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Equine arteritis virus (EAV) is the causal agent of equine viral arteritis, a disease of equids. During natural outbreaks of the disease, EAV can cause abortion in pregnant mares and persistent infection in stallions. Understanding how host cellular proteins interact with viral RNA and viral proteins, as well as their role in viral infection, will enable better characterization of the pathogenesis of EAV and establishment of persistent infection in stallions. Accordingly, we hypothesized that both viral factors and host genetically related factors could influence the outcome of EAV infection in horses. To test this hypothesis, we first combined contemporary molecular biology techniques with dual color flow cytometric analysis to characterize the interactions of viral structural proteins and the equine peripheral blood mononuclear cells in vitro. Results from this study demonstrated that interactions between GP2, GP3, GP4, GP5 and M envelope proteins of EAV play a major role in determining the CD14+ monocyte tropism while the tropism of CD3+ T lymphocytes is determined by GP2, GP4, GP5 and M envelope proteins but not the GP3 protein. Secondly, a genome wide association study using SNP genotyping identified a common haplotype associated with the in vitro CD3+ T lymphocyte/resistance to EAV infection among four breeds of horses. Subsequently, these studies were extended to establish a possible correlation between the in vitro susceptibility of CD3+ T lymphocytes to EAV and establishment of persistent infection in stallions. Interestingly, carrier stallions with susceptible CD3+ T lymphocyte phenotype to EAV may represent those at higher risk of becoming persistently infected. Finally, the precise effect of EAV on the immune system of horses, innate and humoral immunity, was studied. Horses were shown to mount a strong humoral antibody response to nonstructural proteins (nsps) 2, 4, 5 and 12 of EAV, whereas nsps 1, 2 and 11 suppressed the type I interferon production. The data presented in this dissertation suggest new directions for future EAV research using genomic and proteomic approaches to study host cell factors involved in EAV attachment and entry and establishment of persistent infection in the stallions.

Equine arteritis virus (EAV) is the causal agent of equine viral arteritis (EVA), a disease of equids. Following EAV infection, up to 70% of stallions may become carriers and continuously shed the virus in their semen for varying time periods. The long-term carrier stallion has an important role in the transmission and maintenance of EAV in horse populations. Recently, it has been demonstrated a correlation between in vitro susceptibility of CD3+ T lymphocytes to EAV infection and establishment of long-term persistent infection among stallions following natural infections. In this study, we investigated whether stallions with in vitro EAV susceptible CD3+ T lymphocytes are at higher risk of becoming long-term carriers compared to those with the resistant phenotype following experimental infection with the KY84 strain of EAV. Furthermore, we investigated whether there is a significant effect of EAV infection on semen quality during acute phase of the infection. The data suggested that the establishment of the long-term carrier state seems to be associated with the in vitro CD3+ T lymphocyte susceptible phenotypes and that reduced semen quality resulted from the combined effect of fever and scrotal edema observed following EAV infection rather than the direct effect of the virus.

Diese Arbeit beschreibt die Farb-Duplex-Sonographie bei Vaskulitiden, insbesondere bei der Arteriitis temporalis (AT), bei der Takayasu-Arteriitis und bei Vaskulitis-Overlap-Syndromen. Dabei wird die erstmals von uns beschriebene charakteristische echoarme Wandschwellung der Temporalarterien (Halo-Phänomen) bei akuter AT erläutert. Dieser Befund wird durch ein Arterienwandödem erklärt und verschwindet unter Therapie mit Kortikosteroiden innerhalb von durchschnittlich 16 Tagen. Zusätzlich lassen sich Stenosen und akute Verschlüsse der Temporalarterien in der Akutphase der Erkrankung darstellen. An den ersten 30 konsekutiven Patienten mit akuter AT führten wir eine kontrollierte, prospektive Studie durch. Diese ergab, dass das Halo-Phänomen bei 73 %, Stenosen und/oder Verschlüsse bei 80 %, und alle