The Mouse Cytomegalovirus Gene m42 Targets Surface Expression of the Protein Tyrosine Phosphatase CD45 in Infected Macrophages

The receptor-like protein tyrosine phosphatase CD45 is expressed on the surface of cells of hematopoietic origin and has a pivotal role for the function of these cells in the immune response. Here we report that following infection of macrophages with mouse cytomegalovirus (MCMV) the cell surface expression of CD45 is drastically diminished. Screening of a set of MCMV deletion mutants allowed us to identify the viral gene m42 of being responsible for CD45 down-modulation. Moreover, expression of m42 independent of viral infection upon retroviral transduction of the RAW264.7 macrophage cell line led to comparable regulation of CD45 expression. In immunocompetent mice infected with an m42 deletion mutant lower viral titers were observed in all tissues examined when compared to wildtype MCMV, indicating an important role of m42 for viral replication in vivo. The m42 gene product was identified as an 18 kDa protein expressed with early kinetics and is predicted to be a tailanchored membrane protein. Tracking of surface-resident CD45 molecules revealed that m42 induces internalization and degradation of CD45. The observation that the amounts of the E3 ubiquitin ligases Itch and Nedd4 were diminished in cells expressing m42 and that disruption of a PY motif in the N-terminal part of m42 resulted in loss of function, suggest that m42 acts as an activator or adaptor for these Nedd4-like ubiquitin ligases, which mark CD45 for lysosomal degradation. In conclusion, the down-modulation of CD45 expression in MCMV-infected myeloid cells represents a novel pathway of virus-host interaction

Clinical assessment, genetic parameters and the role of elastin in chronic progressive lymphedema in the Belgian Draught Horse

