Effects of Overcheck Use on Stress Parameters and Welfare Implications in Driving Horses.

Three sequential studies were performed to evaluate the effects of tying horses while wearing overchecks (strap from the bridle to backpad). In an observational study, horses (n = 305) wore high (HC), low (LC), or no overchecks (NC) with frequencies of 29.2%, 51.8% and 19.0% respectively. Study 1 (Latin square, n = 6) consisted of a 90-min tie test (90TT) with treatments (HC, LC, NC) x periods (1-3). Horses wearing HC had higher plasma cortisol (P < 0.01) when compared to LC and NC. Muscle soreness (MSS) and tightness (MTS) were evaluated pre and 24 hr post 90TT and were higher (P < 0.01) 24 hrs after 90TT regardless of treatment. In study 2, in order to determine if acclimation to tying with overchecks could reduce the cortisol response in study 1, horses (n = 6) were tied 60 minutes/day for 2 weeks followed by a 60-min TT (60TT). Neither plasma cortisol nor heart rate were elevated above baseline levels, suggesting adaptation to novel stressors. Tying with restrictive HC appears to be a stressor, which may be reduced if an adaptation period is provided.

Assessing epidemiology of leprosy and socio-economic distribution of cases.

Leprosy still represents a serious health problem in a number of countries, including Brazil. Although leprosy has been associated with poverty for a long time, it is still difficult to accurately define this relationship. Here, we evaluated in an endemic municipality the progress from 1995 to 2015 of epidemiological indicators to establish if there were any strong associations between social indicators and the occurrence of leprosy. An ecological study was conducted using the SINAN database (Brazilian leprosy-national notifiable diseases information system) in combination with georeferencing of leprosy cases. The georeferencing used the ArcGis programme and occurrence of cases was evaluated in relation to the Health Vulnerability Index (HVI), an indicator that categorises socio-economic and sanitation factors. The data identified a marked decrease in the overall prevalence of leprosy, a reduction in the new case-detection rate and a reduction in the number of cases with grade 2 disabilities (albeit with transient peaks in 2007 and 2015). Logistic regression analysis showed association of detection rates with elevated HVI. Thus, while the epidemiological indicators point to the elimination of leprosy, there is evidence of hidden cases and an association between higher rates of leprosy detection and greater social vulnerability remain.

Distinct Roles of Th17 and Th1 Cells in Inflammatory Responses Associated with the Presentation of Paucibacillary Leprosy and Leprosy Reactions.

It is well established that helper T cell responses influence resistance or susceptibility to Mycobacterium leprae infection, but the role of more recently described helper T cell subsets in determining severity is less clear. To investigate the involvement of Th17 cells in the pathogenesis of leprosy, we determined the immune profile with variant presentations of leprosy. Firstly, IL-17A, IFN-γ and IL-10 were evaluated in conjunction with CD4+ T cell staining by confocal microscopy of lesion biopsies from tuberculoid (TT) and lepromatous leprosy (LL) patients. Secondly, inflammatory cytokines were measured by multiplex assay of serum samples from Multibacillary (MB, n = 28) and Paucibacillary (PB, n = 23) patients and household contacts (HHC, n = 23). Patients with leprosy were also evaluated for leprosy reaction occurrence: LR+ (n = 8) and LR- (n = 20). Finally, peripheral blood mononuclear cells were analysed by flow cytometry used to determine the phenotype of cytokine-producing cells. Lesions from TT patients were found to have more CD4+ IL-17A+ cells than those from LL patients. Higher concentrations of IL-17A and IL-1ß were observed in serum from PB than MB patients. The highest serum IFN-γ concentrations were, however, detected in sera from MB patients that developed leprosy reactions (MB LR+ ). Together, these results indicate that Th1 cells were associated with both the PB presentation and also with leprosy reactions. In contrast, Th17 cells were associated with an effective inflammatory response that is present in the PB forms but were not predictive of leprosy reactions in MB patients.

Specific antibody responses as indicators of treatment efficacy for visceral leishmaniasis.

