Does bovine besnoitiosis affect the sexual function of chronically infected bulls?

Bovine besnoitiosis is a reemerging disease in Europe. The clinically Besnoitia besnoiti infection in bulls is characterized by fever, nasal discharge, and orchitis in the acute phase and by scleroderma in the chronic phase. However, in many bulls, B besnoiti infection remains at a subclinical stage. Bull infertility is an economically relevant consequence of besnoitiosis infection. It is not clear, however, if semen quality returns to normal levels when infected animals have clinically recovered. The aim of this study was to examine the relationship between chronic besnoitiosis and bull sexual function in a region of eastern France, where the disease is reemerging, by comparing semen quality and genital lesions in 11 uninfected, 17 subclinically infected, and 12 clinically infected bulls. The presence of anti-B besnoiti antibodies was detected by Western blot test. Semen was collected by electroejaculation. Bulls clinically infected with B besnoiti showed significantly more genital tract alterations than uninfected or subclinically infected bulls. No relationship was evidenced between besnoitiosis infectious status and semen quality, whereas a significant relationship was noted between genital lesions and semen score. This means that in the absence of moderate to severe genital lesions, chronic bovine besnoitiosis is unlikely to alter semen quality. However, as the presence of infected animals could lead to spread of the disease, culling or separation of clinically infected bulls from the remaining healthy animals is strongly recommended.

No detection of Besnoitia besnoiti DNA in the semen of chronically infected bulls.

Bovine besnoitiosis is a chronic and debilitating disease observed in many European countries that may cause important economic losses in cattle. The recent widespread of the parasite in Europe had led the European Food Safety Authority to declare bovine besnoitiosis as a re-emerging disease in Europe. Many aspects of the epidemiology of bovine besnoitiosis such as the main routes of transmission are still unclear and need to be further studied. Among the different hypotheses, a sexual transmission has not yet been investigated. Therefore, the aim of this study was to evaluate the presence of Besnoitia besnoiti DNA in the semen of naturally infected bulls by using a highly sensitive method (real-time qPCR). Both pre-sperm and sperm fractions of 40 bulls, including seronegative (n = 11), seropositive subclinically (n = 17), and seropositive clinically (n = 12) infected animals, were collected by electroejaculation and analyzed by real-time qPCR. No B. besnoiti DNA was detected in 27 pre-sperm and 28 sperm fractions of the 40 examined bulls, suggesting that the transmission of B. besnoiti infection by the semen of chronically infected bulls is very unlikely.

Impaired endothelial function is not associated with arterial stiffness in adults with type 1 diabetes.

AIM: This study investigated the relationship between endothelial dysfunction (ED) and arterial stiffness (AS) in adults with type 1 diabetes and no clinical cardiovascular (CV) disease. METHODS: A total of 68 patients with type 1 diabetes and 68 age- and gender-matched healthy (non-diabetic) subjects were evaluated. ED was assessed by reactive hyperaemia peripheral arterial tonometry (RH-PAT) and by serum concentrations of soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1) and E-selectin. AS was assessed by aortic pulse wave velocity (aPWV). All statistical analyses were stratified by gender. RESULTS: Adults with type 1 diabetes had RH-PAT index scores similar to those of their matching controls [men: 1.55 (1.38-1.98)% versus 1.61 (1.40-2.17)%, P=0.556; women: 2.07 (1.55-2.31)% versus 2.08 (1.79-2.49)%; P=0.215]. However, after adjusting for potential confounders, type 1 diabetes emerged as the main determinant of the RH-PAT index in women. Also, differences between genders in both the controls and type 1 diabetes patients disappeared. Men with diabetes had higher serum concentrations of E-selectin, and women had higher serum concentrations of sICAM-1, sVCAM-1 and E-selectin than their respective controls. However, after adjusting for potential confounders, only the differences in sICAM-1 (women) and E-selectin (both genders) remained significant. No association was found between aPWV and the RH-PAT index and ED markers after adjusting for CV risk factors. CONCLUSION: ED was increased in adults with type 1 diabetes compared with age-matched non-diabetic subjects. Also, gender differences in ED were lost in type 1 diabetes. However, ED was not associated with AS after adjusting for potential confounders. These findings suggest that ED occurs earlier than AS in type 1 diabetes.

