Specificity of the rapid rK39 antigen-based immunochromatographic test Kalazar Detect(r) in patients with cutaneous leishmaniasis in Brazil

The aim of this study was to evaluate the specificity of a rapid immunochromatographic test that was developed to detect antibodies against the rK39 antigen for the diagnosis of visceral leishmaniasis (VL). This evaluation was performed using sera from patients with a confirmed diagnosis of active cutaneous leishmaniasis. The sera from 272 patients with a confirmed diagnosis of localised cutaneous leishmaniasis (CL) who resided in an area endemic for Leishmania braziliensis in Brazil were obtained before the initiation of antileishmanial treatment. Kalazar Detect(r)(InBios, Seattle, WA) recombinant K39 antigen-based immunochromatographic strips were used according to the manufacturer's instructions. The test results were evaluated independently by two examiners in sequential order. The positive controls for the test included five serum samples from five patients with parasitologically confirmed diagnosis of VL caused by Leishmania infantum in Brazil. Overall, 100% of the samples obtained from patients with CL were negative, confirming the absence of a serological cross-reaction for individuals with cutaneous disease when these patients were evaluated using the rapid test. The lack of a cross-reaction in patients who were infected by parasites of the same genus highlights the specificity of the rK39 antigen for the diagnosis of VL in areas with the sympatric circulation of L. braziliensis and L. infantum.

The N-terminal DNA-binding 'zinc finger' of the oestrogen and glucocorticoid receptors determines target gene specificity.

Steroid hormone receptors activate specific gene transcription by binding as hormone-receptor complexes to short DNA enhancer-like elements termed hormone response elements (HREs). We have shown previously that a highly conserved 66 amino acid region of the oestrogen (ER) and glucocorticoid (GR) receptors, which corresponds to part of the receptor DNA binding domain (region C) is responsible for determining the specificity of target gene activation. This region contains two sub-regions (CI and CII) analogous to the 'zinc-fingers' of the transcription factor TFIIIA. We show here that CI and CII appear to be separate domains both involved in DNA binding. Furthermore, using chimaeric ERs in which either the first (N-terminal) (CI) or second (CII) 'zinc finger' region has been exchanged with that of the GR, indicates that it is the first 'zinc finger' which largely determines target gene specificity. We suggest that receptor recognition of the HRE is analogous to that of the helix-turn-helix DNA binding motif in that the receptor binds to DNA as a dimer with the first 'zinc finger' lying in the major groove recognizing one half of the palindromic HRE, and that protein-DNA interaction is stabilized through non-specific DNA binding and dimer interactions contributed by the second 'zinc finger'.

Causal mechanisms underlying host specificity in bat ectoparasites.

In parasites, host specificity may result either from restricted dispersal capacity or from fixed coevolutionary host-parasite adaptations. Knowledge of those proximal mechanisms leading to particular host specificity is fundamental to understand host-parasite interactions and potential coevolution of parasites and hosts. The relative importance of these two mechanisms was quantified through infection and cross-infection experiments using mites and bats as a model. Monospecific pools of parasitic mites (Spinturnix myoti and S. andegavinus) were subjected either to individual bats belonging to their traditional, native bat host species, or to another substitute host species within the same bat genus (Myotis). The two parasite species reacted differently to these treatments. S. myoti exhibited a clear preference for, and had a higher fitness on, its native host, Myotis myotis. In contrast, S. andegavinus showed no host choice, although its fitness was higher on its native host M. daubentoni. The causal mechanisms mediating host specificity can apparently differ within closely related host-parasite systems.

Fab antibodies capable of blocking T cells by competitive binding have the identical specificity but a higher affinity to the MHC-peptide-complex than the T cell receptor.

In transplant rejection, graft versus host or autoimmune diseases T cells are mediating the pathophysiological processes. Compared to unspecific pharmacological immune suppression specific inhibition of those T cells, that are involved in the disease, would be an alternative and attractive approach. T cells are activated after their T cell receptor (TCR) recognizes an antigenic peptide displayed by the Major Histocompatibility Complex (MHC). Molecules that interact with MHC-peptide-complexes in a specific fashion should block T cells with identical specificity. Using the model of the SSX2 (103-111)/HLA-A*0201 complex we investigated a panel of MHC-peptide-specific Fab antibodies for their capacity blocking specific T cell clones. Like TCRs all Fab antibodies reacted with the MHC complex only when the SSX2 (103-111) peptide was displayed. By introducing single amino acid mutations in the HLA-A*0201 heavy chain we identified the K66 residue as the most critical binding similar to that of TCRs. However, some Fab antibodies did not inhibit the reactivity of a specific T cell clone against peptide pulsed, artificial targets, nor cells displaying the peptide after endogenous processing. Measurements of binding kinetics revealed that only those Fab antibodies were capable of blocking T cells that interacted with an affinity in the nanomolar range. Fab antibodies binding like TCRs with affinities on the lower micromolar range did not inhibit T cell reactivity. These results indicate that molecules that block T cells by competitive binding with the TCR must have the same specificity but higher affinity for the MHC-peptide-complex than the TCR.