drei pathologischen Befunde bei 93 % der Patienten mit der klinischen Diagnose einer akuten AT nachweisbar waren. Die Sensitivität gegenüber der Temporalarterien-Histologie betrug jeweils 76 %, 86 % und 95 %. Die Spezifität des Halo-Phänomens betrug 100 % und von Stenosen/Verschlüssen 93 % gegenüber der klinischen Diagnose. Inzwischen hat der Autor dieser Arbeit 742 Untersuchungen an 630 Patienten durchgeführt. Davon hatten 80 Patienten eine akute AT und 104 weitere Patienten eine akute Polymyalgia rheumatica (PMR). Sensitivität und Spezifität sind vergleichbar mit den Ergebnissen der zuvor genannten kontrollierten Studie. Dabei liegt die Sensitivität der Sonographie sogar über derjenigen der Histologie, da längere Gefäßabschnitte untersucht werden. Bei eindeutigem klinischen und sonographischen Befund erscheint es möglich, auf die Biopsie zu verzichten. Außerdem wird der sonographische Befund der peripheren Riesenzellarteriits an der A. brachialis dargestellt. Zusätzlich fanden wir bei 10 von 33 konsekutiven Patienten mit akuter AT entzündliche Veränderungen an vielen anderen Arterien. Diese Befunde belegen, dass die akute AT stärker generalisiert ist, als bisher angenommen. Selbst bei einer Wegener`schen Granulomatose kann es zum Befall großer Gefäße, wie der Arteria carotis interna kommen, der sich sonographisch darstellen lässt, wie ein weiterer Patient zeigt. Die PMR kann gemeinsam mit der AT vorkommen. Umgekehrt war bei 7 von 102 Patienten mit PMR ohne klinische Zeichen einer AT die Sonographie der Temporalarterien wegweisend für die Diagnose einer begleitenden AT. Bei der Takayasu-Arteriitis finden sich ebenfalls charakteristische, allerdings hellere Wandschwellungen. Drei Patientinnen werden beschrieben, bei denen die Sonographie bereits im prästenotischen Frühstadium für die Diagnose wegweisend war. Die Duplex-Sonographie ist eine faszinierende Methode, wenn sie bei Patienten mit Verdacht auf eine Vaskulitis großer Gefäße angewandt wird, weil es mit ihr schnell, nicht-invasiv und mit vorhandener Technik gelingt, eine Diagnose zu stellen. / Color Duplex sonography is an excellent tool in the diagnosis of vasculitides, particularly for giant cell arteritis (GCA), for Takayasu`s artertis, and for vasculitis overlap syndromes. We described a characteristic dark wall-swelling of the temporal arteries (halo) in acute GCA that is due to an edema. It resolves within about 16 days with corticosteroid therapy. Additionally stenoses and occlusions of the temporal arteries are found in acute disease. A prospective, controlled study was performed on 30 patients with acute GCA. Sensitivity was 73 % (halo), 80 % (stenoses and/or occlusions), and 93 % (at least one of the three findings) for the clinical diagnosis, and 76 %, 86 %, and 95 % for positive histology. Specificity for the clinical diagnosis was 100 % (halo) and 93 % (stenoses, occlusions). Until now the author has performed 742 investigations in 630 patients including 80 patients with acute GCA and 104 more patients with acute polymyalgia rheumatica (PMR). Sensitivity and specificity are comparable to the results of the study. Sensitivity of sonography is even superior to that of histology because a longer segment of the artery can be investigated. In cases with typical clinical and ultrasound findings biopsy may not be necessary any more. Additionally we described the ultrasound image of large-vessel GCA in the brachial artery. In 10 of 33 consecutive patients with acute GCA we found inflammatory changes in many arteries other than the temporal arteries. Thus a generalized vasculitis occurs more frequently in GCA than assumed up to now. Even in Wegener`s granulomatosis large vessels like the internal carotid artery may be involved. We described the ultrasound image of one patient. In 102 patients with PMR and no clinical symptoms of GCA sonography of the temporal arteries delineated typical findings of GCA. In Takayasu`s arteritis sonography also shows a characteristic wall swelling which is brighter than in GCA. We described three patients in which sonography aided in establishing the diagnosis already in the early prestenotic stage of the disease. Duplex sonography is a fascinating method that can be used in the diagnosis of patients with suspected large vessel vasculitis like GCA and Takayasu`s arteritis. It is fast, non-invasive, and the equipment is widely available.