Adult subjects with Prader-Willi syndrome show more low-grade systemic inflammation than matched obese subjects.

AIM: Adult subjects with Prader-Willi syndrome (PWS) may show several conditions that are associated with an activation of innate immunity such as obesity, deficient GH secretion or hypogonadism. Our aim was to study whether obese adult PWS subjects show an additional low-grade systemic inflammation (LGSI) in relation to obese adult non-PWS subjects and lean healthy control subjects before and after a standardized liquid meal. METHODS: Seven obese adult PWS subjects, 7 matched obese non-PWS subjects and 7 lean healthy control subjects were studied for 6 h from the administration of a standard liquid meal. RESULTS: Compared to non-PWS, PWS subjects showed higher plasma concentrations of C-reactive protein (CRP) (p=0.030), complement component C3 (p=0.018), interleukin(IL)-18 (p=0.048), and IL-6 (p=0.041) that persisted post-prandially elevated for CRP (p<0.0001), C3 (p=0.015), and IL-18 (p=0.003). Tumor necrosis factor(TNF)-alpha did not differ between the 3 groups. These results were independent from IGF-I levels, homeostasis model assessment index, and body mass index (BMI). In male subjects with PWS, testosterone levels correlated to IL-18 (r=-0,646, p=0.041). CONCLUSIONS: Compared to matched non-PWS subjects, the obese PWS subjects in this study showed an additional LGSI that persisted postprandially and was independent from BMI, insulin resistance, and deficient GH secretion. However, in PWS males, high IL-18 levels were related to low testosterone concentrations.

Lower heart rate variability is associated with higher plasma concentrations of IL-6 in type 1 diabetes.

OBJECTIVE: In type 1 diabetes, cardiovascular autonomic neuropathy (CAN) is associated with cardiovascular risk factors related to insulin resistance, which in turn are associated with low-grade systemic inflammation. Reduced heart rate variability (HRV) is considered one of the first indicators of CAN. Since the autonomic nervous system interacts with systemic inflammation, we evaluated CAN to study its possible association with low-grade systemic inflammation. DESIGN: Cross-sectional study of a group of 120 subjects diagnosed with type 1 diabetes mellitus 14 years before. METHODS: Information recorded: 1) clinical characteristics: sex, age, body mass index, waist-to-hip ratio (WHR), blood pressure (BP), smoking, alcohol intake, insulin dose, HbA1c, and lipid profile; 2) plasma levels of soluble fractions of tumour necrosis factor alpha receptors 1 and 2, IL-6, and C-reactive protein; 3) insulin resistance by estimation of the glucose disposal rate (eGDR); and 4) tests for CAN: HRV in response to deep breathing (E/I ratio), HRV in response to the Valsalva maneuver, and changes in systolic BP responding to standing. RESULTS: A significant negative correlation was found between E/I ratio and plasma concentrations of IL-6 (r=-0.244, P=0.032), which remained significant after adjusting for potential confounding factors (age, sex, HbA1c, WHR, diastolic BP, triglycerides, HDL-cholesterol, retinopathy, nephropathy, peripheral neuropathy, insulin dose, and smoking; r=-0.231, P=0.039). No other significant associations were found between inflammation-related proteins, tests for CAN, and eGDR. CONCLUSIONS: These findings suggest a link between low-grade inflammation and early alterations of CAN in type 1 diabetes and may be of importance in the pathogenesis of CAN and/or its clinical implications.

Association between increased CCL2 (MCP-1) expression in lesions and persistence of disease activity in giant-cell arteritis.