Sensitivity and specificity of parallel or serial serological testing for detection of canine Leishmania infection

In Brazil, human and canine visceral leishmaniasis (CVL) caused byLeishmania infantum has undergone urbanisation since 1980, constituting a public health problem, and serological tests are tools of choice for identifying infected dogs. Until recently, the Brazilian zoonoses control program recommended enzyme-linked immunosorbent assays (ELISA) and indirect immunofluorescence assays (IFA) as the screening and confirmatory methods, respectively, for the detection of canine infection. The purpose of this study was to estimate the accuracy of ELISA and IFA in parallel or serial combinations. The reference standard comprised the results of direct visualisation of parasites in histological sections, immunohistochemical test, or isolation of the parasite in culture. Samples from 98 cases and 1,327 noncases were included. Individually, both tests presented sensitivity of 91.8% and 90.8%, and specificity of 83.4 and 53.4%, for the ELISA and IFA, respectively. When tests were used in parallel combination, sensitivity attained 99.2%, while specificity dropped to 44.8%. When used in serial combination (ELISA followed by IFA), decreased sensitivity (83.3%) and increased specificity (92.5%) were observed. Serial testing approach improved specificity with moderate loss in sensitivity. This strategy could partially fulfill the needs of public health and dog owners for a more accurate diagnosis of CVL.

New host, geographic records, and histopathologic studies ofAngiostrongylus spp (Nematoda: Angiostrongylidae) in rodents from Argentina with updated summary of records from rodent hosts and host specificity assessment

To date, 21 species of the genus Angiostrongylus (Nematoda: Angiostrongylidae) have been reported around the world, 15 of which are parasites of rodents. In this study, new host, geographic records, and histopathologic studies of Angiostrongylus spp in sigmodontine rodents from Argentina, with an updated summary of records from rodent hosts and host specificity assessment, are provided. Records of Angiostrongylus costaricensis from Akodon montensis andAngiostrongylus morerai from six new hosts and geographical localities in Argentina are reported. The gross and histopathologic changes in the lungs of the host species due to angiostrongylosis are described. Published records of the genus Angiostrongylus from rodents and patterns of host specificity are presented. Individual Angiostrongylusspecies parasitise between one-19 different host species. The most frequent values of the specificity index (STD) were between 1-5.97. The elevated number of host species (n = 7) of A. morerai with a STD = 1.86 is a reflection of multiple systematic studies of parasites from sigmodontine rodents in the area of Cuenca del Plata, Argentina, showing that an increase in sampling effort can result in new findings. The combination of low host specificity and a wide geographic distribution of Angiostrongylus spp indicates a troubling epidemiological scenario although, as yet, no human cases have been reported.

Probing the different chaperone activities of the bacterial HSP70-HSP40 system using a thermolabile luciferase substrate.

During mild heat-stress, a native thermolabile polypeptide may partially unfold and transiently expose water-avoiding hydrophobic segments that readily tend to associate into a stable misfolded species, rich in intra-molecular non-native beta-sheet structures. When the concentration of the heat-unfolded intermediates is elevated, the exposed hydrophobic segments tend to associate with other molecules into large stable insoluble complexes, also called "aggregates." In mammalian cells, stress- and mutation-induced protein misfolding and aggregation may cause degenerative diseases and aging. Young cells, however, effectively counteract toxic protein misfolding with a potent network of molecular chaperones that bind hydrophobic surfaces and actively unfold otherwise stable misfolded and aggregated polypeptides. Here, we followed the behavior of a purified, initially mostly native thermolabile luciferase mutant, in the presence or absence of the Escherichia coli DnaK-DnaJ-GrpE chaperones and/or of ATP, at 22 °C or under mild heat-stress. We concomitantly measured luciferase enzymatic activity, Thioflavin-T fluorescence, and light-scattering to assess the effects of temperature and chaperones on the formation, respectively, of native, unfolded, misfolded, and/or of aggregated species. During mild heat-denaturation, DnaK-DnaJ-GrpE+ATP best maintained, although transiently, high luciferase activity and best prevented heat-induced misfolding and aggregation. In contrast, the ATP-less DnaK and DnaJ did not maintain optimal luciferase activity and were less effective at preventing luciferase misfolding and aggregation. We present a model accounting for the experimental data, where native, unfolded, misfolded, and aggregated species spontaneously inter-convert, and in which DnaK-DnaJ-GrpE+ATP specifically convert stable misfolded species into unstable unfolded intermediates.

