Differential expression of microRNAs in bovine papillomavirus type 1 transformed equine cells.

Bovine papillomavirus (BPV) types 1 and 2 play an important role in the pathogenesis of equine sarcoids (ES), the most common cutaneous tumour affecting horses. MicroRNAs (miRNAs), small non-coding RNAs that regulate essential biological and cellular processes, have been found dysregulated in a wide range of tumours. The aim of this study was to identify miRNAs associated with ES. Differential expression of miRNAs was assessed in control equine fibroblasts (EqPalFs) and EqPalFs transformed with the BPV-1 genome (S6-2 cells). Using a commercially available miRNA microarray, 492 mature miRNAs were interrogated. In total, 206 mature miRNAs were differentially expressed in EqPalFs compared with S6-2 cells. Aberrant expression of these miRNAs in S6-2 cells can be attributed to the presence of BPV-1 genomes. Furthermore, we confirm the presence of 124 miRNAs previously computationally predicted in the horse. Our data supports the involvement of miRNAs in the pathogenesis of ES.

Paediatric continuing medical education needs and preferences of UNRWA physicians in Jordan.

Most physicians who work in the United Nations Relief and Works Agency (UNRWA) infant and child health programme in Jordan are general practitioners with no postgraduate training in paediatrics. Furthermore, in resource-poor or remote settings, the ability to deliver live continuing medical education (CME) is often limited. A questionnaire exploring the resources available for accessing CME, preferences for types of CME, current sources of CME and topics of interest in the field of paediatric care was sent to all 92 physicians practising in UNRWA clinics in Jordan. Of the 89 respondents 80% had attended live medical lectures for CME and 70% CME meetings. Despite most physicians having access to the Internet only 52.8% were interested in Internet-based courses for accessing CME. There was a statistically significant relationship between year of graduation from medical school and preference for Internet-based CME. Implications for CME participation and paediatric CME topics are discussed.

Paediatric continuing medical education needs and preferences of UNRWA physicians in Jordan

إن معظم الأطباء الذين يعملون في برنامج صحة الرضع والأطفال التابع لوكالة الأمم المتحدة لغوث وتشغيل اللاجئن الأونروا في الأردن هم ممارسون عامون لم يدرَّبوا بعد التخرج عى طب الأطفال. وعاوة عى ذلك فإن القدرة على تقديم تعليم طبي مستمر مباشر - في الأماكن شحيحة الموارد أو في المناطق النائية - غالباً ما تكون محدودة. فقد أُرسل لجميع الأطباء ال 92 الذين يمارسون العمل في عيادات الأونروا في الأردن استبيان لاستقصاء الموارد المتاحة والمصادر الحالية للتعليم الطبي المستمر، واستكشاف ما يفضلونه من أناط التعليم الطبي المستمر، والوقوف عى الموضوعات ذات الاهتام في مجال الرعاية الطبية للأطفال. فذكر % 80 من المجيبن ال 89 أنهم حضروا محاضرات طبية مباشرة للتعليم الطبي المستمر، كما ذكر % 70 أنهم حضروا اجتماعات للتعليم الطبي المستمر. وعلى الرغم من أن معظم الأطباء تتوافر لديهم إمكانية الوصول إلى الإنترنت فإن % 52.8 منهم فقط كان مهتاً بدورات شبكة الإنترنت للحصول عى التعليم الطبي المستمر. وكانت هناك علاقة ذات دلالة إحصائية بين سنة التخرج من كلية الطب وبين تفضيل التعليم الطبي المستمر عن طريق شبكة الإنترنت. إن الآثار المترتبة عى المشاركة في التعليم الطبي المستمر ومواضيع التعليم الطبي المستمر في مجال طب الأطفال هي قيد المناقشة.

Most physicians who work in the United Nations Relief and Works Agency (UNRWA) infant and child health programme in Jordan are general practitioners with no postgraduate training in paediatrics. Furthermore, in resource-poor or remote settings, the ability to deliver live continuing medical education (CME) is often limited. A questionnaire exploring the resources available for accessing CME, preferences for types of CME, current sources of CME and topics of interest in the field of paediatric care was sent to all 92 physicians practising in UNRWA clinicsin Jordan. Of the 89 respondents 80% had attended live medical lectures for CME and 70% CME meetings. Despite most physicians having access to the Internet only 52.8% were interested in Internet-based courses for accessing CME. There was a statistically significant relationship between year of graduation from medical school and preference for Internet-based CME. Implications for CME participation and paediatric CME topics are discussed.

