The γδTCR combines innate immunity with adaptive immunity by utilizing spatially distinct regions for agonist selection and antigen responsiveness.

T lymphocytes expressing γδ T cell antigen receptors (TCRs) comprise evolutionarily conserved cells with paradoxical features. On the one hand, clonally expanded γδ T cells with unique specificities typify adaptive immunity. Conversely, large compartments of γδTCR+ intraepithelial lymphocytes (γδ IELs) exhibit limited TCR diversity and effect rapid, innate-like tissue surveillance. The development of several γδ IEL compartments depends on epithelial expression of genes encoding butyrophilin-like (Btnl (mouse) or BTNL (human)) members of the B7 superfamily of T cell co-stimulators. Here we found that responsiveness to Btnl or BTNL proteins was mediated by germline-encoded motifs within the cognate TCR variable γ-chains (Vγ chains) of mouse and human γδ IELs. This was in contrast to diverse antigen recognition by clonally restricted complementarity-determining regions CDR1-CDR3 of the same γδTCRs. Hence, the γδTCR intrinsically combines innate immunity and adaptive immunity by using spatially distinct regions to discriminate non-clonal agonist-selecting elements from clone-specific ligands. The broader implications for antigen-receptor biology are considered.

Butyrophilin-2A1 Directly Binds Germline-Encoded Regions of the Vγ9Vδ2 TCR and Is Essential for Phosphoantigen Sensing.

Vγ9Vδ2 T cells respond in a TCR-dependent fashion to both microbial and host-derived pyrophosphate compounds (phosphoantigens, or P-Ag). Butyrophilin-3A1 (BTN3A1), a protein structurally related to the B7 family of costimulatory molecules, is necessary but insufficient for this process. We performed radiation hybrid screens to uncover direct TCR ligands and cofactors that potentiate BTN3A1's P-Ag sensing function. These experiments identified butyrophilin-2A1 (BTN2A1) as essential to Vγ9Vδ2 T cell recognition. BTN2A1 synergised with BTN3A1 in sensitizing P-Ag-exposed cells for Vγ9Vδ2 TCR-mediated responses. Surface plasmon resonance experiments established Vγ9Vδ2 TCRs used germline-encoded Vγ9 regions to directly bind the BTN2A1 CFG-IgV domain surface. Notably, somatically recombined CDR3 loops implicated in P-Ag recognition were uninvolved. Immunoprecipitations demonstrated close cell-surface BTN2A1-BTN3A1 association independent of P-Ag stimulation. Thus, BTN2A1 is a BTN3A1-linked co-factor critical to Vγ9Vδ2 TCR recognition. Furthermore, these results suggest a composite-ligand model of P-Ag sensing wherein the Vγ9Vδ2 TCR directly interacts with both BTN2A1 and an additional ligand recognized in a CDR3-dependent manner.

Ctf4 Links DNA Replication with Sister Chromatid Cohesion Establishment by Recruiting the Chl1 Helicase to the Replisome.

DNA replication during S phase is accompanied by establishment of sister chromatid cohesion to ensure faithful chromosome segregation. The Eco1 acetyltransferase, helped by factors including Ctf4 and Chl1, concomitantly acetylates the chromosomal cohesin complex to stabilize its cohesive links. Here we show that Ctf4 recruits the Chl1 helicase to the replisome via a conserved interaction motif that Chl1 shares with GINS and polymerase α. We visualize recruitment by EM analysis of a reconstituted Chl1-Ctf4-GINS assembly. The Chl1 helicase facilitates replication fork progression under conditions of nucleotide depletion, partly independently of Ctf4 interaction. Conversely, Ctf4 interaction, but not helicase activity, is required for Chl1's role in sister chromatid cohesion. A physical interaction between Chl1 and the cohesin complex during S phase suggests that Chl1 contacts cohesin to facilitate its acetylation. Our results reveal how Ctf4 forms a replisomal interaction hub that coordinates replication fork progression and sister chromatid cohesion establishment.

Impact of AlphaFold on structure prediction of protein complexes: The CASP15-CAPRI experiment.

