A promising new ELISA diagnostic test for cattle babesiosis based on Babesia bigemina Apical Membrane Antigen-1.

Babesiosis due to Babesia bigemina is a relevant tick‑borne disease, affecting cattle worldwide. Many surface proteins of the pathogen including the Apical Membrane Antigen 1 (AMA‑1) ‑ have been analysed for vaccine and diagnostic purposes. This study focused on B. bigemina AMA‑1 and on its use for the assessment of diagnostic tests. After bioinformatic analyses, AMA‑1 codifying region was amplified and cloned into an expression vector used to induce protein synthesis in Escherichia coli cells. AMA‑1 was purified by affinity chromatography and used to set up the best condition for an ELISA protocol. Bovine field sera positive to B. bigemina were used to evaluate the presence of anti‑AMA‑1 antibodies. In order to verify the assay specificity, sera positive to Babesia bovis or to the piroplasm Theileria annulata were also included. Significant differences were obtained between sera negative to both B. bigemina and B. bovis and samples positive to B. bigemina, to B. bovis or to both pathogens. No significant reaction was observed with T. annulata positive sera. The results showed that AMA‑1 protein is suitable to be used as antigen in diagnostic assays for babesiosis diagnosis in cattle, as it does not show any cross reaction with anti-T. annulata antibodies

Invariant NKT cells are expanded in peripheral blood but are undetectable in salivary glands of patients with primary Sj\uf6gren's syndrome.

OBJECTIVES: Invariant NKT (iNKT) cells play a role in regulating the function of autoreactive B cells before their entry into germinal centres. Absence and/or reduction of iNKT cells have been demonstrated in patients with systemic lupus erythematosus (SLE) together with an increase of autoreactive B cell activity. Primary Sj\uf6gren's syndrome (pSS) is a systemic autoimmune disease in which lymphocyte infiltration and organisation in lymphoid structures of inflamed salivary glands occurs. The aim of the study was to investigate the percentage and function of iNKT in the salivary glands and peripheral blood of patients with pSS. METHODS: Minor salivary gland biopsies were obtained from patients with pSS and with non-specific chronic sialoadenitis (nSS). Flow cytometry analysis of CD1d/\u3b1-GalactosylCeramide (\u3b1-GalCer) tetramer positive cells, producing IFN-\u3b3 and IL-17, and quantitative gene expression analysis by TaqMan real-time PCR for V\u3b124 were performed on salivary glands biopsies and peripheral blood samples obtained from patients and controls. Flow cytometry and immunofluorescence analysis for autoreactive B lymphocytes and ELISA for anti-SSA antibodies (Ab) detection were also performed. RESULTS: An increase of iNKT was detected ex vivo in peripheral blood of pSS patients; after \u3b1-GalCer stimulation this subset produce IL-17 and IFN-iNKT were undetectable in the salivary glands of pSS patients and anti-SSA specific B cells were found in target tissue. Invariant NKT cells were able to inhibit autoantibody production by B cells obtained from salivary glands of pSS. CONCLUSIONS: Impaired iNKT migration to inflamed sites might induce the activation of autoreactive B cells specific for SSA-antigen in salivary glands of pSS patients

A Prospective Observational Study on the Role of Immunohistochemical Expression of Orphanin in Laryngeal Squamous Cell Carcinoma Recurrence

To date, histological biomarkers expressed by laryngeal cancer are poorly known. The identification of biomarkers associated with laryngeal squamous cell carcinoma (SCC), would help explain the tumorogenesis and prevent the possible recurrence of the lesion after treatment. For this reason, the aim of this study is to investigate, for the first time, the Orphanin expression in 48 human specimens of laryngeal SCC and evaluate its possible correlation with patients prognosis. We analyzed pathological specimens from 48 patients with laryngeal SCC to detect the presence of Orphanin by using an immunohistochemistry test. We compared the findings with healthy tissue acquired from patients who underwent surgery for mesenchymal benign tumours of the larynx. The specimens were stained with anti-Orphanin monoclonal antibodies. Results were processed through a computerised image analysis system to determine a scale of staining intensity. All the tumoural specimens examined showed a significant immunoreaction for Orphanin when compared with healthy tissues (p < 0.05) but with a different immune reactivity related to clinical-pathological features. A high Orphanin expression was not significantly related to Histological Grading (HG), TNM, and stage (p > 0.05). In the multivariate analysis, the Orphanin expression was significantly related only to the malignant recurrence (p < 0.05). Our study suggests that Orphanin could have a role in tumorigenesis by increasing the recurrence of cancer; therefore, it should be further explored as a possible biomarker for laryngeal cancer