La plupart des médecins qui travaillent pour le programme de santé du nourisson et de l’enfant en Jordanie à l’Office de secours et de travaux des Nations Unis pour les réfugiés de Palestine dans le Proche-Orient (UNRWA) sont des médecins généralistes sans spécialisation en pédiatrie. Par ailleurs, dans un contexte de ressources limitées ou dans des zones isolées, la capacité à dispenser une formation médicale continue (FMC) est souvent limitée. Un questionnaire étudiant les ressources disponibles qui permettent d’accéder à la formation médicale continue, les types de formation préférés, les sources actuelles de formation médicale continue et les sujets d’intérêt dans le domaine des soins pédiatriques a été envoyé à l’ensemble des 92 médecins exerçant dans des cliniques de l’UNRWA en Jordanie. Sur un total de 89 répondants, 80 % avaient assisté en personne à des conférences médicales dans le cadre de la formation médicale continue et 70 % à des réunions de formation médicale continue. Si la plupart des médecins avaient accès à l'Internet, seuls 52,8 % étaient intéressés par des cours en ligne permettant d’accéder à la formation médicale continue. Il existait une relation statistiquement significative entre l’année de fin d’études en faculté de médecine et la préférencepour une formation médicale continue sur l'Internet. Les implications pour une participation à la formation médicale continue et les sujets de formation médicale continue en pédiatrie sont en cours de discussion.

The human and canine TERT promoters function equivalently in human and canine cells.

Telomerase targeted cancer gene therapy is being exploited for treatment of human cancer. The high incidence and many comparative aspects of human and canine cancer and the compliance and dedication of dog owners to treat cancer makes the canine pet population a good clinical model for investigating and developing new cancer therapeutics. Here, we report that the human telomerase promoter operates in canine cells, suggesting that human telomerase promoter-driven cancer therapy can be used to treat cancer in canines. Therefore, the canine pet population can act as a clinical model for new drug development based on telomerase therapeutics.

Metabolic syndrome in patients with hypertension attending a family practice clinic in Jordan.

Metabolic syndrome is being reported more frequently in the Eastern Mediterranean region. Patients with hypertension attending family practice clinics in the University of Jordan Hospital between February and July 2006 were assessed for the frequency of metabolic syndrome and its individual components. Of 345 patients studied, 65% had metabolic syndrome. Females were more likely to meet Adult Treatment Panel-III criteria for the diagnosis. Diabetes mellitus was the most frequent component of metabolic syndrome in males, while low serum high-density lipoprotein cholesterol and high waist circumference ranked first and second in females. Primary care providers should be alert to the importance of screening patients with hypertension for metabolic syndrome to prevent and manage these combined conditions.

Bovine papillomavirus type 1 E2 and E7 proteins down-regulate Toll Like Receptor 4 (TLR4) expression in equine fibroblasts.

BPV-1 and less commonly BPV-2 are associated with the pathogenesis of equine skin tumours termed sarcoids. We recently documented the transcriptional changes that are induced by BPV-1 in equine fibroblasts using microarray analyses. TLR4 expression was found to be significantly down-regulated by BPV-1. In the present study, we show that TLR4 expression is significantly decreased following the exogenous expression of BPV-1 E2 and E7 in primary equine fibroblasts. The results were confirmed by the demonstration of increased TLR4 expression following siRNA suppression of BPV-1 E2 and E7 viral gene expression. These data imply that BPV-1 is able to subvert the innate immune response by downregulation of TLR4.

Metabolic syndrome in patients with hypertension attending a family practice clinic in Jordan

Metabolic syndrome is being reported more frequently in the Eastern Mediterranean region. Patients with hypertension attending family practice clinics in the University of Jordan Hospital between February and July 2006 were assessed for the frequency of metabolic syndrome and its individual components. Of 345 patients studied, 65% had metabolic syndrome. Females were more likely to meet Adult Treatment Panel-III criteria for the diagnosis. Diabetes mellitus was the most frequent component of metabolic syndrome in males, while low serum high-density lipoprotein cholesterol and high waist circumference ranked first and second in females. Primary care providers should be alert to the importance of screening patients with hypertension for metabolic syndrome to prevent and manage these combined conditions

Oncolytic gene therapy for canine cancers: teaching old dog viruses new tricks.

The use of viruses to treat cancer has been studied for decades. With the advancement of molecular biology, viruses have been modified and genetically engineered to optimize their ability to target cancer cells. Canine viruses, such as distemper virus and adenovirus, are being exploited for the treatment of canine cancer as the dog has proven to be a good comparative model for human cancer research and proof of concept investigations. In this review, we introduce the concept of oncolytic viruses and describe some of the preliminary attempts to use oncolytic viruses for the treatment of canine cancer.

Transcriptional changes induced by bovine papillomavirus type 1 in equine fibroblasts.