We present the results for CAPRI Round 54, the 5th joint CASP-CAPRI protein assembly prediction challenge. The Round offered 37 targets, including 14 homodimers, 3 homo-trimers, 13 heterodimers including 3 antibody-antigen complexes, and 7 large assemblies. On average ~70 CASP and CAPRI predictor groups, including more than 20 automatics servers, submitted models for each target. A total of 21 941 models submitted by these groups and by 15 CAPRI scorer groups were evaluated using the CAPRI model quality measures and the DockQ score consolidating these measures. The prediction performance was quantified by a weighted score based on the number of models of acceptable quality or higher submitted by each group among their five best models. Results show substantial progress achieved across a significant fraction of the 60+ participating groups. High-quality models were produced for about 40% of the targets compared to 8% two years earlier. This remarkable improvement is due to the wide use of the AlphaFold2 and AlphaFold2-Multimer software and the confidence metrics they provide. Notably, expanded sampling of candidate solutions by manipulating these deep learning inference engines, enriching multiple sequence alignments, or integration of advanced modeling tools, enabled top performing groups to exceed the performance of a standard AlphaFold2-Multimer version used as a yard stick. This notwithstanding, performance remained poor for complexes with antibodies and nanobodies, where evolutionary relationships between the binding partners are lacking, and for complexes featuring conformational flexibility, clearly indicating that the prediction of protein complexes remains a challenging problem.

Application of deep learning methods: From molecular modelling to patient classification.

We are now well into the information driven age with complex, heterogeneous, datasets in the biological sciences continuing to grow at a rapid pace. Moreover, distilling of such datasets, to find new governing principles, are underway. Leading the surge are new and exciting algorithmic developments in computer simulation and machine learning, most notably for the latter, those centred on deep learning. However, practical applications of cell centric computations within the biological sciences, even when carefully benchmarked against existing experimental datasets, remain challenging. Here we discuss the application of deep learning methodologies to support our understanding of cell functionality and as an aid to patient classification. Whilst comprehensive end-to-end deep learning approaches that utilise knowledge of the cell and its molecular components to aid human disease classification are yet to be implemented, important for opening the door to more effective molecular and cell-based therapies, we illustrate that many deep learning applications have been developed to tackle components of such an ambitious pipeline. We end our discussion on what the future may hold, especially how an integrated framework of computer simulations and deep learning, in conjunction with wet-bench experimentation, could enable to reveal the governing principles underlying cell functionalities within the tissue environments cells operate.

Response to thermal and infection stresses in an American vector of visceral leishmaniasis.

Lutzomyia longipalpis is known as one of the primary insect vectors of visceral leishmaniasis. For such ectothermic organisms, the ambient temperature is a critical life factor. However, the impact of temperature has been ignored in many induced-stress situations of the vector life. Therefore, this study explored the interaction of Lu. longipalpis with temperature by evaluating its behaviour across a thermal gradient, thermographic recordings during blood-feeding on mice, and the gene expression of heat shock proteins (HSP) when insects were exposed to extreme temperature or infected. The results showed that 72 h after blood ingestion, Lu. longipalpis became less active and preferred relatively low temperatures. However, at later stages of blood digestion, females increased their activity and remained at higher temperatures. Real-time imaging showed that the body temperature of females can adjust rapidly to the host and remain constant until the end of blood-feeding. Insects also increased the expression of HSP90(83) during blood-feeding. Our findings suggest that Lu. longipalpis interacts with temperature by using its behaviour to avoid temperature-induced physiological damage during the gonotrophic cycle. However, the expression of certain HSP might be triggered to mitigate thermal stress in situations where a behavioural response is not the best option.

Comparison of loop extrusion and diffusion capture as mitotic chromosome formation pathways in fission yeast.

Underlying higher order chromatin organization are Structural Maintenance of Chromosomes (SMC) complexes, large protein rings that entrap DNA. The molecular mechanism by which SMC complexes organize chromatin is as yet incompletely understood. Two prominent models posit that SMC complexes actively extrude DNA loops (loop extrusion), or that they sequentially entrap two DNAs that come into proximity by Brownian motion (diffusion capture). To explore the implications of these two mechanisms, we perform biophysical simulations of a 3.76 Mb-long chromatin chain, the size of the long Schizosaccharomyces pombe chromosome I left arm. On it, the SMC complex condensin is modeled to perform loop extrusion or diffusion capture. We then compare computational to experimental observations of mitotic chromosome formation. Both loop extrusion and diffusion capture can result in native-like contact probability distributions. In addition, the diffusion capture model more readily recapitulates mitotic chromosome axis shortening and chromatin compaction. Diffusion capture can also explain why mitotic chromatin shows reduced, as well as more anisotropic, movements, features that lack support from loop extrusion. The condensin distribution within mitotic chromosomes, visualized by stochastic optical reconstruction microscopy (STORM), shows clustering predicted from diffusion capture. Our results inform the evaluation of current models of mitotic chromosome formation.