OBJECTIVE: Patients with giant-cell arteritis (GCA) usually respond dramatically to corticosteroid treatment. However, recurrences are frequent and corticosteroid requirements are highly variable among patients. The aim of our study was to identify genes potentially involved in disease persistence. METHODS: Gene expression was explored with cDNA arrays in temporal artery biopsies from six GCA patients with relapsing disease and six patients who easily achieved sustained remission. Differentially expressed genes of interest were subsequently analysed by quantitative real-time polymerase chain reaction (PCR) and immunohistochemistry in temporal artery biopsies from 35 patients with biopsy-proven GCA and nine controls. RESULTS: CCL2 (MCP-1) was up-regulated in temporal artery samples from relapsing individuals. In the extended series of patients, CCL2 mRNA concentration in lesions was significantly higher than in controls (31 +/- 15.6 vs 0.44 +/- 0.10, P = 0.0001). In addition, CCL2 was more abundant in patients who experienced two or more relapses during the first year compared with those who endured sustained remission (127 +/- 82 vs 11 +/- 5.5, P = 0.0233) and correlated with the cumulated prednisolone dose (R = 0.533, P = 0.0024). CCL2 mRNA concentration correlated with IL-1beta (R = 0.45, P = 0.02), tumour necrosis factor-alpha (TNF-alpha) (R = 0.47, P = 0.013) and IL-6 (R = 0.52, P = 0.0053) mRNA. However, circulating CCL2 determined by ELISA was decreased in patients with strong systemic inflammatory response, suggesting that reduction in circulating CCL2 may reinforce the local gradient in lesions. CONCLUSION: Increased CCL2 (MCP-1) expression in lesions is associated with persistence of disease activity in GCA.

Tissue production of pro-inflammatory cytokines (IL-1beta, TNFalpha and IL-6) correlates with the intensity of the systemic inflammatory response and with corticosteroid requirements in giant-cell arteritis.

OBJECTIVES: To investigate proinflammatory cytokine expression in temporal arteries from patients with giant-cell arteritis (GCA) and to analyse its relationship with the intensity of the initial systemic inflammatory reaction and response to corticosteroid therapy. METHODS: Quantification of interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), and interleukin-6 (IL-6) mRNA by real-time quantitative PCR in temporal artery samples from 36 patients with biopsy-proven GCA and 11 controls. Immunohistochemical detection of IL-1beta, TNFalpha, and IL-6 in temporal artery sections from 74 patients with GCA and 15 controls. Clinical and biochemical parameters of inflammation as well as the time (weeks) required to reach a maintenance prednisone dose <10 mg/day were recorded. RESULTS: IL-1beta (13.8 +/- 2.5 vs 5.4 +/- 1.3 relative units, P = 0.012) and IL-6 transcripts (34 +/- 13.7 vs 7.8 +/- 4.5 relative units, P = 0.034) were significantly more abundant in patients with a strong systemic inflammatory response compared with those with no inflammatory parameters. Immunohistochemical scores for IL-1beta (2.7 +/- 0.3 vs 1.9 +/- 0.2, P = 0.018), TNFalpha (3.2 +/- 0.2 vs 2.4 +/- 0.3, P = 0.028) and IL-6 (3 +/- 0.2 vs 2.1 +/- 0.3, P = 0.023) were also significantly higher in patients with strong systemic inflammatory reaction. A significant correlation was found between the amount of tissue TNFalpha mRNA and the time required to reach a maintenance dose of prednisone <10 mg/day (r = 0.586, P = 0.001). CONCLUSION: GCA patients with a strong systemic inflammatory response, who have been previously shown to be more resistant to corticosteroid therapy, have elevated tissue expression of proinflammatory cytokines IL-1beta, TNFalpha and IL-6. High production of TNFalpha is associated with longer corticosteroid requirements.

Tissue targeting and disease patterns in systemic vasculitis.

The systemic vasculitides include a group of diseases with highly heterogeneous organ distribution and disease expression patterns. The mechanisms mediating tissue targeting in systemic vasculitis are largely unknown. Mechanical forces may contribute to the distribution of lesions in immune complex-mediated vasculitis. The site at which the antigen is encountered may be crucial in determining the location of inflammatory infiltrates in some vasculitides. Co-existence of different immunopathogenic mechanisms with variable dominance may generate diversity in disease presentation patterns. Heterogeneous and incompletely understood triggering mechanisms attract inflammatory cells to the site of interest through sophisticated molecular mechanisms: interplay between leukocyte receptors and endothelial ligands, and interactions between chemokines and chemokine receptors. Even with a similar distribution of lesions, patients with vasculitis may display highly variable clinical manifestations. Variations in genes involved in immune response might determine the severity of disease, the intensity of the systemic inflammatory response, the degree of vessel occlusion and the response to therapy.

Genomic characterization of CD84 reveals the existence of five isoforms differing in their cytoplasmic domains.