Preference, specificity and cheating in the arbuscular mycorrhizal symbiosis.

Arbuscular mycorrhizal symbioses are mutualistic interactions between fungi and most plants. There is considerable interest in this symbiosis because of the strong nutritional benefits conferred to plants and its influence on plant diversity. Until recently, the symbiosis was assumed to be unspecific. However, two studies have now revealed that although it can be largely unspecific with the fungal community composition changing seasonally, in certain ecosystems it can also be highly specific and might potentially allow plants to cheat the arbuscular mycorrhizal network that connects plants below ground.

PHYTOCHROME KINASE SUBSTRATE 1 is a phototropin 1 binding protein required for phototropism.

Phototropism, or plant growth in response to unidirectional light, is an adaptive response of crucial importance. Lateral differences in low fluence rates of blue light are detected by phototropin 1 (phot1) in Arabidopsis. Only NONPHOTOTROPIC HYPOCOTYL 3 (NPH3) and root phototropism 2, both belonging to the same family of proteins, have been previously identified as phototropin-interacting signal transducers involved in phototropism. PHYTOCHROME KINASE SUBSTRATE (PKS) 1 and PKS2 are two phytochrome signaling components belonging to a small gene family in Arabidopsis (PKS1-PKS4). The strong enhancement of PKS1 expression by blue light and its light induction in the elongation zone of the hypocotyl prompted us to study the function of this gene family during phototropism. Photobiological experiments show that the PKS proteins are critical for hypocotyl phototropism. Furthermore, PKS1 interacts with phot1 and NPH3 in vivo at the plasma membrane and in vitro, indicating that the PKS proteins may function directly with phot1 and NPH3 to mediate phototropism. The phytochromes are known to influence phototropism but the mechanism involved is still unclear. We show that PKS1 induction by a pulse of blue light is phytochrome A-dependent, suggesting that the PKS proteins may provide a molecular link between these two photoreceptor families.

Tissue specificity in DNA repair: lessons from trinucleotide repeat instability.

DNA must constantly be repaired to maintain genome stability. Although it is clear that DNA repair reactions depend on cell type and developmental stage, we know surprisingly little about the mechanisms that underlie this tissue specificity. This is due, in part, to the lack of adequate study systems. This review discusses recent progress toward understanding the mechanism leading to varying rates of instability at expanded trinucleotide repeats (TNRs) in different tissues. Although they are not DNA lesions, TNRs are hotspots for genome instability because normal DNA repair activities cause changes in repeat length. The rates of expansions and contractions are readily detectable and depend on cell identity, making TNR instability a particularly convenient model system. A better understanding of this type of genome instability will provide a foundation for studying tissue-specific DNA repair more generally, which has implications in cancer and other diseases caused by mutations in the caretakers of the genome.

Substrate metabolism, nutrient balance and obesity development in children and adolescents: a target for intervention?

Obesity results from the organism's inability to maintain energy balance over a long term. Childhood obesity and its related factors and pathological consequences tend to persist into adulthood. A cluster of factors, including high energy density in the diet (high fat intake), low energy expenditure, and disturbed substrate oxidation, favour the increase in fat mass. Oxidation of three major macronutrients and their roles in the regulation of energy balance, particularly in children and adolescents, are discussed. Total glucose oxidation is not different between obese and lean children; exogenous glucose utilization is higher whereas endogenous glucose utilization is lower in obese compared with lean children. Carbohydrate composition of the diet determines carbohydrate oxidation regardless of fat content of the diet. Both exogenous and endogenous fat oxidation are higher in obese than in lean subjects. The influence of high fat intake on accumulation of fat mass is operative rather over a long term. Several future directions are addressed, such that a combination of increased physical activity and modification in diet composition, in terms of energy density and glycemic index, is recommended for children and adolescents.