Prediction of protein assemblies, the next frontier: The CASP14-CAPRI experiment.

We present the results for CAPRI Round 50, the fourth joint CASP-CAPRI protein assembly prediction challenge. The Round comprised a total of twelve targets, including six dimers, three trimers, and three higher-order oligomers. Four of these were easy targets, for which good structural templates were available either for the full assembly, or for the main interfaces (of the higher-order oligomers). Eight were difficult targets for which only distantly related templates were found for the individual subunits. Twenty-five CAPRI groups including eight automatic servers submitted ~1250 models per target. Twenty groups including six servers participated in the CAPRI scoring challenge submitted ~190 models per target. The accuracy of the predicted models was evaluated using the classical CAPRI criteria. The prediction performance was measured by a weighted scoring scheme that takes into account the number of models of acceptable quality or higher submitted by each group as part of their five top-ranking models. Compared to the previous CASP-CAPRI challenge, top performing groups submitted such models for a larger fraction (70-75%) of the targets in this Round, but fewer of these models were of high accuracy. Scorer groups achieved stronger performance with more groups submitting correct models for 70-80% of the targets or achieving high accuracy predictions. Servers performed less well in general, except for the MDOCKPP and LZERD servers, who performed on par with human groups. In addition to these results, major advances in methodology are discussed, providing an informative overview of where the prediction of protein assemblies currently stands.

Extracellular matrix anisotropy is determined by TFAP2C-dependent regulation of cell collisions.

The isotropic or anisotropic organization of biological extracellular matrices has important consequences for tissue function. We study emergent anisotropy using fibroblasts that generate varying degrees of matrix alignment from uniform starting conditions. This reveals that the early migratory paths of fibroblasts are correlated with subsequent matrix organization. Combined experimentation and adaptation of Vicsek modelling demonstrates that the reorientation of cells relative to each other following collision plays a role in generating matrix anisotropy. We term this behaviour 'cell collision guidance'. The transcription factor TFAP2C regulates cell collision guidance in part by controlling the expression of RND3. RND3 localizes to cell-cell collision zones where it downregulates actomyosin activity. Cell collision guidance fails without this mechanism in place, leading to isotropic matrix generation. The cross-referencing of alignment and TFAP2C gene expression signatures against existing datasets enables the identification and validation of several classes of pharmacological agents that disrupt matrix anisotropy.

Enhanced sampling of protein conformational states for dynamic cross-docking within the protein-protein docking server SwarmDock.

The formation of specific protein-protein interactions is often a key to a protein's function. During complex formation, each protein component will undergo a change in the conformational state, for some these changes are relatively small and reside primarily at the sidechain level; however, others may display notable backbone adjustments. One of the classic problems in the protein-docking field is to be able to a priori predict the extent of such conformational changes. In this work, we investigated three protocols to find the most suitable input structure conformations for cross-docking, including a robust sampling approach in normal mode space. Counterintuitively, knowledge of the theoretically best combination of normal modes for unbound-bound transitions does not always lead to the best results. We used a novel spatial partitioning library, Aether Engine (see Supplementary Materials), to efficiently search the conformational states of 56 receptor/ligand pairs, including a recent CAPRI target, in a systematic manner and selected diverse conformations as input to our automated docking server, SwarmDock, a server that allows moderate conformational adjustments during the docking process. In essence, here we present a dynamic cross-docking protocol, which when benchmarked against the simpler approach of just docking the unbound components shows a 10% uplift in the quality of the top docking pose.

Leishmania proteophosphoglycans regurgitated from infected sand flies accelerate dermal wound repair and exacerbate leishmaniasis via insulin-like growth factor 1-dependent signalling.