Acute visceral leishmaniasis (VL) is caused by infection with parasites of the Leishmania donovani complex and may be fatal if not treated. Early diagnosis and efficacious treatment are the keys to effective VL management and control. Novel regimens are being developed to overcome limitations in VL treatment options, which are currently restricted by high costs, severe systemic side effects, and unresponsiveness. Although simple and accurate serological tests are available to help confirm VL, none are suitable to monitor treatment efficacy and cure. Here, we confirm that serum antibody responses to the diagnostic antigens rK39 and rK28 are unaltered by treatment, but demonstrate that antibodies produced against two antigens, rK26 and rK18, can be used as an indirect measure of parasite clearance. The levels of anti-rK18 and -rK26 antibodies were high in patients at initial diagnosis but declined in patients treated with either SSG (Ethiopia) or AmBisome (Bangladesh). Taken together, we propose that serological tests which measure antibodies to rK26 and rK18 merit consideration as potential markers of treatment success and cure.

Synergistic antigen combinations for the development of interferon gamma release assays for paucibacillary leprosy.

The development of immunodiagnostic tests for paucibacillary leprosy (PB) is based on Mycobacterium leprae specific-cell mediated immunity (CMI)/IFN-γ production. Recently, novel M. leprae protein antigens that stimulate CMI have been described. This study evaluated different M. leprae antigen combinations in whole blood assay (WBA). Five study groups were tested (20 per group): newly diagnosed, untreated PB patients and multibacillary leprosy patients (MB); household contacts of MB patients (HHC); healthy endemic controls (EC); pulmonary tuberculosis patients (TB). WBA (heparinized, 24 h 37 °C 5% CO2) were stimulated with: 10 µg/ml of each individual M. leprae recombinant protein (rML) and five combinations of rML (46f + LID-1, ML0276 + LID-1, ML2055 + ML1632 + ML2044, ML0276 + 46f, ML2055 + LID-1)-M. leprae cell sonicate (MLCS, 10 µg/ml), PHA (1 µg/ml), and PBS alone. Human IFN-γ ELISA (QuantiFERON-TB Gold/QFT-G, Cellestis) was performed using stimulated plasma (arbitrary cut-off = 50 pg/ml). Three out of five antigen combinations (46f + LID-1, ML0276 + LID-1, ML2055 + ML1632 + ML2044) were able to increase the levels of IFN-γ production in WBA in a larger number of responders among both PB leprosy and contacts. However, the magnitude of IFN-γ responses was higher among contacts. The antigen combination (46f + ML0276) stimulated IFN-γ only in symptomatic PB leprosy patients and not in asymptomatic contacts. Few controls (EC, TB) responded to combinations (0-15%), indicating the specificity of the response in an endemic area with high BCG coverage. The synergistic effect of new combinations of M. leprae proteins upon IFN-γ production in WBA indicates their potential use for the development of an interferon gamma release assay/IGRA for the diagnosis of PB leprosy.

Biomarkers for intracellular pathogens: establishing tools as vaccine and therapeutic endpoints for visceral leishmaniasis.

Visceral leishmaniasis in South Asia is a serious disease affecting children and adults. Acute visceral leishmaniasis develops in only a fraction of those infected individuals, the majority being asymptomatic with the potential to transmit infection and develop disease. We followed 56 individuals characterized as being asymptomatic by seropositivity with rk39 rapid diagnostic test in a hyperendemic district of Bangladesh to define the utility of Leishmania-specific antibodies and DNA in identifying infection. At baseline, 54 of the individuals were seropositive with one or more quantitative antibody assays and antibody levels persisted at follow up. Most seropositive individuals (47/54) tested positive by quantitative PCR at baseline, but only 16 tested positive at follow up. The discrepancies among the different tests may shed light on the dynamics of asymptomatic infections of Leishmania donovani, as well as underscore the need for standard diagnostic tools for active surveillance as well as assessing the effectiveness of prophylactic and therapeutic interventions.

Alteration of the serum biomarker profiles of visceral leishmaniasis during treatment.