Innate Immune Response to Tick-Borne Pathogens: Cellular and Molecular Mechanisms Induced in the Hosts.

Many pathogens are transmitted by tick bites, including Anaplasma spp., Ehrlichia spp., Rickettsia spp., Babesia and Theileria sensu stricto species. These pathogens cause infectious diseases both in animals and humans. Different types of immune effector mechanisms could be induced in hosts by these microorganisms, triggered either directly by pathogen-derived antigens or indirectly by molecules released by host cells binding to these antigens. The components of innate immunity, such as natural killer cells, complement proteins, macrophages, dendritic cells and tumor necrosis factor alpha, cause a rapid and intense protection for the acute phase of infectious diseases. Moreover, the onset of a pro-inflammatory state occurs upon the activation of the inflammasome, a protein scaffold with a key-role in host defense mechanism, regulating the action of caspase-1 and the maturation of interleukin-1β and IL-18 into bioactive molecules. During the infection caused by different microbial agents, very similar profiles of the human innate immune response are observed including secretion of IL-1α, IL-8, and IFN-α, and suppression of superoxide dismutase, IL-1Ra and IL-17A release. Innate immunity is activated immediately after the infection and inflammasome-mediated changes in the pro-inflammatory cytokines at systemic and intracellular levels can be detected as early as on days 2-5 after tick bite. The ongoing research field of "inflammasome biology" focuses on the interactions among molecules and cells of innate immune response that could be responsible for triggering a protective adaptive immunity. The knowledge of the innate immunity mechanisms, as well as the new targets of investigation arising by bioinformatics analysis, could lead to the development of new methods of emergency diagnosis and prevention of tick-borne infections

An item and construct bias analysis of two language versions of a verbal analogies scale

The Woodcock Muñoz Language Survey is a test of cognitive academic language proficiency that has been adapted from English into Xhosa by a South African team of researchers. This study was primarily concerned with the Verbal Analogies Scale of the Woodcock Muñoz Language Survey and aimed to extend previous research on the equivalence of the two language versions of the scale. The study employed a monolingual two-group design consisting of 150 mainly English-speaking and 149 mainly Xhosa learners in Grades 6 and 7. The first research objective was to investigate item bias (or differential item functioning items) in the Visual Analogies Scale across the Xhosa and English versions using logistic regression and Mantel–Haenszel statistical techniques. Five items were identified as differential item functioning. The second objective was to evaluate the construct equivalence of the two versions by conducting a factor analysis after removing the differential item functioning items from the scale. Two factors were identified. The first factor displayed significant loadings across both language versions. The second factor was stable for the English version but not for the Xhosa version. Results were supported by calculating a Tucker’s phi coefficient for both factors. It was therefore concluded that Factor 1 is structurally equivalent across the two language versions but that Factor 2 was not structurally equivalent. Thus, the detection and removal of differential item functioning items did not result in structural equivalence.Department of HE and Training approved lis

Type 3 innate lymphoid cells producing IL-17 and IL-22 are expanded in the gut, in the peripheral blood, synovial fluid and bone marrow of patients with ankylosing spondylitis