Leishmania parasites are transmitted to vertebrate hosts by female phlebotomine sand flies as they bloodfeed by lacerating the upper capillaries of the dermis with their barbed mouthparts. In the sand fly midgut secreted proteophosphoglycans from Leishmania form a biological plug known as the promastigote secretory gel (PSG), which blocks the gut and facilitates the regurgitation of infective parasites. The interaction between the wound created by the sand fly bite and PSG is not known. Here we nanoinjected a sand fly egested dose of PSG into BALB/c mouse skin that lead to the differential expression of 7,907 transcripts. These transcripts were transiently up-regulated during the first 6 hours post-wound and enriched for pathways involved in inflammation, cell proliferation, fibrosis, epithelial cell differentiation and wound remodelling. We found that PSG significantly accelerated wound healing in vitro and in mice; which was associated with an early up-regulation of transcripts involved in inflammation (IL-1ß, IL-6, IL-10, TNFα) and inflammatory cell recruitment (CCL2, CCL3, CCL4, CXCL2), followed 6 days later by enhanced expression of transcripts associated with epithelial cell proliferation, fibroplasia and fibrosis (FGFR2, EGF, EGFR, IGF1). Dermal expression of IGF1 was enhanced following an infected sand fly bite and was acutely responsive to the deposition of PSG but not the inoculation of parasites or sand fly saliva. Antibody blockade of IGF1 ablated the gel's ability to promote wound closure in mouse ears and significantly reduced the virulence of Leishmania mexicana infection delivered by an individual sand fly bite. Dermal macrophages recruited to air-pouches on the backs of mice revealed that IGF1 was pivotal to the PSG's ability to promote macrophage alternative activation and Leishmania infection. Our data demonstrate that through the regurgitation of PSG Leishmania exploit the wound healing response of the host to the vector bite by promoting the action of IGF1 to drive the alternative activation of macrophages.

Matrix feedback enables diverse higher-order patterning of the extracellular matrix.

The higher-order patterning of extra-cellular matrix in normal and pathological tissues has profound consequences on tissue function. Whilst studies have documented both how fibroblasts create and maintain individual matrix fibers and how cell migration is altered by the fibers they interact with, a model unifying these two aspects of tissue organization is lacking. Here we use computational modelling to understand the effect of this interconnectivity between fibroblasts and matrix at the mesoscale level. We created a unique adaptation to the Vicsek flocking model to include feedback from a second layer representing the matrix, and use experimentation to parameterize our model and validate model-driven hypotheses. Our two-layer model demonstrates that feedback between fibroblasts and matrix increases matrix diversity creating higher-order patterns. The model can quantitatively recapitulate matrix patterns of tissues in vivo. Cells follow matrix fibers irrespective of when the matrix fibers were deposited, resulting in feedback with the matrix acting as temporal 'memory' to collective behaviour, which creates diversity in topology. We also establish conditions under which matrix can be remodelled from one pattern to another. Our model elucidates how simple rules defining fibroblast-matrix interactions are sufficient to generate complex tissue patterns.

Experimental infection of Leishmania (Mundinia) martiniquensis in BALB/c mice and Syrian golden hamsters.

Our objective was to investigate clinical progression, presence of parasites and DNAs, parasite loads, and histological alterations in BALB/c mice and Syrian golden hamsters after intraperitoneal inoculation with Leishmania (Mundinia) martiniquensis promastigotes with a goal to choosing an appropriate animal model for visceral leishmaniasis. Infections were monitored for 16 weeks. Infected BALB/c mice were asymptomatic during the infection course. Parasite DNAs were detected in the liver at week 8 of infection, followed by clearance in most animals at week 16; whereas in the spleen, parasite DNAs were detected until week 16. These results are correlated to those obtained measuring parasite loads in both organs. No parasite DNA and no alteration in the bone marrow were observed indicating that no dissemination occurred. These results suggest the control of visceralization of L. martiniquensis by BALB/c mice. In hamsters, weight loss, cachexia, and fatigue were observed after week 11. Leishmania martiniquensis parasites were observed in tissue smears of the liver, spleen, and bone marrow by week 16. Parasite loads correlated with those from the presence of parasites and DNAs in the examined tissues. Alterations in the liver with nuclear destruction and cytoplasmic degeneration of infected hepatocytes, presence of inflammatory infiltrates, necrosis of hepatocytes, and changes in splenic architecture and reduction and deformation of white pulp in the spleen were noted. These results indicate a chronic form of visceral leishmaniasis indicating that the hamster is a suitable animal model for the study of pathological features of chronic visceral leishmaniasis caused by L. martiniquensis.

Refinement of protein-protein complexes in contact map space with metadynamics simulations.