Chronic progressive lymphedema (CPL) is an incurable disorder that affects several related draught horse breeds worldwide, including the Belgian Draught Horse. Clinical signs most typically occur at the lower part of the limbs, including a progressive soft tissue swelling, fibrosis and deformation associated with skin surface aberrance. A partial genetic susceptibility is suspected. In the Belgian Draught Horse, there is no uniform CPL diagnosis methodology, which hampers the calculation of disease prevalence and the estimation of genetic parameters. If CPL is hereditary, ideally, selection should be based on molecular markers. Therefore, in present work, the necessary steps were taken to investigate the current state of matter on CPL in the native population of Belgian Draught Horses, and, to explore the genetic background of the disease susceptibility by means of quantitative and molecular genetic research.A CPL evaluation system (using clinical scores) was assessed, standardizing clinical diagnosis. Using this system, clinical scores were collected in Belgian Draught Horses at official contests (Belgium) and stable visits (Flanders, Belgium) from 2009 to 2011. Clinical CPL prevalence approximated 61% in our total sampling group including a large proportion of young and subclinically affected horses. In a subset of horses older than 3 years of age, implying more phenotypic certainty (CPL = chronic disorder), prevalence amounted to 86%. First lesions occurred from 1 year of age in a very mild condition, whereas distinct lesions were generally seen from 3 years of age. Clinical severity increased with age, though faster in stallions than in mares. Factors possibly associated with clinical scores, skin fold thickness and hair diameter, were recorded as well. The latter 2 traits are objectively measurable and could enhance future multivariable genetic models and/or function as a direct diagnostic aid next to clinical examination. Factors (e.g. age and gender) possibly associated with clinical scores, skin fold thickness and hair diameter were identified using statistical (mixed) models, which served as a basis for the quantitative genetic part of the project.A laboratory test for CPL would confirm clinical scores and assist in diagnosis. Furthermore, such test values offer an extra objective trait in genetic models. An enzyme-linked immunosorbent assay (ELISA) was described previously and proposed as a diagnostic aid for CPL in draught horses. Using this ELISA, blood anti‑elastin antibodies (AEAb) were demonstrated previously in Belgian Draught Horses whereby a clinically healthy group had significantly lower AEAb than 3 clinically affected groups (mild, moderate and severe symptoms). We evaluated the use of this AEAb ELISA as an in vitro diagnostic aid in individual, clinically examined Belgian Draught Horses. Test reproducibility was assessed performing assays in 2 different laboratories. Factors (e.g. gender and pregnancy) possibly associated with AEAb were analyzed using statistical (mixed) models. Even when a high AEAb cut‑off was handled to obtain a reasonable specificity of 90%, a low sensitivity (21%) of AEAb for CPL diagnosis was obtained. Therefore, the described AEAb ELISA procedure was shown to be of no use as a diagnostic aid for CPL per horse, hence the obtained AEAb values were not included in further analyses.Genetic parameters for CPL were estimated using a multi-trait animal model, including clinical scores, skin fold thickness and hair diameter. A separate bi‑variate analysis was performed to estimate the genetic correlation between clinical scores of fore and hind limbs. Due to the uncertainty of the clinical scores in younger (possibly subclinically) affected horses, analyses were based on 2 sets of data. A restricted dataset (D_3+) was formed, excluding records from horses under 3 years of age from the complete dataset (D_full). Both fixed (e.g. age) and random (permanent environment and date of recording) effects were included. Heritabilities (s.e.) for clinical scores for D_full and D_3+ were 0.11 (0.06) and 0.26 (0.05) respectively. A large proportion of the variance in clinical scores was attributed to the permanent environmental effect in D_full, but less in D_3+. Date of recording explained a proportion of variance (s.e.) from 0.09 (0.03) to 0.61 (0.08). Additive genetic correlations between clinical scores and both skin fold thickness and hair diameter showed that the latter 2 traits cannot be used as a direct diagnostic aid for CPL, whereas their use in the full model has to be examined further on a larger dataset. The additive genetic correlation between scores of fore and hind limbs was high (93%). Due to the moderate heritability of the clinical scores, and, in the absence of molecular markers, selection against CPL should focus on estimated breeding values (from repeated clinical examinations of only the fore limbs).Histological skin elastin alterations have been demonstrated in draught horses of susceptible breeds compared to those of a non‑susceptible breed. Therefore, elastin gene (ELN) aberrance was suggested. We sequenced the full ELN in clinically affected Belgian Draught Horses. The equine ELN reference of a Thoroughbred mare was analyzed in silico. ELN regions in the Belgian Draught Horse that included polymorphisms were confirmed in a lymphedema negative breed (Vlaams Paard). If Vlaams Paard polymorphisms were different from those in the Belgian