Until recently, chemotherapy for visceral leishmaniasis (VL; also known as kala-azar) was severely limited by factors such as high cost, route of administration, generation of side effects and potential for resistance. Although largely effective, chemotherapies have become available with the introduction of new drugs and multi-drug regimens for VL. These could be further improved by the identification of biomarkers that are altered during effective treatment. The identification of such biomarkers in the circulation would also simplify efficacy trials. In this study, we determined immunological signatures within the serum of ethnically and geographically distinct VL patients (from Bangladesh and Brazil). Our results indicate that inflammatory and regulatory cytokines (IFNγ, TNFα, IL-10, IL-17), as well as levels of growth factors (FGF, VEGF), are elevated within the serum of VL patients from these sites. The examination of samples from Brazilian VL patients during and beyond standard treatment with meglumine antimoniate identified multiple parameters that revert to levels comparable to those of healthy endemic control individuals. The consolidation of these results provides a 'response to treatment' signature that could be used within efficacy trials to rapidly and simply determine successful interruption of VL.

Evaluation of various cytokines elicited during antigen-specific recall as potential risk indicators for the differential development of leprosy.

Leprosy is a dermato-neurological disease caused by Mycobacterium leprae infection that manifests across a wide range of clinical and immunological outcomes. Diagnosis is still currently based on clinical manifestations and simple tests are needed. This study investigated whether biomarkers induced by defined M. leprae proteins in 24-h whole blood assays (WBA) could discriminate active leprosy patients from at-risk contacts. Newly diagnosed, untreated paucibacillary (PB; tuberculoid leprosy/borderline tuberculoid [TT/BT]) and multibacillary (MB; borderline lepromatous/lepromatous leprosy [BL/LL]) leprosy patients, as well as healthy household contacts (HHC) of MB patients, were recruited in central western Brazil (Goiânia/Goiás). Cell-based responses to the ML0276, ML1623, ML0405, ML1632, 92f, and ML1011 antigens were measured by Luminex 14-plex assays detecting eotaxin, IFNγ, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-15, IL-17A, IL-23, IL-31, IP-10, and TNFα. Our data reinforce that IFNγ is currently the best indicator of the antigen-specific cellular immune response of TT/BT leprosy and demonstrate that the same antigens promote the secretion of IL-4 in blood from BL/LL leprosy patients. While none of the biomarkers tested could discriminate leprosy patients from HHC, our data indicate that, although most HHC antigen-specific responses are qualitatively similar to TT/BT patients, some HHC can respond similarly to BL/LL patients.

Specific IgG antibody responses may be used to monitor leprosy treatment efficacy and as recurrence prognostic markers.

Although curable, leprosy requires better diagnostic and prognostic tools to accompany therapeutic strategies. We evaluated the serum samples of leprosy patients from Venezuela and Brazil for reactivity against the specific recombinant proteins, ML0405 and ML2331, and the LID-1 fusion protein that incorporates both of these antigens. Antigen-specific IgG was highest in lepromatous leprosy patients (LL) and decreased across the disease spectrum, such that only a small subset of true tuberculoid patients (TT) tested positive. The impact of multidrug therapy (MDT) on these antibody responses was also examined. Several years after treatment, the vast majority of Venezuelan patients did not possess circulating anti-LID-1, anti-ML0405, and anti-ML2331 IgG, and the seropositivity of the remaining cases could be attributed to irregular treatment. At discharge, the magnitude and proportion of positive responses of Brazilian patients against the proteins and phenolic glycolipid (PGL)-I were lower for most of the clinical forms. The monthly examination of IgG levels in LL patient sera after MDT initiation indicated that these responses are significantly reduced during treatment. Thus, responses against these antigens positively correlate with bacillary load, clinical forms, and operational classification at diagnosis. Our data indicate that these responses could be employed as an auxiliary tool for the assessment of treatment efficacy and disease relapse.

Preschool vision screening using the MTI-Photoscreener.