Accurate protein-protein complex prediction, to atomic detail, is a challenging problem. For flexible docking cases, current state-of-the-art docking methods are limited in their ability to exhaustively search the high dimensionality of the problem space. In this study, to obtain more accurate models, an investigation into the local optimization of initial docked solutions is presented with respect to a reference crystal structure. We show how physics-based refinement of protein-protein complexes in contact map space (CMS), within a metadynamics protocol, can be performed. The method uses 5 times replicated 10 ns simulations for sampling and ranks the generated conformational snapshots with ZRANK to identify an ensemble of n snapshots for final model building. Furthermore, we investigated whether the reconstructed free energy surface (FES), or a combination of both FES and ZRANK, referred to as CSα , can help to reduce snapshot ranking error.

Blind prediction of homo- and hetero-protein complexes: The CASP13-CAPRI experiment.

We present the results for CAPRI Round 46, the third joint CASP-CAPRI protein assembly prediction challenge. The Round comprised a total of 20 targets including 14 homo-oligomers and 6 heterocomplexes. Eight of the homo-oligomer targets and one heterodimer comprised proteins that could be readily modeled using templates from the Protein Data Bank, often available for the full assembly. The remaining 11 targets comprised 5 homodimers, 3 heterodimers, and two higher-order assemblies. These were more difficult to model, as their prediction mainly involved "ab-initio" docking of subunit models derived from distantly related templates. A total of ~30 CAPRI groups, including 9 automatic servers, submitted on average ~2000 models per target. About 17 groups participated in the CAPRI scoring rounds, offered for most targets, submitting ~170 models per target. The prediction performance, measured by the fraction of models of acceptable quality or higher submitted across all predictors groups, was very good to excellent for the nine easy targets. Poorer performance was achieved by predictors for the 11 difficult targets, with medium and high quality models submitted for only 3 of these targets. A similar performance "gap" was displayed by scorer groups, highlighting yet again the unmet challenge of modeling the conformational changes of the protein components that occur upon binding or that must be accounted for in template-based modeling. Our analysis also indicates that residues in binding interfaces were less well predicted in this set of targets than in previous Rounds, providing useful insights for directions of future improvements.

Comparative genomics of Leishmania (Mundinia).

BACKGROUND: Trypanosomatids of the genus Leishmania are parasites of mammals or reptiles transmitted by bloodsucking dipterans. Many species of these flagellates cause important human diseases with clinical symptoms ranging from skin sores to life-threatening damage of visceral organs. The genus Leishmania contains four subgenera: Leishmania, Sauroleishmania, Viannia, and Mundinia. The last subgenus has been established recently and remains understudied, although Mundinia contains human-infecting species. In addition, it is interesting from the evolutionary viewpoint, representing the earliest branch within the genus and possibly with a different type of vector. Here we analyzed the genomes of L. (M.) martiniquensis, L. (M.) enriettii and L. (M.) macropodum to better understand the biology and evolution of these parasites. RESULTS: All three genomes analyzed were approximately of the same size (~ 30 Mb) and similar to that of L. (Sauroleishmania) tarentolae, but smaller than those of the members of subgenera Leishmania and Viannia, or the genus Endotrypanum (~ 32 Mb). This difference was explained by domination of gene losses over gains and contractions over expansions at the Mundinia node, although only a few of these genes could be identified. The analysis predicts significant changes in the Mundinia cell surface architecture, with the most important ones relating to losses of LPG-modifying side chain galactosyltransferases and arabinosyltransferases, as well as ß-amastins. Among other important changes were gene family contractions for the oxygen-sensing adenylate cyclases and FYVE zinc finger-containing proteins. CONCLUSIONS: We suggest that adaptation of Mundinia to different vectors and hosts has led to alternative host-parasite relationships and, thereby, made some proteins redundant. Thus, the evolution of genomes in the genus Leishmania and, in particular, in the subgenus Mundinia was mainly shaped by host (or vector) switches.

Axenic amastigote cultivation and in vitro development of Leishmania orientalis.