Simultaneous co-expression of memory- and effector-related genes by individual human CD8 T cells depends on antigen specificity and differentiation

Purpose/Objective: Phenotypic and functional T cell properties are usually analyzed at the level of defined cell populations. However, large differences between individual T cells may have important functional consequences. To answer this issue, we performed highly sensitive single-cell gene expression profiling, which allows the direct ex vivo characterization of individual virus- and tumor-specific T cells from healthy donors and melanoma patients. Materials and methods: HLA-A*0201-positive patients with stage III/ IV metastatic melanoma were included in a phase I clinical trial (LUD- 00-018). Patients received monthly low-dose of the Melan-AMART- 1 26_35 unmodified natural (EAAGIGILTV) or the analog A27L (ELAGIGILTV) peptides, mixed CPG and IFA. Individual effector memory CD28+ (EM28+) and EM28- tetramer-specific CD8pos T cells were sorted by flow cytometer. Following direct cell lysis and reverse transcription, the resulting cDNA was precipitated and globally amplified. Semi-quantitative PCR was used for gene expression and TCR BV repertoire analyses. Results: We have previously shown that vaccination with the natural Melan-A peptide induced T cells with superior effector functions as compared to the analog peptide optimized for enhanced HLA binding. Here we found that natural peptide vaccination induced EM28+ T cells with frequent co-expression of both memory/homing-associated genes (CD27, IL7R, EOMES, CXCR3 and CCR5) and effector-related genes (IFNG, KLRD1, PRF1 and GZMB), comparable to protective EBV- and CMV-specific T cells. In contrast, memory/homing- and effectorassociated genes were less frequently co-expressed after vaccination with the analog peptide. Conclusions: These findings reveal a previously unknown level of gene expression diversity among vaccine- and virus-specific T cells with the simultaneous co-expression of multiple memory/homing- and effector- related genes by the same cell. Such broad functional gene expression signatures within antigen-specific T cells may be critical for mounting efficient responses to pathogens or tumors. In summary, direct ex vivo high-resolution molecular characterization of individual T cells provides key insights into the processes shaping the functional properties of tumor- and virus-specific T cells.

Molecular insights for optimizing T cell receptor specificity against cancer.

Cytotoxic CD8 T cells mediate immunity to pathogens and they are able to eliminate malignant cells. Immunity to viruses and bacteria primarily involves CD8 T cells bearing high affinity T cell receptors (TCRs), which are specific to pathogen-derived (non-self) antigens. Given the thorough elimination of high affinity self/tumor-antigen reactive T cells by central and peripheral tolerance mechanisms, anti-cancer immunity mostly depends on TCRs with intermediate-to-low affinity for self-antigens. Because of this, a promising novel therapeutic approach to increase the efficacy of tumor-reactive T cells is to engineer their TCRs, with the aim to enhance their binding kinetics to pMHC complexes, or to directly manipulate the TCR-signaling cascades. Such manipulations require a detailed knowledge on how pMHC-TCR and co-receptors binding kinetics impact the T cell response. In this review, we present the current knowledge in this field. We discuss future challenges in identifying and targeting the molecular mechanisms to enhance the function of natural or TCR-affinity optimized T cells, and we provide perspectives for the development of protective anti-tumor T cell responses.

Metabolic compartmentalization in the human cortex and hippocampus: evidence for a cell- and region-specific localization of lactate dehydrogenase 5 and pyruvate dehydrogenase.

BACKGROUND: For a long time now, glucose has been thought to be the main, if not the sole substrate for brain energy metabolism. Recent data nevertheless suggest that other molecules, such as monocarboxylates (lactate and pyruvate mainly) could be suitable substrates. Although monocarboxylates poorly cross the blood brain barrier (BBB), such substrates could replace glucose if produced locally.The two key enzymatiques systems required for the production of these monocarboxylates are lactate dehydrogenase (LDH; EC1.1.1.27) that catalyses the interconversion of lactate and pyruvate and the pyruvate dehydrogenase complex that irreversibly funnels pyruvate towards the mitochondrial TCA and oxydative phosphorylation. RESULTS: In this article, we show, with monoclonal antibodies applied to post-mortem human brain tissues, that the typically glycolytic isoenzyme of lactate dehydrogenase (LDH-5; also called LDHA or LDHM) is selectively present in astrocytes, and not in neurons, whereas pyruvate dehydrogenase (PDH) is mainly detected in neurons and barely in astrocytes. At the regional level, the distribution of the LDH-5 immunoreactive astrocytes is laminar and corresponds to regions of maximal 2-deoxyglucose uptake in the occipital cortex and hippocampus. In hippocampus, we observed that the distribution of the oxidative enzyme PDH was enriched in the neurons of the stratum pyramidale and stratum granulosum of CA1 through CA4, whereas the glycolytic enzyme LDH-5 was enriched in astrocytes of the stratum moleculare, the alveus and the white matter, revealing not only cellular, but also regional, selective distributions. The fact that LDH-5 immunoreactivity was high in astrocytes and occurred in regions where the highest uptake of 2-deoxyglucose was observed suggests that glucose uptake followed by lactate production may principally occur in these regions. CONCLUSION: These observations reveal a metabolic segregation, not only at the cellular but also at the regional level, that support the notion of metabolic compartmentalization between astrocytes and neurons, whereby lactate produced by astrocytes could be oxidized by neurons.