Introdução: A prevalência de Síndrome Metabólica (SM) tende a ser alta em pacientes com doenças reumáticas, sendo as doenças cardiovasculares a principal causa de óbito nestas condições. Objetivos: Determinar a prevalência de SM em pacientes com Arterite de Takayasu (AT) e sua associação com fatores de risco, níveis de adipocinas e de citocinas. Métodos: Foi realizado um estudo transversal incluindo 45 mulheres com AT e 47 controles saudáveis pareados por idade e índice de massa corporal (IMC). Resultados: A prevalência de SM (critérios da IDF/AHA) foi maior em pacientes com AT comparada aos controles (33,34 vs. 8,51%, p = 0,003). Pacientes com TA apresentaram maior frequência de hipertensão (p < 0,001) e dislipidemia (p = 0,001) e maiores níveis de insulina (p = 0,021), HOMA-IR (p = 0,024), apolipoproteína E (p = 0,029), resistina (p = 0,018) e PCR (p < 0,001) comparada aos controles saudáveis, com níveis comparáveis de adiponectina e PAI-1 (p > 0,05). Análise adicional de pacientes com AT com e sem SM revelou um maior frequência de sobrepeso/obesidade (66,66 vs. 26,66%, p = 0,022), escore de Framingham >-1 (p=0,032) e menores níveis de adiponectina (20,37+-21,16 vs. 38,64+-22,62ug/ml, p=0,022) no primeiro grupo. Não foram encontradas diferenças quanto à duração de doença, atividade, uso de glicorticóides, níveis de resistina e PAI-1 nos dois grupos de pacientes com AT (p > 0,05). Pacientes com e sem SM não demonstraram diferenças em relação aos níveis plasmáticos de citocinas (IL-12, IL-1a, IL-6 e TNFalfa). Foi evidenciada correlação de Pearson positive entre IL-6 e PCR somente nos pacientes com SM (r=0.57; p=0.050). Conclusão: Alta prevalência de SM foi observada em pacientes com AT, sendo que esta comorbidade parece identificar um subgrupo de pacientes com sobrepeso/obesidade com alto risco cardiovascular sem associação com o status de doença. Estudos longitudinais são necessários para observar o impacto do controle de fatores de risco modificáveis na qualidade de vida e sobrevida dos pacientes com AT / Introduction: The prevalence of Metabolic Syndrome (MetS) tends to be high among rheumatic patients, and cardiovascular disease is the leading cause of death in these conditions. Objective: To determine the prevalence of MetS in Takayasu Arteritis patients (TA) and its association with risk factors and adipokines and cytokines levels. Methods: A cross sectional study was conducted in 45 consecutive TA women with 47 age- and body mass index (BMI)-matched healthy controls. Results: The prevalence of MetS (IDF/AHA criteria) was higher in TA compared to controls (33.34 vs. 8.51%, p=0.003). TA patients had higher frequency hypertension (p < 0.001), dyslipidemia (p=0.001), insulin (p=0.021), HOMA-IR (p=0.024), apoliprotein E (p=0.029), resistin (p=0.018) and CRP (p < 0.001) compared to healthy subjects, with similar levels of adiponectin and PAI-1 (p > 0.05). Further analysis of TA patients with and without MetS revealed a higher frequency of overweightness/obesity (66.66 vs. 26.66%, p=0.022), Framingham score >-1 (p=0.032), and lower adiponectin levels (20.37+-21.16 vs. 38.64±22.62ug/ml, p=0.022) in the former group. No differences were found regarding disease duration, activity, glucocorticoid use, resistin and PAI-1 levels in these two groups of TA patients (p > 0.05). Patients with and without MetS showed no differences respect to cytokines levels (IL-12, IL-1a, IL-6 and TNFalfa). IL-6 had a positive Pearson correlation with CRP only in TA patients with MetS (r=0.57; p=0.050). Conclusion: A high prevalence of MetS was observed in TA patients and this comorbidity seems to identify a subgroup of overweight/obese patients with high cardiovascular risk without a significant association with disease status. Further longitudinal studies are necessary to observe the impact of controlling this modifiable risk factor in the quality of life and survival of TA patients

Target cell lysis is the hallmark of immune effector responses of cytotoxic T lymphocytes (CTL), natural killer (NK) cells, and monocytes. The most commonly used assay to measure target cell lysis is the 51Cr release assay and is considered the ‘gold standard’. However, this assay has many disadvantages that limit its use by most laboratories. Thus, several alternative assays have been developed. Some of these alternative assays are more sensitive, easy to perform and do not use radioactive elements. In this study, four of these assays were evaluated for their ability to detect antigen- specific CTL responses in equine blood. Three long-term equine arteritis virus (EAV) carrier stallions, two vaccinated stallions and one naïve stallion were included in this study. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood collected of these stallions to be used as effector cells. The PBMCs were stimulated with EAV in vitro for 7-10 days to generate antigen-specific effector cells. The granzyme B assay, the Carboxyfluorescein succinimidyl ester (CFSE)/7-Aminoactinomycin D (7AAD) assay and the Lactate dehydrogenase (LDH) assay were performed using these effector cells and autologous equine dermal cells (isolated from each stallion) as target cells. The first two assays (i.e., granzyme B and CFSE/7AAD assays) were difficult to optimize for this study because they work well with non-adherent targets and require immediate flow cytometry analysis. The LDH assay, however detected CTL lysis in one of the two vaccinated stallions at day 99 post vaccination and no response was detected in PBMCs isolated from carrier stallions and control stallion. Based on these findings, the LDH assay is the most suitable assay since it works well with adherent target cells, it produces quantitative data, and is ideal for high-throughput screening.