Background The aim of the study was to better characterise the immunological origin and the behaviour of interleukin (IL)-23-responsive innate lymphoid cells (ILCs) in the gut, synovial fluid (SF) and bone marrow (BM) of patients with ankylosing spondylitis (AS).Methods ILC1, ILC2 and ILC3 cells were determined and characterised by confocal microscopy and flow cytometry in ileal and BM biopsies, in peripheral blood (PB) and SF mononuclear cells obtained from patients with AS and controls. Mucosal vascular addressin cell adhesion molecule 1 (MADCAM-1), IL-7, IL-15 and aggregates of lymphoid tissue inducer cells (LTi) were evaluated by immunohistochemistry. The in vitro ability of epithelial cells in driving the differentiation of ILC3 and the effect of tumour necrosis factor inhibitors (TNFi) on the frequency of ILC3 and the expression of MADCAM1 were also assessed.Results ILC3 characterised as Lyn(-)RORc(-)Tbet(+) NKp44(+) cells were significantly expanded in the gut, SF and BM of patients with AS compared with controls, produced high levels of IL-17 and IL-22 and expressed alpha 4 beta 7. MADcAM1 was overexpressed in BM and ileal high endothelial venules. IL-7 was significantly increased in AS gut, especially in the context of Paneth cells, and accompanied by the presence of aggregates of c-kit/IL-7R(+) cells (LTi). In in vitro experiments, epithelial cells from patients with AS actively induced differentiation of ILC3 from LTi. TNFi efficacy was accompanied by a significant decrease in the percentage of intestinal and circulating ILC3 and in the expression of MADCAM1.Conclusions Gut-derived IL-17(+) and IL-22(+) ILC3 are expanded in the peripheral blood, SF and inflamed BM of patients with AS, suggesting the presence of an active homing axis between the gut and the inflamed sacroiliac joints

Detection of natural killer T cells in mice infected with Rickettsia conorii

Little information is available regarding the role of natural killer T (NKT) cells during the early stage of Rickettsia conorii infection. Herein, C3H/HeN mice were infected with the Malish 7 strain of R. conorii. Splenocytes from these mice were analysed in the early stage of the infection by flow cytometry and compared with uninfected controls. Our results showed an increase in NKT cells in infected mice. Additionally, NKT interleukin (IL)-17(+) cells increased three days after infection, together with a concurrent decrease in the relative amount of NKT interferon (IFN)-\u3b3(+) cells. We also confirmed a higher amount of NK IFN-\u3b3(+) cells in infected mice. Taken together, our data showed that NKT cells producing Il-17 increased during the early stage of rickettsial infection. These results suggest a connection between IL-17(+) NKT cells and vasculitis, which is the main clinical symptom of rickettsiosi

Immune response to tick-borne hemoparasites: Host adaptive immune response mechanisms as potential targets for therapies and vaccines

Tick-transmitted pathogens cause infectious diseases in both humans and animals. Different types of adaptive immune mechanisms could be induced in hosts by these microorganisms, triggered either directly by pathogen antigens or indirectly through soluble factors, such as cytokines and/or chemokines, secreted by host cells as response. Adaptive immunity effectors, such as antibody secretion and cytotoxic and/or T helper cell responses, are mainly involved in the late and long-lasting protective immune response. Proteins and/or epitopes derived from pathogens and tick vectors have been isolated and characterized for the immune response induced in different hosts. This review was focused on the interactions between tick-borne pathogenic hemoparasites and different host effector mechanisms of T-and/or B cell-mediated adaptive immunity, describing the efforts to define immunodominant proteins or epitopes for vaccine development and/or immunotherapeutic purposes. A better understanding of these mechanisms of host immunity could lead to the assessment of possible new immunotherapies for these pathogens as well as to the prediction of possible new candidate vaccine antigens

ABERRANT EXPRESSION OF IL-22RA1 ON HEMATOPOIETIC CELLS AS IMMUNOLOGICALLY SIGNATURE OF PRIMARY SJOGREN\u2019S SYNDROME AND SJOGREN-ASSOCIATED NON-HODGKIN LYMPHOMAS