Draught Horse, they were confirmed in Thoroughbred Horses. Formerly unknown regions in the ELN reference were annotated de novo. We confirmed polymorphisms previously reported and described new ones. No polymorphisms were unique in the Belgian Draught Horses. However, completeness of the equine ELN reference can be disputed, based on the high similarity between the genomic region 3 of the equine reference ELN and human reference cDNA. Furthermore, exploratory sequencing of a genomic fragment 3 of ELN revealed high similarity (81%identical nucleotides) to human ELN exon 31, suggesting a longer equine ELN coding region. Although this study does not support ELN as a candidate for CPL, future perspectives for equine cDNA characterization and new possibilities in the application of ELN as a candidate in elastin related pathologies are offered.In conclusion, CPL in the Belgian Draught Horse is a multifactorial disorder. Horses are born clinically healthy with a genetic load and pass through a subclinical stage. Clinical disease onset can occur early in life, already from 1 year of age, CPL signs afterwards progressively deteriorate. Clinical diagnosis should ideally be supported with a laboratory diagnostic aid that also enables early detection. This however does not exist. Clinical scores are influenced by permanent environmental effect as well as date of examination. Histology showed marked dermal elastin aberrance in susceptible breeds and molecular analysis could not refute ELN as a candidate for CPL, mainly due to uncertainty on the horse ELN reference sequence. Therefore, future perspectives reside in ELN cDNA characterization for the assessment of elastin related disorders in horses. In the absence of molecular markers, and, due to the moderate heritability coefficients for clinical scores, selection against CPL in the Belgian Draught Horse should ideally be based on estimated breeding values. The necessary data are clinical scores, obtained by a scoring system from repetitive clinical examinations of the fore limbs.SUMMARY i SAMENVATTING iv LIST OF ABBREVIATIONS vii LIST OF PUBLICATIONS ix GENERAL INTRODUCTION AND OBJECTIVES 1 1 STATE-OF-THE-ART 9 1.1 Chronic progressive lymphedema in draught horses 10 1.1.1 History and terminology 10 1.1.2 The lymph system: basic principles 13 1.1.3 Pathogenesis 14 1.1.4 Clinical symptoms 19 1.1.5 Diagnosis 20 1.1.6 Treatment 24 1.1.7 Factors associated with lymphedema occurrence and severity 26 1.2 Quantitative genetic analysis of CPL susceptibility 27 1.2.1 Introduction 27 1.2.2 Heritability of CPL 35 1.3 Molecular genetic research of chronic progressive lymphedema 38 1.3.1 Introduction 38 1.3.2 Molecular genetic research in man 39 1.3.3 Molecular genetic research in draught horses 63 1.3.4 The elastin gene 65 2 CHRONIC PROGRESSIVE LYMPHEDEMA IN BELGIAN DRAUGHT HORSES IN BELGIUM: CLINICAL PHENOTYPING, PREVALENCE AND RISK FACTOR ANALYSIS 78 2.1. Abstract 81 2.2. Introduction 82 2.3. Materials and methods 83 2.3.1. Clinical phenotyping 83 2.3.2. Traits possibly associated to clinical scores 84 2.3.3. Population 85 2.3.4. Statistical analysis 86 2.4. Results 87 2.4.1. Clinical phenotyping 87 2.4.2. Population 88 2.4.3. Descriptive statistics 88 2.4.4. CPL prevalence and severity 89 2.4.5. Univariate linear regression analysis (Table 2.4) 90 2.4.6. Multivariable linear regression (Table 2.5.) 92 2.5. Discussion 94 2.6. Conclusion 95 3 GENETIC PARAMETERS FOR CHRONIC PROGRESSIVE LYMPHEDEMA IN BELGIAN DRAUGHT HORSES 97 3.1. Abstract 98 3.2. Introduction 99 3.3. Materials and methods 100 3.3.1. Traits and exploratory analysis 100 3.3.2. Animals and datasets 100 3.3.3. Genetic models 101 3.4. Results 102 3.4.1. Descriptive statistics and exploratory analysis 102 3.4.2. Estimated heritabilities and genetic correlations 102 3.4.3. Relationship of clinical severity between fore and hind limbs 103 3.5. Discussion 104 3.6. Conclusion 106 4 ASSESMENT OF ANTI-ELASTIN ANTIBODIES FOR USE AS A DIAGNOSTIC AID FOR CHRONIC PROGRESSIVE LYMPHEDEMA IN BELGIAN DRAUGHT HORSES 107 4.1. Abstract 108 4.2. Introduction 109 4.3. Materials and methods 110 4.3.1. Study sample 110 4.3.2. ELISA 111 4.3.3. Statistical methods 111 4.4. Results 112 4.4.1. Reproducibility 112 4.4.2. Descriptive statistics 113 4.4.3. Full model analysis and influence of gender and pregnancy 114 4.4.4. Diagnostic accuracy 115 4.5. Discussion 116 4.6. Conclusion 117 5 THE ELASTIN GENE IN CHRONIC PROGRESSIVE LYMPHEDEMA AFFECTED BELGIAN DRAUGHT HORSES AS A PARAMETER FOR ELASTIN DEPENDENT DISORDERS 118 5.1. Abstract 119 5.2. Introduction 120 5.3. Materials and methods 121 5.3.1. Animals 121 5.3.2. In silico analysis 121 5.3.3. Experimental procedures and sequence analysis 122 5.4. Results 127 5.4.1. Animals and phenotyping 127 5.4.2. In silico analysis 127 5.4.3. Sequence analysis 128 5.5. Discussion 132 5.6. Conclusion 134 6 GENERAL DISCUSSION 135 6.1. General discussion 136 6.1.1. Introduction 136 6.1.2. Establishing a clear phenotype 138 6.1.3. Genetic approaches 143 6.2. Conclusions and future perspectives 149 7. REFERENCES 151Acknowledgements: Studbook of the Belgian Draught Horse (KMBT, Brussels, Belgium); Ghent University, Faculty of Veterinary Medicine, Department of Surgery and anaesthesiology of domestic animals, Department of Pathology, bacteriology and poultry diseases and Department of Medical imaging of domestic animals; Algemeen Medisch Laboratorium (AML, Hoboken, Belgium)nrpages: 171status: publishe