A major goal of this research was to evaluate a variety of cell lines for theirpermissiveness to equine arteritis virus (EAV) infection and then identify the mechanismthat restricts EAV infection in certain cell lines. The cell lines BHK-21, RK-13, andC2C12 were found to support productive infection with EAV strain VBS53, whereasHela, Hep-2, and L-M cell lines exhibited limited susceptibility to infection with thisvirus. In the course of the study, it was found that the Hela cell line became moresusceptible to infection with EAV strain VBS53 after extended serial passage. Therespective cell lines were referred to as Hela High (passage 170-221) and Hela Low(passage 95-115) lines. While the Hela High cell line was more susceptible than the HelaLow cell line, it was still considerably less susceptible than the BHK-21 cell line to EAVinfection. Subsequent studies demonstrated that infection with EAV strain VBS53 wasrestricted at the entry step in Hela, Hep-2, and L-M cell lines.The second major goal of this research was to establish an in vitro model ofpersistent EAV infection using cell culture and then use the persistently infected culturesas a tool to study virus-host cell interactions, and to investigate virus and host cellevolution. Persistent infection was successfully established in the Hela High cell line withthe VBS53 strain of EAV. Properties of the persistently infected Hela High cell line werecharacterized. Virus evolution with respect to virus growth characteristics, ability of thevirus to initiate secondary persistent infection, and genetic changes during persistentEAV infection in Hela cells was investigated. Neutralization phenotypic changes of viruses were observed during the course of persistent EAV infection in Hela cells. Reverse genetics studies identified that amino acid 98 of the GP5 protein is a new neutralization determinant of EAV. Using an in vitro assay, it was found that EAV probably became progressively less virulent during the course of persistent infection in Hela cells. The potential changes in pathogenicity of EAV during persistent infection of Hela cells need to be verified by inoculation of horses.

Introdução: A prevalência de Síndrome Metabólica (SM) tende a ser alta em pacientes com doenças reumáticas, sendo as doenças cardiovasculares a principal causa de óbito nestas condições. Objetivos: Determinar a prevalência de SM em pacientes com Arterite de Takayasu (AT) e sua associação com fatores de risco, níveis de adipocinas e de citocinas. Métodos: Foi realizado um estudo transversal incluindo 45 mulheres com AT e 47 controles saudáveis pareados por idade e índice de massa corporal (IMC). Resultados: A prevalência de SM (critérios da IDF/AHA) foi maior em pacientes com AT comparada aos controles (33,34 vs. 8,51%, p = 0,003). Pacientes com TA apresentaram maior frequência de hipertensão (p < 0,001) e dislipidemia (p = 0,001) e maiores níveis de insulina (p = 0,021), HOMA-IR (p = 0,024), apolipoproteína E (p = 0,029), resistina (p = 0,018) e PCR (p < 0,001) comparada aos controles saudáveis, com níveis comparáveis de adiponectina e PAI-1 (p > 0,05). Análise adicional de pacientes com AT com e sem SM revelou um maior frequência de sobrepeso/obesidade (66,66 vs. 26,66%, p = 0,022), escore de Framingham >-1 (p=0,032) e menores níveis de adiponectina (20,37+-21,16 vs. 38,64+-22,62ug/ml, p=0,022) no primeiro grupo. Não foram encontradas diferenças quanto à duração de doença, atividade, uso de glicorticóides, níveis de resistina e PAI-1 nos dois grupos de pacientes com AT (p > 0,05). Pacientes com e sem SM não demonstraram diferenças em relação aos níveis plasmáticos de citocinas (IL-12, IL-1a, IL-6 e TNFalfa). Foi evidenciada correlação de Pearson positive entre IL-6 e PCR somente nos pacientes com SM (r=0.57; p=0.050). Conclusão: Alta prevalência de SM foi observada em pacientes com AT, sendo que esta comorbidade parece identificar um subgrupo de pacientes com sobrepeso/obesidade com alto risco cardiovascular sem associação com o status de doença. Estudos longitudinais são necessários para observar o impacto do controle de fatores de risco modificáveis na qualidade de vida e sobrevida dos pacientes com AT / Introduction: The prevalence of Metabolic Syndrome (MetS) tends to be high among rheumatic patients, and cardiovascular disease is the leading cause of death in these conditions. Objective: To determine the prevalence of MetS in Takayasu Arteritis patients (TA) and its association with risk