Bovine papillomavirus type 1 (BPV-1) and, less commonly, BPV-2 are associated with the pathogenesis of common equine skin tumors termed sarcoids. In an attempt to understand the mechanisms by which BPV-1 induces sarcoids, we used gene expression profiling as a screening tool to identify candidate genes implicated in disease pathogenesis. Gene expression profiles of equine fibroblasts transformed by BPV-1 experimentally or from explanted tumors were compared with those of control equine fibroblasts to identify genes associated with expression of BPV-1. Analysis of the microarray data identified 81 probe sets that were significantly (P < 0.01) differentially expressed between the BPV-1-transformed and control cell lines. Expression of several deregulated genes, including MMP-1, CXCL5, FRA-1, NKG7, TLR4, and the gene encoding the major histocompatibility complex class I (MHC-I) protein, was confirmed using other BPV-1-transformed cell lines. Furthermore, expression of these genes was examined using a panel of 10 sarcoids. Increased expression of MMP-1, CXCL5, FRA-1, and NKG7 was detected in a subset of tumors, and TLR4 and MHC I showed robust down-regulation in all tumors. Deregulated expression was confirmed at the protein level for MMP-1 and MHC-I. The present report identifies genes modulated by BPV-1 transformation and will help identify the molecular mechanisms involved in disease pathogenesis.

Establishment and characterization of equine fibroblast cell lines transformed in vivo and in vitro by BPV-1: model systems for equine sarcoids.

It is now widely recognized that BPV-1 and less commonly BPV-2 are the causative agents of equine sarcoids. Here we present the generation of equine cell lines harboring BPV-1 genomes and expressing viral genes. These lines have been either explanted from sarcoid biopsies or generated in vitro by transfection of primary fibroblasts with BPV-1 DNA. Previously detected BPV-1 genome variations in equine sarcoids are also found in sarcoid cell lines, and only variant BPV-1 genomes can transform equine cells. These equine cell lines are morphologically transformed, proliferate faster than parental cells, have an extended life span and can grow independently of substrate. These characteristics are more marked the higher the level of viral E5, E6 and E7 gene expression. These findings confirm that the virus has an active role in the induction of sarcoids and the lines will be invaluable for further studies on the role of BPV-1 in sarcoid pathology.

Vaccination of sarcoid-bearing donkeys with chimeric virus-like particles of bovine papillomavirus type 1.

Equine sarcoids are fibroblastic skin tumours affecting equids worldwide. While the pathogenesis is not entirely understood, infection with bovine papillomavirus (BPV) type 1 (and less commonly type 2) has been implicated as a major factor in the disease process. Sarcoids very seldom regress and in fact often recrudesce following therapy. Nothing is known about the immune response of the equine host to BPV. Given that the viral genes are expressed in sarcoids, it is reasonable to assume that vaccination of animals against the expressed viral proteins would lead to the induction of an immune response against the antigens and possible tumour rejection. To this end we vaccinated sarcoid-bearing donkeys in a placebo-controlled trial using chimeric virus-like particles (CVLPs) comprising BPV-1 L1 and E7 proteins. The results show a tendency towards enhanced tumour regression and reduced progression in the vaccinated group compared to control animals. Although promising, further studies are required with larger animal groups to definitely conclude that vaccination with CVLPs is a potential therapy for the induction of sarcoid regression.

Preliminary studies of particle-mediated gene delivery to the joints of dogs.

This paper describes a preliminary evaluation of particle-mediated bombardment via the Helios gene gun for the delivery of therapeutic genes to synovial cells in culture. A reporter gene, enhanced green fluorescent protein, was delivered to rabbit synovial fibroblasts (HIG-82) using gold particle (1.0 microm) bombardment to evaluate transfection efficiency at helium pressures of 100 and 150 psi. Transfection of cells occurred at these pressures despite some cell death. The in vitro delivery of gold particles to samples of synovial membrane and articular cartilage from a freshly euthanased dog was also studied to examine depth of penetration of gold particles (1.0 microm) at helium pressures of 250 and 500 psi. Light microscopical examination of histological sections of the synovial membrane showed that particles of gold had penetrated the lining cells of the synovium. However, no gold particles had penetrated the articular cartilage even at 500 psi.

Identification and functional analysis of sequence variants in the long control region and the E2 open reading frame of bovine papillomavirus type 1 isolated from equine sarcoids.

BPV-1 DNA is the predominant viral type detected in equine sarcoids and represents the only reported natural cross species infection of papillomaviruses. In this study, nucleotide variations in the LCR and the E2 regions of equine sarcoid-associated BPV-1 were characterised by sequence analysis. Variants particular to sarcoid BPV-1 were identified in both the LCR and E2 sequence. The functionality of the most common LCR variant was examined in equine and bovine cells. These studies showed that the activity of the variant LCR was higher in equine cells than bovine cells; the activity of the variant LCR in the presence of the E2 variant was similar to the reference/wild-type sequences in equine cells, whereas in bovine cells the variant function was reduced by 50%. These data suggest the viral BPV variants commonly detected in sarcoids have an enhanced function in equine cells compared to their function in bovine cells.