Chronic progressive lymphoedema in draught horses

The objective of this review is to summarize and evaluate the current knowledge of chronic progressive lymphoedema (CPL) in draught horses. Clinical signs of this multifactorial disorder are mainly restricted to the lower limbs, comprising progressively deteriorating skin swelling and deformation. Although typical lesions were first reported in the beginning of the 20th century, CPL was only recognised as a specific syndrome in 2003, and since then research has driven forward. Despite the high prevalence in some breeds and the serious economic impact, the pathogenesis is not fully understood and the available treatment options remain symptomatic and non-curative. There is a need to improve diagnostic techniques and to develop selection tools.status: publishe

Assessment of the elastin gene in chronic progressive lymphedema affected Belgian Draught Horses

Several large draught horse breeds, including Belgian draught horses, are susceptible to chronic progressive lymphedema (CPL). External features of this incurable disorder arise secondary, as a result of poor lymphatic drainage in skin and subcutis. Clinical symptoms predominantly occur at the level of the lower limbs, including a progressive soft tissue swelling and fibrosis, associated with skin anomalies (scaling, dermatitis, ulcerations) and deformations (skinfolds and nodules). Initial symptoms are often missed in younger horses (from 1.5 years of age), however, with disease progression, deterioration of lesions and increased disability frequently justify early euthanasia in older horses (from 6 years of age). With an approximate disease prevalence of 70% in Belgian draught horses, urgent measures are needed. A genetic susceptibility for CPL is assumed, but so far, molecular genetic research in susceptible draught horse breeds has not identified exact causative factors. Unravelling a genetic component in CPL-pathology might lead to genetic selection towards a lower disease susceptibility and prevalence. It was shown that the extracellular matrix protein “elastin” supports lymph uptake and flow. In CPL-susceptible draught horses, quantitative and qualitative alterations of skin elastin have been associated with the severity of their clinical symptoms. Therefore, reduced efficiency of lymphatic support by elastin might explain CPL-susceptibility. Moreover, different genetically based skin disorders in man have been associated to mutations in ELN. We hypothesized that (a) mutation(s) in ELN is a causative factor for CPL-susceptibility in Belgian draught horses. Therefore, the sequence of all exons and 60 % of introns was determined in three clinically affected Belgian draught horses, by means of Sanger sequencing. The consensus sequence was compared to the equine reference sequence (www.ensembl.org, accession ENSECAG00000011106). Found polymorphisms were confirmed by sequence determination of the corresponding region in three horses of a not CPL-susceptible draught horse breed (Vlaams Paard). Found polymorphisms as well as their nature and location throughout ELN will be discussed.status: publishe

Expression of interleukin-4-receptor and chemokine CCL5 in PBMC is not related to insect bite hypersensitivity in horses

Insect bite hypersensitivity (IBH) in horses represents a type I or sometimes a type IV hypersensitivity to salivary antigens from numerous Culicoides spp and some other insects. Until now, there has been no curative treatment available, but there are clear indications that the susceptibility to IBH is partly heritable. Identification of equine genes that are associated with susceptibility to IBH could lead to the development of a marker assisted selection method. The aim of this study was to investigate the expression of functional candidate genes in relation to IBH. Based on literature, the interleukin-4 receptor (IL4R) and chemokine CCL5 (CCL5) genes were selected for examination by quantitative reverse transcription-PCR (qRT-PCR). Expression levels were determined in 16 horses: 8 IBH-positive horses, all showing clinical IBH symptoms at sampling; and 8 IBH-negative horses, stabled at the same location (case-control set up). Peripheral blood mononuclear cells (PBMC) were isolated from whole- blood samples, and expression levels of IL4R and CCL5 were measured. No differential expression was found for IL4R and CCL5 in PBMC between IBH-positive and IBH-negative horses (P =.58 and P = .63). Expression of CCL5 and IL4R in PBMC is not related to insect bite hypersensitivity in warmblood horses. Research toward a marker-assisted selection procedures could reduce IBH prevalence in horse populations by means of selection.status: publishe

Genetic parameters for chronic progressive lymphedema in the Belgian Draught Horse