factors and adipokines and cytokines levels. Methods: A cross sectional study was conducted in 45 consecutive TA women with 47 age- and body mass index (BMI)-matched healthy controls. Results: The prevalence of MetS (IDF/AHA criteria) was higher in TA compared to controls (33.34 vs. 8.51%, p=0.003). TA patients had higher frequency hypertension (p < 0.001), dyslipidemia (p=0.001), insulin (p=0.021), HOMA-IR (p=0.024), apoliprotein E (p=0.029), resistin (p=0.018) and CRP (p < 0.001) compared to healthy subjects, with similar levels of adiponectin and PAI-1 (p > 0.05). Further analysis of TA patients with and without MetS revealed a higher frequency of overweightness/obesity (66.66 vs. 26.66%, p=0.022), Framingham score >-1 (p=0.032), and lower adiponectin levels (20.37+-21.16 vs. 38.64±22.62ug/ml, p=0.022) in the former group. No differences were found regarding disease duration, activity, glucocorticoid use, resistin and PAI-1 levels in these two groups of TA patients (p > 0.05). Patients with and without MetS showed no differences respect to cytokines levels (IL-12, IL-1a, IL-6 and TNFalfa). IL-6 had a positive Pearson correlation with CRP only in TA patients with MetS (r=0.57; p=0.050). Conclusion: A high prevalence of MetS was observed in TA patients and this comorbidity seems to identify a subgroup of overweight/obese patients with high cardiovascular risk without a significant association with disease status. Further longitudinal studies are necessary to observe the impact of controlling this modifiable risk factor in the quality of life and survival of TA patients

Equine arteritis virus (EAV) has a global impact on the equine industry being the causative agent of equine viral arteritis (EVA), a reproductive, respiratory, and systemic disease of equids. A distinctive feature of EAV infection is that it establishes long-term persistent infection in the reproductive tract of stallions and is continuously shed in the semen (carrier state). Recent studies showed that long-term persistence is associated with a specific allele of the CXCL16 gene (CXCL16S). However, the cellular and molecular mechanisms underlying the establishment and maintenance of persistent infection are yet to be determined. The studies were undertaken herein unequivocally demonstrated that the ampulla is the main EAV tissue reservoir rather than immunologically privileged tissues (i.e., testes) and that EAV has specific tropism for stromal cells and CD8+ T and CD21+ B lymphocytes but not glandular epithelium in the reproductive tract. Furthermore, persistent EAV infection is associated with a significant humoral, mucosal antibody and inflammatory response at the site of persistence, characterized by induction of high levels of neutralizing antibodies (IgG1), mucosal anti-EAV-specific IgA, IgG1, IgG3/5, and IgG4/7 with variable neutralizing efficacy; and moderate, multifocal lymphoplasmacytic ampullitis, with significant infiltration of T lymphocytes (mainly CD8+ and low numbers of FOXP3+ lymphocytes), CD21+ B lymphocytes, diverse Ig-secreting plasma cells, and Iba-1+ and CD83+ tissue macrophages/dendritic cells. Moreover, EAV long-term persistent infection is associated with a CD8+ T lymphocyte transcriptional profile with upregulation of T-cell exhaustion-related transcripts and homing chemokines/chemokine receptors (CXCL9-11/CXCR3 and CXCL16/CXCR6), orchestrated by a specific subset of transcription factors (EOMES, PRDM1, BATF, NFATC2, STAT1, IRF1, TBX21), which are associated with the presence of the susceptibility allele (CXCL16S). Finally, these studies have determined that long-term EAV persistence is associated with the downregulation of a specific seminal exosome-associated miRNA (eca-mir-128) along with an enhanced expression of CXCL16 in the reproductive tract, a putative target of eca-mir-128. These findings provide evidence that this miRNA plays a crucial role in the regulation of the CXCL16/CXCR6 axis in the reproductive tract of persistently infected stallions, a chemokine axis strongly implicated in EAV persistence. The findings presented herein suggest that complex host-pathogen interactions shape the outcome of EAV infection in the stallion and that EAV employs complex immune evasion mechanisms favoring persistence in the reproductive tract. Further studies to identify specific mechanisms mediating the modulation of the CXCL16/CXCR6 axis and viral immune evasion in the reproductive tract of the EAV long-term carrier stallion are warranted.