Amblyopia is a condition that, if detected and treated early, can improve vision for most children. Thus, both pediatric and ophthalmologic groups have acknowledged the need for preschool vision screening. However, vision screening is the exception rather than the rule for preschoolers, since traditional methods of vision screening are often inappropriate for the preschool population and almost impossible for those children who are preverbal or nonverbal, developmentally delayed, and/or have chronic illnesses or disabilities. This study evaluated the use of a photoscreener to detect vision problems in a preschool population. Fifty-one children ages 3 to 5 years were evaluated using the MTI Photoscreener. Results were compared with a complete ophthalmologic examination, including cycloplegia. The sensitivity and specificity calculated for this study was 83% and 68%, respectively. Findings conclude that the MTI Photoscreener detected a broad range of vision problems, seemed to require less time, and seemed more acceptable to preschoolers when compared with the traditional vision screening methods performed by registered nurses. Although the sensitivity and specificity rates for this study were less than desired, it is likely that both could be improved with additional photo interpretation training.

Inhibiting amyloid precursor protein C-terminal cleavage promotes an interaction with presenilin 1.

Presenilin 1 (PS1) plays a pivotal role in the production of the amyloid-beta protein, which is central to the pathogenesis of Alzheimer's disease. It has been demonstrated that PS1 regulates the gamma-secretase proteolysis of the amyloid precursor protein (APP) C-terminal fragment (APP-C100), which is the final step in amyloid-beta protein production. The mechanism and detailed pathway of this PS1 activity has yet to be fully resolved, but it may be due to a presenilin-controlled trafficking of the APP fragment or possibly an inherent PS1 proteolytic activity. We have investigated the possibility of a direct interaction of PS1 and the APP-C100 within the high molecular mass presenilin complex. However, the APP-C100 is rapidly degraded, and if it forms, then any PS1.APP complex is likely to be very transitory. To circumvent this problem, we have utilized the protease inhibitor N-acetyl-leucyl-norleucinal (LLnL) and the lysosomotropic agent NH(4)Cl, which inhibits the turnover of the APP-C100. Under these conditions, levels of the fragment increased appreciably, and as shown by glycerol gradient analysis, the APP-C100 shifted to a higher molecular mass complex that overlapped with PS1. Immunoprecipitation studies demonstrated that a significant population of the APP-C100 co-precipitated with PS1. These findings suggest that PS1 may mediate the shuttling of APP fragments and/or facilitate their presentation for gamma-secretase cleavage through a direct interaction.

Differential effects of UVA1 and UVB radiation on Langerhans cell migration in mice.

The UVB (280-315 nm)- and UVA1 (340-400 nm)-induced migration of Langerhans cells (LC) from the epidermis and accumulation of dendritic cells (DC) in the lymph nodes draining the exposed skin site of C3H/HeN mice have been investigated. One minimum erythemal dose (MED) of UVB (1.5 kJ/m2) and of UVA1 (500 kJ/m2) were chosen, which have been shown previously to suppress delayed hypersensitivity (DTH). UVB irradiation resulted in a reduction in epidermal LC numbers, local to the site of the exposure, which was most apparent 12 h after exposure, but, in contrast, UVA1 had no significant effect even at 72 h after exposure. UVA1 did not exert any protection against the UVB-mediated depletion in LC numbers. The reduction in local LC following UVB exposure was prevented by systemic (intraperitoneal) treatment of mice with neutralising antibodies to either tumor necrosis factor (TNF)-alpha or interleukin (IL)-beta 2 h prior to the irradiation. It has been reported previously that UVB exposure caused an increase in the number of dendritic cells (DC) in the lymph nodes draining the irradiated skin site. In the present study we have shown that UVA1 had a similar effect. Pretreatment of the mice with neutralising antibodies to IL-1beta (by intraperitoneal injection) substantially inhibited DC accumulation induced by both UV regimens. However, anti-TNF-alpha antibodies affected only the UVB-induced increase, and did not alter the elevation in DC numbers observed following UVA1 exposure. These results indicate that UVB causes the migration of LC from the epidermis and an accumulation of DC in the draining lymph nodes by a mechanism that requires both TNF-alpha and IL-1beta. In contrast, UVAI does not cause LC migration from the epidermis and the accumulation of DC in the draining lymph nodes observed following UVA1 exposure requires IL-1beta, but not TNF-alpha. It is likely therefore that UVA1 acts through a different mechanism from UVB and may target a cutaneous antigen presenting cell other than LC, such as the dermal DC.