CD84, a new member of the immunoglobulin superfamily, shows high homology with several molecules belonging to the CD2 family of differentiation antigens. By screening a peripheral blood leukocyte cDNA library four CD84 isoforms were obtained differing in their 3' sequence. A reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed that these isoforms were normally found on leukocytes and a new isoform was identified. To establish the nature of the five isoforms obtained (CD84a, CD84b, CD84c, CD84d and CD84e) the genomic structure of the CD84 gene was determined. Our results show that it is composed of at least eight exons, with an exon coding for the 5' UTR and the leader peptide, two exons coding for each of the two immunoglobulin-like domains, an exon encoding the transmembrane portion and four exons coding for the cytoplasmic domains. The isoforms are generated by several mechanisms: alternative use of exons, reading frame shift, use of a cryptic splice site or absence of splicing. The differential expression of several potentially phosphorylatable residues on the different isoforms could be a way to regulate its possible activity in signal transduction.

Fibronectin upregulates gelatinase B (MMP-9) and induces coordinated expression of gelatinase A (MMP-2) and its activator MT1-MMP (MMP-14) by human T lymphocyte cell lines. A process repressed through RAS/MAP kinase signaling pathways.

T-lymphocyte migration into tissues requires focal degradation of the basement membrane. In this study, we show that transient adherence to fibronectin induces the production of activated forms of matrix metalloproteinase-2 (MMP-2) and MMP-9, as well as downregulation of tissue inhibitor of metalloproteinase-2 (TIMP-2) by T-cell lines. MMP-2 activation was likely achieved by inducing a coordinated expression of membrane-type matrix metalloproteinase-1 (MMP-14), a major activator of MMP-2. Blocking monoclonal antibodies against alpha4, alpha5, and alphav integrins strongly reduced MMP-2 and MMP-9 production induced by fibronectin. Disrupting actin cytoskeleton organization by cytochalasin D strongly enhanced fibronectin-induced MMP-2 and MMP-9 expression. Inhibiting Src tyrosine kinases with herbimycin A reduced MMP-2 and MMP-9 production with no effect on cell attachment. By contrast, G-protein inhibition by pertussis toxin, or transfection with a dominant negative mutant of Ha-Ras strongly increased fibronectin-induced MMP-2 and MMP-9. Inhibition of PI3 kinase, MAPkinase (MEK1), or p38 MAPkinase by wortmannin, PD 98059, or SB 202190, respectively, strongly promoted fibronectin-induced MMP2 and MMP-9. Cells at high density lost their ability to synthesize MMP-2 and MMP-9 in response to fibronectin and MMP expression was restored by transfection with a dominant-negative mutant of Ha-Ras or by treatment with wortmannin, PD 98059, or SB 202190. Our findings suggest that adhesion to fibronectin transduces both stimulatory (through Src-type tyrosin kinases) and inhibitory signals (through Ras/MAPKinase signaling pathways) for MMP-2 and MMP-9 expression by T lymphocytes and that their relative predominance is regulated by additional stimuli related to cell adhesion, motility, and growth.

Circulating soluble adhesion molecules in patients with giant cell arteritis. Correlation between soluble intercellular adhesion molecule-1 (sICAM-1) concentrations and disease activity.

OBJECTIVE: To evaluate whether changes in concentrations of circulating adhesion molecules are related to disease activity in patients with giant cell arteritis (GCA). METHODS: A sandwich ELISA was used to measure soluble intercellular adhesion molecule-1 (sICAM-1), sICAM-3, vascular cell adhesion molecule-1 (sVCAM-1), E-selectin (sE-selectin), and L-selectin (sL-selectin) in serum and plasma samples from patients with GCA. A cross sectional study was performed on 64 GCA patients at different activity stages and on 35 age and sex matched healthy donors. Thirteen of these patients were evaluated at the time of diagnosis and serially during follow up. RESULTS: At the time of diagnosis, sICAM-1 concentrations were significantly higher in active GCA patients than in controls (mean (SD) 360.55 (129.78) ng/ml versus 243.25 (47.43) ng/ml, p < 0.001). In contrast, sICAM-3, sVCAM-1, sE-selectin, and sL-selectin values did not differ from those obtained in normal donors. With corticosteroid administration, a decrease in sICAM-1 concentrations was observed, reaching normal values when clinical remission was achieved (263.18 (92.7) ng/ml globally, 293.59 (108.39) ng/ml in the group of patients in recent remission, and 236.83 (70.02) ng/ml in those in long term remission). In the 13 patients followed up longitudinally, sICAM-1 values also normalised with clinical remission (225.87 (64.25) ng/ml in patients in recent remission, and 256.29 (75.15) ng/ml in those in long term remission). CONCLUSIONS: Circulating sICAM-1 concentrations clearly correlate with clinically apparent disease activity in GCA patients. Differences with results previously found in patients with other vasculitides may indicate that different pathogenic mechanisms contribute to vascular inflammation in different disorders.