Inhibition of telomerase in canine cancer cells following telomestatin treatment.

Telomere shortening in normal somatic cells has been proposed as a major barrier to unlimited cellular proliferation. Telomerase is an enzyme capable of maintaining telomere length, and thus bypassing this barrier. In human beings, telomerase activity is restricted to cancer cells and cells of stem or germ cell lineages. Dogs represent a potentially useful clinical model for the development of telomerase-based therapies because telomerase activity is also restricted to cancer cells and stem cells in this species. We examined the ability of telomestatin to inhibit telomerase activity in telomerase-positive D17 and CMT7 canine cancer cell lines. At a concentration of 2 microM, telomestatin treatment resulted in a decrease in telomerase activity, telomere shortening, growth inhibition and apoptosis in telomerase-positive cancer cells. These effects were not seen in telomerase-negative skin fibroblasts or negative controls. These results confirm that telomestatin specifically inhibits telomerase activity in canine cancer cells and strengthens the usefulness of dogs as a model for testing telomerase-based therapies.

Telomerase reverse transcriptase (TERT) expression and proliferation in canine brain tumours.

Telomerase is a ribonucleoprotein enzyme complex that synthesizes telomere DNA. It is detected in 85-90% of malignant tumours in humans, but not in most somatic cells. Because telomerase plays a critical role in cell immortality, it represents an important target for anticancer therapies. We have previously shown that the dog is a potentially useful model for evaluating telomerase-based therapeutics. In this present study we analysed 93 canine brain tumours for telomerase reverse transcriptase (TERT) expression by immunohistochemistry. TERT immunoreactivity was detected in 16 of 50 grade 1 (32%) and 29 of 43 grade 2 tumours (67.4%), demonstrating a statistically significant association with histological grade (P = 0.00012). A subset of 51 tumours was also assessed for MIB-1 expression. The MIB-1 labelling index (LI) was found to correlate significantly with tumour grade, with a mean MIB-1 LI of 1.5% for grade 1 tumours, as compared with a mean MIB-1 LI of 21.7% for grade 2 tumours (P << 0.001). The MIB-1 LI was also significantly associated with TERT expression in all brain tumours (P << 0.001). These data further support the dog as a model for the preclinical development of telomerase-based therapeutics in brain tumours.

Telomerase activity and telomerase reverse transcriptase catalytic subunit expression in canine lymphoma: correlation with Ki67 immunoreactivity.

Increased telomerase activity (TA) has been found in human and canine solid tumours, stem cells and somatic tissues with enhanced proliferative potential. The relationship between TA in normal and malignant lymphoid tissues remains unclear. The TA and the expression of canine telomerase reverse transcriptase catalytic subunit (dogTERT) messenger RNA (mRNA) were analyzed in malignant lymph nodes from 30 dogs with lymphoma, from two dogs with non-neoplastic illness and from two clinically normal dogs, demonstrating a statistically significant difference between TA in lymphoma lymph nodes (n = 30) and normal nodes (n = 4) but no significant difference in dogTERT mRNA expression. In addition, the expression of telomerase reverse transcriptase catalytic subunit (TERT) protein and Ki67 was analyzed in malignant lymph nodes from 10 dogs with lymphoma and from two clinically normal dogs by immunohistochemistry. TERT expression was associated with Ki67 in all lymphoma nodes (n = 10), and differences were illustrated between TERT and Ki67 expression between lymphoma (n = 10) and non-lymphoma (n = 2) nodes. This data support further investigation of telomerase in canine haematopoietic neoplasia through large-scale prospective studies.

The canine telomerase catalytic subunit (dogTERT): characterisation of the gene promoter and identification of proximal core sequences necessary for specific transcriptional activity in canine telomerase positive cell lines.

Telomerase biology is complicated by studies that show that telomere expression and telomere biology differs between species, and that existing animal models do not closely resemble the human situation. We have previously reported a description of telomere/telomerase biology in the dog and have suggested this as an alternative model system. To further elucidate telomerase biology in this species we have cloned and characterised the canine reverse transcriptase (dogTERT) promoter. We demonstrate that core promoter activity is contained within a region extending approximately 300 bp upstream of the ATG codon. Transient transfections in telomerase-positive canine cell lines and telomerase negative fibroblasts showed that the promoter is only active in telomerase positive cell lines. Sequence analysis demonstrated that the 5' regulatory region is GC-rich and contains no TATA or CAAT box, similar to the human TERT promoter. Motif searches revealed the presence of multiple transcription factor binding sites common to both the human and canine TERT promoters, including a single E-box, Sp1, AP1, MZF-2 and ER/Sp1 binding sites. These findings suggest that the dogTERT gene shares similar transcriptional control to hTERT. Identification of the core promoter necessary for activity may allow the use of naturally occurring cancers in dogs as a preclinical testing ground for telomerase targeted therapies in human cancer patients.