The effects of ultraviolet radiation on the human immune system.

The adverse outcome of increased ultraviolet (UV) irradiation on human health is currently of concern. While many experiments have been carried out in rodent models, fewer have been designed to test the effects of UV exposure in human subjects. This review concentrates on the modulations induced in the human immune system by UV, and outlines changes in antigen presentation by Langerhans cells and macrophages, in the activities of natural killer cells and T cells, and in cytokine regulation. Precautionary measures which might be taken to help protect people against the immunosuppressive action of UV irradiation are considered.

Presenilin mutations associated with Alzheimer disease cause defective intracellular trafficking of beta-catenin, a component of the presenilin protein complex.

The presenilin proteins are components of high-molecular-weight protein complexes in the endoplasmic reticulum and Golgi apparatus that also contain beta-catenin. We report here that presenilin mutations associated with familial Alzheimer disease (but not the non-pathogenic Glu318Gly polymorphism) alter the intracellular trafficking of beta-catenin after activation of the Wnt/beta-catenin signal transduction pathway. As with their effect on betaAPP processing, the effect of PS1 mutations on trafficking of beta-catenin arises from a dominant 'gain of aberrant function' activity. These results indicate that mistrafficking of selected presenilin ligands is a candidate mechanism for the genesis of Alzheimer disease associated with presenilin mutations, and that dysfunction in the presenilin-beta-catenin protein complexes is central to this process.

Presenilins interact with armadillo proteins including neural-specific plakophilin-related protein and beta-catenin.

Missense substitutions in the presenilin 1 (PS1) and presenilin 2 (PS2) proteins are associated with early-onset familial Alzheimer's disease. We have used yeast-two-hybrid and coimmunoprecipitation methods to show that the large cytoplasmic loop domains of PS1 and PS2 interact specifically with three members of the armadillo protein family, including beta-catenin, p0071, and a novel neuronal-specific armadillo protein--neural plakophilin-related armadillo protein (NPRAP). The PS1:NPRAP interaction occurs between the arm repeats of NPRAP and residues 372-399 at the C-terminal end of the large cytoplasmic loop of PS1. The latter residues contain a single arm-like domain and are highly conserved in the presenilins, suggesting that they form a functional armadillo protein binding site for the presenilins.

The presenilin 1 protein is a component of a high molecular weight intracellular complex that contains beta-catenin.

The presenilin (PS) genes associated with Alzheimer disease encode polytopic transmembrane proteins which undergo physiologic endoproteolytic cleavage to generate stable NH2- and COOH-terminal fragments (NTF or CTF) which co-localize in intracellular membranes, but are tightly regulated in their stoichiometry and abundance. We have used linear glycerol velocity and discontinuous sucrose gradient analysis to investigate the distribution and native conformation of PS1 and PS2 during this regulated processing in cultured cells and in brain. The PS1 NTF and CTF co-localize in the endoplasmic reticulum (ER) and in the Golgi apparatus, where they are components of a approximately 250-kDa complex. This complex also contains beta-catenin but not beta-amyloid precursor protein (APP). In contrast, the PS1 holoprotein precursor is predominantly localized to the rough ER and smooth ER, where it is a component of a approximately 180-kDa native complex. PS2 forms similar but independent complexes. Restricted incorporation of the presenilin NTF and CTF along with a potentially functional ligand (beta-catenin) into a multimeric complex in the ER and Golgi apparatus may provide an explanation for the regulated accumulation of the NTF and CTF.

Characterization and epitope mapping of several new anti-P-glycoprotein monoclonal antibodies.