Belgian draught horses (BDH) are susceptible to chronic progressive lymphedema (CPL). The main cause is a failure of the lymphatic system of skin and subcutis, resulting in excessive protein rich interstitial fluid (lymphedema). Symptoms are mainly restricted to skin anomalies and deformations of the lower limbs. Initial signs (from 1.5 yr) are often missed in younger horses, however, with disease progression, increased disability frequently justifies early euthanasia in older horses (from 6 yr). As a genetic background is suggested and disease prevalence in BDH approaches 70%, urgent measures are needed. The aim of this study was to estimate genetic parameters for CPL in a BDH population. Analyses were performed on two datasets, comprising clinical examinations (CPL-scores), collected at official horse contests and stable visits throughout Belgium. The first dataset comprised all collected data, whereas in the second data set, scores for horses younger than 3 years were omitted. Age (years), coat color and sex were included as fixed effects, whereas date of recording was a random effect. Variance component estimates were obtained using the REML method (VCE6) by means of an animal model. Heritability coefficients (standard error, SE) for CPL ranged from 0.25 (0.07) to 0.46 (0.09). Phenotypic and genetic correlations (SE) for CPL-scores between fore -and hind limbs were high and respectively counted 0.85 and 0.96 (0.02). Date of recording was a factor of importance, where the proportion of variance (SE) ranged from 0.16 (0.05) to 0.22 (0.04). To improve reliability of estimated breeding values, continued collection of CPL-scores is advised. This study indicates the potential to reduce chronic progressive lymphedema occurrence in BDH through selection.status: publishe

Early detection of chronic progressive lymphedema susceptibility in Belgian draught horse stallions by means of ELISA

Belgian draught horses are susceptible to chronic progressive lymphoedema (CPL), a disorder with genetic predisposition. Breeding decisions require early diagnosis. Healthy individuals have minor elastin (ELN) turnover, eliciting only basic antibody formation (anti-ELN antibodies/AEAb’s). In CPL-affected draught horses, AEAb levels have been shown to correlate with clinical severity. It was hypothesized that AEAb levels in young horses, with no/minor clinical signs, indicate CPL susceptibility. Therefore, plasma samples, distal limb radiographs and clinical scores were obtained from stallions (< 36 months), and these data were linked to disease progression at least 2 years later. If initial AEAb levels correlate with the later disease status, this test could allow for early identification.status: publishe

Genetic parameters for chronic progressive lymphedema in Belgian Draught Horses

Genetic parameters for chronic progressive lymphedema (CPL)-associated traits in Belgian Draught Horses were estimated, using a multitrait animal model. Clinical scores of CPL in the four limbs/horse (CPLclin), skinfold thickness and hair samples (hair diameter) were studied. Due to CPLclin uncertainty in younger horses (progressive CPL character), a restricted data set (D_3+) was formed, excluding records from horses under 3 years from the complete data set (D_full). Age, gender, coat colour and limb hair pigmentation were included as fixed, permanent environment and date of recording as random effects. Higher CPLclin certainty (D_3+) increased heritability coefficients of, and genetic correlations between traits, with CPLclin heritabilities (SE) for the respective data sets: 0.11 (0.06) and 0.26 (0.05). A large proportion of the CPLclin variance was attributed to the permanent environmental effect in D_full, but less in D_3+. Date of recording explained a proportion of variance from 0.09 +/- 0.03 to 0.61 +/- 0.08. Additive genetic correlations between CPLclin and both skinfold thickness and hair diameter showed the latter two traits cannot be used as a direct diagnostic aid for CPL. Due to the relatively low heritability of CPLclin, selection should focus on estimated breeding values (from repeated clinical examinations) to reduce CPL occurrence in the Belgian Draught Horse

Foamy virus–mediated gene transfer to canine repopulating cells

Foamy virus (FV) vectors are particularly attractive gene-transfer vectors for stem-cell gene therapy because they form a stable transduction intermediate in quiescent cells and can efficiently transduce hematopoietic stem cells. Here, we studied the use of FV vectors to transduce long-term hematopoietic repopulating cells in the dog, a clinically relevant large animal model. Mobilized canine peripheral blood (PB) CD34(+) cells were transduced with an enhanced green fluorescent protein (EGFP)–expressing FV vector in an 18-hour transduction protocol. All 3 dogs studied had rapid neutrophil engraftment to greater than 500/μL with a median of 10 days. Transgene expression was detected in all cell lineages (B cells, T cells, granulocytes, red blood cells, and platelets), indicating multilineage engraftment of transduced cells. Up to 19% of blood cells were EGFP(+), and this was confirmed at the DNA level by real-time polymerase chain reaction (PCR) and Southern blot analysis. These transduction rates were higher than the best results we obtained previously with lentiviral vectors in a similar transduction protocol. Integration site analysis also demonstrated polyclonal repopulation and the transduction of multipotential hematopoietic repopulating cells. These data suggest that FV vectors should be useful for stem-cell gene therapy, particularly for applications in which short transduction protocols are critical