Background: Interleukin (IL)-22 is a potent mediator of cellular inflammatory responses that has been recently reported to play a role in the pathogenesis of primary Sjogren's Syndrome (p-SS) (1, 2) and of T and B lymphomas. IL-22 biological activity is initiated by binding to a cell-surface complex composed of two subunits, IL-22R1 and IL-10R2 receptor chains, and further regulated by interactions with a soluble binding protein, IL-22BP. Unlike the IL-10R2, which is constitutively expressed in many human tissues, IL-22R1 is not detectable in immune cells. Objectives: Aim of this study was to better characterize the role of IL-22 axis in the pathogenesis of p-SS and p-SSassociated lymphomas. Methods: Minor salivary gland biopsies were obtained from 30 patients with p-SS and 20 with non-specific chronic sialoadenitis (n-SS) and evaluated by RT-PCR for IL-22, IL-22R1 and IL-22BP expression. The protein expression of IL-22, IL- 22R1 and p-STAT3 was evaluated by immunohistochemistry and IL-22R1-expressing cells were caharcterized by confocal microscopy. Flow cytometry analysis of IL-22R1 expression was also conducted on peripheral blood mononuclear cells (PBMCs) from p-SS (n=30), n-SS (n=20), SLE (n=20) and rheumatoid arthritis (n=20) patients and normal controls (n=30). PBMCs isolated from 10 p-SS and 10 controls were also cultured with (or without) recombinant IL- 22 and the modulation of the transcripts for pro-inflammatory cytokines was assessed by RT-PCR. Paraffin embedded sections of non-Hodgkin lymphomas from 5 p-SS patients were finally evaluated for the expression of IL-22R1. Results: IL-22, STAT3 and IL-22BP but not IL-22R1 transcript levels were significantly up-regulated in the inflamed salivary glands of p-SS but not in n-SS. Immunohistochemistry confirmed the increased salivary expression of IL-22 and p-STAT3 also demonstrating a significant increased protein levels of IL-22R1 in p-SS. Confocal microscopy analysis showed that IL-22R1 was aberrantly and strongly expressed in monocytes/macrophages infiltrating the p-SS salivary glands and these cells also co-expressed p-STAT3, suggesting the occurrence of autocrine activation of p-STAT3. CD68+ cells obtained from the peripheral blood also aberrantly expressed IL-22R1 in p-SS patients. IL-22R1 expression on cells of hematopoietic origin was never observed in control disease patients, n-SS and healthy controls. The stimulation with recombinant IL-22 of PBMCs from p-SS but not controls significantly up-regulated the expression of IL-17 and IL-22. Non-Hodgkin lymphoma tissues from p-SS patients were also characterized by the aberrant expression of IL- 22R1 on macrophages and neoplastic B cells. Conclusions: The aberrant expression of IL-22RA1 on cells of hematopoietic origin seems to be a specific immunological signature of patients with p-SS and p-SS associated lymphomas and suggests a fundamental role of IL-22 signaling in the pathogenesis of p-SS and its neoplastic evolution. Targeting of IL-22 pathway may represent a successful therapeutic strategy in p-SS patients. References: 1. Ciccia F, et al. Ann RheumDis 2012. 2. Ciccia F, et al. Ann Rheum Dis 2012

Using Cognitive Interviewing for the Semantic Enhancement of Multi-Lingual Versions of Personality Questionnaires

We discuss the use of cognitive interviewing with bilinguals as an integral part of cross-cultural adaptation of personality questionnaires. The aim is to maximize semantic equivalence to increase the likelihood of items maintaining the intended structure and meaning in the target language. We refer to this part of adaptation as semantic enhancement, and integrate cognitive interviewing within it as a tool for scrutinizing translations, the connotative meaning, and the psychological impact of items across languages. During the adaptation of a work-based personality questionnaire from English to Arabic, Chinese (Mandarin), and Spanish, we cognitively interviewed 12 bilingual participants about 136 items in different languages (17% of all items), of which 67 were changed. A content analysis categorizing the reasons for amending items elicited eleven errors that affect two identified forms of semantic equivalence. We provide the resultant coding scheme as a framework for designing cognitive interviewing protocols and propose a procedure for implementing them. We discuss implications for theory and practic