Monoclonal antibodies (MAbs) were raised against partially purified Class I P-glycoprotein from multidrug-resistant Chinese hamster ovary CHRB30 cells. Fifteen stable monoclonal hybridoma cell lines were established, and the secreted antibodies were classified into 8 groups on the basis of banding pattern on immunoblots of P-glycoprotein digested with cyanogen bromide or partially digested with proteases. One representative of each group was tested further for several activities. Six of the 8 recognized human P-glycoprotein in the multidrug-resistant SKVLBI cell line. None of the antibodies recognized P-glycoprotein in unfixed cells, suggesting that all recognize cytoplasmic epitopes or extracellular epitopes not accessible in native P-glycoprotein. All 8 antibodies were able to immunoprecipitate P-glycoprotein from non-denaturing detergent solution. The linear epitopes of the antibodies were mapped to 11-27 amino acids. Two of the antibodies had epitopes in the linker region, 3 in the N-terminal nucleotide binding domain, 2 in the C-terminal nucleotide binding domain and 1 in the predicted cytoplasmic loop between predicted transmembrane helices 8 and 9. These antibodies, with known epitopes, could have uses for P-glycoprotein detection, structure/function studies, purification and quantitation.

Topological determinants of internal transmembrane segments in P-glycoprotein sequences.

P-glycoprotein (Pgp) is a polytopic membrane protein responsible for multidrug resistance in cancer cells. Previously, we have used a coupled cell-free translation/translocation system to investigate the membrane orientation of Pgp sequences and have made the unexpected observation that predicted transmembrane (TM) segments from both the NH2-terminal and COOH-terminal halves inserted in microsomal membranes in two different orientations (Zhang, J.-T., Duthie, M., and Ling, V. (1993) J. Biol. Chem. 268, 15101-15110). How these topological forms of Pgp are regulated is not known. In the present study, we have used site-directed mutagenesis to investigate if the amino acids surrounding the internal TM segments of Pgp may affect their orientation. We discovered that the charged amino acids flanking TM4 are important in determining the membrane orientation of the NH2-terminal half molecule of Pgp. This is a novel observation demonstrating the existence of internal topogenic sequences in a mammalian polytopic membrane protein. These findings thus suggest A) that the topological structure of a mammalian polytopic membrane protein does not integrate into the membrane simply by following the lead of the first inserted TM segment but that internal TMs may have independent topogenic information and B) that the TM segments in a multi-spanning membrane protein may be more dynamic than have been previously anticipated, i.e. mutations in the amino acids surrounding internal TMs could drastically change the overall topology of the molecule.

Membrane topology of the N-terminal half of the hamster P-glycoprotein molecule.

P-glycoprotein (Pgp) is a tandemly duplicated plasma membrane protein containing 12 predicted transmembrane (TM) segments and two cytoplasmic ATP-binding domains. Pgp appears to be responsible for multi-drug resistance in cancer cells. A detailed knowledge of the topological structure of Pgp will be required for understanding its mechanism of action. Previously, we have investigated the membrane orientation of Pgp using a cell free translation/translocation system supplemented with canine pancreatic microsomal membranes. We observed unexpectedly that the C-terminal half of the Pgp molecules was present in two different topological orientations (Zhang, J.-T., and Ling, V. (1991) J. Biol. Chem. 266, 18224-18232). In the present study, using a similar approach, we have investigated in detail the topological structure of the N-terminal half of the Pgp molecule. Again, two orientations were observed. One has all six predicted TM segments in the membrane bilayer, the other has only four TM segments in the bilayer with predicted TM3 and TM5 in a cytoplasmic and extracellular location, respectively. Although the primary sequence of Pgp appears to be a tandem duplication, the new topological structure of N-terminal half is not a simple tandem duplication of that in the C-terminal half. Thus it appears that the insertion and orientation of Pgp TM segments are dictated by specific localized sequences. These results, together with our previous findings, raise the possibility that Pgp in the native membrane may be present in different topological orientations and this feature may be important for its function.

Identification of distinct P-glycoprotein gene sequences in rat.

In higher vertebrates, P-glycoprotein is usually encoded by a small family of genes. We have determined that the rat contains three P-glycoprotein genes and have cloned distinct genomic fragments containing the putative 3' untranslated regions of these P-glycoprotein genes. Sequence analysis indicates that the rat P-glycoprotein genes belong to the three P-glycoprotein classes identified in mammals. These cloned sequences will be useful for delineating the expression of P-glycoprotein genes in the rat. We have also isolated a fourth clone which contains only a short, but highly conserved P-glycoprotein domain. This clone appears not be a member of the P-glycoprotein gene family, and its relationship to P-glycoprotein is unknown.