Regulation of ICAM-3 and other adhesion molecule expressions on eosinophils in vitro. Effects of dexamethasone.

BACKGROUND: ICAM-3 has been recently identified as the third leukocyte-function associated-1 (LFA-1) ligand. ICAM-3 is expressed in eosinophils, but its regulation has not been studied. The objective of this study was to investigate the differential expression of ICAM-3 and other adhesion molecules (AM) on the surface of eosinophils. We also evaluated the effects of dexamethasone on AM expression. METHODS: Normodense eosinophils were isolated from peripheral blood and incubated with calcium ionophore A23187 (calcimycin) with and without dexamethasone. Expression of AM was assessed by flow cytometry and expressed as fluorescence mean intensity (FMI). RESULTS: Peripheral blood eosinophils constitutively expressed low levels of ICAM-1 and ICAM-2 (<10 FMI), moderate levels (10-50 FMI) of CD29 and L-selectin, and high levels (>50 FMI) of ICAM-3, LFA-1, and Mac-1. Calcium ionophore (1 microM) significantly increased Mac-1 and ICAM-1 expression at 6 and 24 h. L-selectin expression decreased at 6 and 24 h, but ICAM-2, ICAM-3, LFA-1, and CD29 expression did not show any significant change after calcium ionophore stimulation. Dexamethasone decreased ICAM-3 and increased L-selectin basal expression, and it caused a dose-related inhibition of calcium ionophore-induced ICAM-1 expression. CONCLUSIONS: These findings suggest that some AM, such as ICAM-1, Mac-1, and L-selectin, may be involved in adhesion during eosinophil activation and that glucocorticoids may prevent airway inflammation by regulating the expression of AM in eosinophils. The role of ICAM-3, a leukocyte AM highly expressed in resting eosinophils, remains to be clarified.

Isolation of two CD50 (ICAM-3)-negative Jurkat T-cell clones and their application for analysis of CD50 function.

The leukocyte differentiation antigen CD50 (intercellular adhesion molecule-3, ICAM-3), mediates cell-cell adhesion through its ligand LFA-1 and is a transducting receptor molecule during T-cell activation. Since CD50 homologues in other species have not yet been identified, the role of this molecule can only be analyzed in human cell models. Thus, to better study CD50 function in T cells, we have obtained two CD50-negative T-cell clones, named CAMY.1 and CAMY.2. These clones were derived from the Jurkat T-cell variant PPL.1. Data from analysis of protein expression, specific mRNA content and calcium mobilization assays have confirmed the absence of functional CD50 molecules on these two clones. Thus, CAMY.1 and CAMY.2 show no CD50 expression by phenotypical and immunoprecipitation analysis. CD50-specific mRNA content is undetectable by Northern blot analysis in these clones and, only, when RT-PCR was performed could specific mRNA be detected. Additionally, CD50 cross-linking on theses clones shows no increase in intracellular calcium. Transfection of CD50 cDNA on CAMY cells restores not only CD50 surface expression, but its functional ability to induce calcium mobilization, CD69 upregulation and cell morphological changes. The CAMY.1 and CAMY.2 clones provide useful model systems to analyze CD50 function in T cells.

CD148, a membrane protein tyrosine phosphatase, is able to induce tyrosine phosphorylation on human lymphocytes.

CD148 is a new cluster of differentiation defined in the VI International Workshop on Leucocyte Differentiation Antigens. It has been identified as the hematopoietic form of a formerly described membrane protein tyrosine phosphatase called HPTP eta/ DEP-1. Previous data have demonstrated that this molecule is able to give rise to [Ca2+]i increase. In the present work we show its capability to induce protein tyrosine phosphorylation in human lymphocytes in spite of its intrinsic protein tyrosine phosphatase activity. The induction of kinase activity suggests the involvement of some protein tyrosine kinase based signaling pathway. The activation of this postulated kinase could be carried out through a direct association or via an adapter molecule.