Leishmania (Mundinia) orientalis is a recently described new species that causes leishmaniasis in Thailand. To facilitate characterization of this new species, an in vitro culture system to generate L. orientalis axenic amastigotes was developed. In vitro culture conditions of the axenic culture-derived amastigotes were optimized by manipulation of temperature and pH. Four criteria were used to evaluate the resulting L. orientalis axenic amastigotes, i.e., morphology, zymographic analysis of nucleases, cyclic transformation, and infectivity to the human monocytic cell line (THP-1) cells. Results revealed that the best culture condition for L. orientalis axenic amastigotes was Grace's insect medium supplemented with FCS 20%, 2% human urine, 1% BME vitamins, and 25 µg/ml gentamicin sulfate, pH 5.5 at 35 °C. For promastigotes, the condition was M199 medium, 10% FCS supplemented with 2% human urine, 1% BME vitamins, and 25 µg/ml gentamicin sulfate, pH 6.8 at 26 °C. Morphological characterization revealed six main stages of the parasites including amastigotes, procyclic promastigotes, nectomonad promastigotes, leptomonad promastigotes, metacyclic promastigotes, and paramastigotes. Also, changes in morphology during the cycle were accompanied by changes in zymographic profiles of nucleases. The developmental cycle of L. orientalis in vitro was complete in 12 days using both culture systems. The infectivity to THP-1 macrophages and intracellular growth of the axenic amastigotes was similar to that of THP-1 derived intracellular amastigotes. These results confirmed the successful axenic cultivation of L. orientalis amastigotes. The axenic amastigotes and promastigotes can be used for further study on infection in permissive vectors and animals.

Planar polarization of the atypical myosin Dachs orients cell divisions in Drosophila.

Tissues can grow in a particular direction by controlling the orientation of cell divisions. This phenomenon is evident in the developing Drosophila wing epithelium, where the tissue becomes elongated along the proximal-distal axis. We show that orientation of cell divisions in the wing requires planar polarization of an atypical myosin, Dachs. Our evidence suggests that Dachs constricts cell-cell junctions to alter the geometry of cell shapes at the apical surface, and that cell shape then determines the orientation of the mitotic spindle. Using a computational model of a growing epithelium, we show that polarized cell tension is sufficient to orient cell shapes, cell divisions, and tissue growth. Planar polarization of Dachs is ultimately oriented by long-range gradients emanating from compartment boundaries, and is therefore a mechanism linking these gradients with the control of tissue shape.

IRaPPA: information retrieval based integration of biophysical models for protein assembly selection.

MOTIVATION: In order to function, proteins frequently bind to one another and form 3D assemblies. Knowledge of the atomic details of these structures helps our understanding of how proteins work together, how mutations can lead to disease, and facilitates the designing of drugs which prevent or mimic the interaction. RESULTS: Atomic modeling of protein-protein interactions requires the selection of near-native structures from a set of docked poses based on their calculable properties. By considering this as an information retrieval problem, we have adapted methods developed for Internet search ranking and electoral voting into IRaPPA, a pipeline integrating biophysical properties. The approach enhances the identification of near-native structures when applied to four docking methods, resulting in a near-native appearing in the top 10 solutions for up to 50% of complexes benchmarked, and up to 70% in the top 100. AVAILABILITY AND IMPLEMENTATION: IRaPPA has been implemented in the SwarmDock server ( http://bmm.crick.ac.uk/∼SwarmDock/ ), pyDock server ( http://life.bsc.es/pid/pydockrescoring/ ) and ZDOCK server ( http://zdock.umassmed.edu/ ), with code available on request. CONTACT: moal@ebi.ac.uk. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Analysis of Leishmania mimetic neoglycoproteins for the cutaneous leishmaniasis diagnosis.

Oligosaccharides are broadly present on Leishmania cell surfaces. They can be useful for the leishmaniases diagnosis and also helpful in identifying new cell markers for the disease. The disaccharide Galα1-3Galß is the immunodominant saccharide in Leishmania cell surface and is the unique non-reducing terminal glycosphingolipids structure recognized by anti-α-Gal. This study describes an enzyme-linked immunosorbent assay (ELISA) used to measure serum levels of anti-α-galactosyl (α-Gal) antibodies in patients with cutaneous leishmaniasis (CL). Optimal ELISA conditions were established and two neoglycoproteins (NGP) containing the Galα1-3Gal terminal fraction (Galα1-3Galß1-4GlcNAc-HAS and Galα1-3Gal-HAS) and one Galα1-3Gal NGP analogue (Galα1-3Galß1-3GlcNAc-HAS) were used as antigens. Means of anti-α-Gal antibody titres of CL patients were significantly higher (P < 0.05) than the healthy individuals for all NGPs tested. Sensitivity and specificity of all NGPs ranged from 62.2 to 78.4% and 58.3 to 96.7%, respectively. In conclusion, the NGPs can be used for CL diagnosis.