domains of CD4 and REI. Link conformations between the domains were taken from the elbow region of antibodies. A possible packing model between each of the seven domains is proposed. Each residue of the model is labelled as to its suitability for site-directed mutagenesis.

Characterisation of the single copy trefoil peptides intestinal trefoil factor and pS2 and their ability to form covalent dimers.

A bacterial recombinant expression system was established to produce biologically active rat Intestinal Trefoil Factor (rITF). Characterisation of purified rITF shows that both monomers and dimers can be observed under reducing and non-reducing conditions, respectively. Site-directed mutagenesis studies show that Cys57 is necessary for rITF dimer formation. Samples of human gastrointestinal tissue following biopsy also demonstrated the presence of reducible human pS2 and ITF covalent dimers. Three-dimensional models for pS2 and ITF support the hypothesis that both pS2 and ITF can exist as disulphide-linked dimers in vivo and that any proposed function for these peptides must take dimer formation into account.

A region encompassing the FERM domain of Jak1 is necessary for binding to the cytokine receptor gp130.

The terminal portion of the Janus kinases (Jaks) contains a divergent FERM (Four-point-one, Ezrin, Radixin, Moesin) homology domain comprising 19 conserved hydrophobic regions. To determine the role of this domain in governing recruitment of Jak1, but not Jak3, to the gp130 subunit of the interleukin-6 family of cytokine receptors, the interaction of three Jak1/Jak3 chimeras with gp130 was investigated. Chimeras 1, 2 and 3 (Jak1 FERM regions 1-19, 1-18 and 1-8/Jak3, respectively) were all enzymically active. Chimeras 1 and 2 interacted with the cytoplasmic domain of gp130, although less efficiently than Jak1. Only chimera 2, however, restored gp130 signalling in Jak1-negative cells. The data are consistent with recruitment of Jak1 to gp130 through the Jak1 FERM domain, but also emphasise the likely requirement for precise Jak/receptor orientation to sustain function.