Safety and efficacy of oxandrolone in growth hormone-treated girls with Turner syndrome: evidence from recent studies and recommendations for use.

There has been no consensus regarding the efficacy and safety of oxandrolone (Ox) in addition to growth hormone (GH) in girls with Turner syndrome (TS), the optimal age of starting this treatment, or the optimal dose. This collaborative venture between Dutch, UK and US centers is intended to give a summary of the data from three recently published randomized, placebo-controlled, double-blind studies on the effects of Ox. The published papers from these studies were reviewed within the group of authors to reach consensus about the recommendations. The addition of Ox to GH treatment leads to an increase in adult height, on average 2.3­4.6 cm. If Ox dosages<0.06 mg/kg/day are used, side effects are modest. The most relevant safety concerns are virilization(including clitoromegaly and voice deepening) and a transient delay of breast development. We advise monitoring signs of virilization breast development and possibly blood lipids during Ox treatment, in addition to regular follow-up assessments for TS. In girls with TS who are severely short for age, in whom very short adult stature is anticipated,or in whom the growth rate is modest despite good compliance with GH, adjunctive treatment with Ox at a dosage of 0.03­0.05 mg/kg/day starting from the age of 8­10 years onward scan be considered.

Cholesteatoma has a high prevalence in Turner syndrome, highlighting the need for earlier diagnosis and the potential benefits of otoscopy training for paediatricians.

AIM: Girls with Turner syndrome are prone to cholesteatoma, a serious suppurative middle ear disease. We aimed to confirm its high prevalence in Turner syndrome, identify risk factors and suggest possible strategies for earlier detection. METHODS: We reviewed 179 girls with Turner syndrome between 1989 and 2012 to identify cases of cholesteatoma. RESULTS: Seven girls (3.9%) had cholesteatoma (index girls) and each was compared with three age-matched girls without cholesteatoma (comparison girls). All the index girls had either the 45,X or 45,X/46X,i(Xq) karyotypes. Nine ears were initially affected, with three recurrences in two girls. Median age at first cholesteatoma presentation was 11.9 years (range: 7.5-15.2), with otorrhoea for three (range: one to seven) months in all 12 affected ears. Index girls had a significantly higher proportion of previous recurrent acute (p = 0.007) and chronic otitis media (p = 0.008), chronic perforation (p = 0.038) aural polyps (p < 0.0001) and tympanic membrane retraction (p = 0.0001) than comparison girls. CONCLUSION: Cholesteatoma has a high prevalence in Turner syndrome. Risk factors include 45,X and 46,XiXq karyotypes; a history of chronic otitis media, tympanic membrane retraction and persistent otorrhoea; and older age. Earlier recognition of ear disease is needed and otoscopy training for paediatricians caring for Turner syndrome patients may be beneficial.

Increased incidence of Campylobacter jejuni-associated Guillain-Barré syndromes in the Greater Paris area.

The role of Campylobacter jejuni as the triggering agent of Guillain-Barré syndrome (GBS) has not been reassessed since the end of the 1990s in France. We report that the number of C. jejuni-related GBS cases increased continuously between 1996 and 2007 in the Paris region (mean annual increment: 7%, P = 0·007).

Longitudinal evaluation of viral interactions in treated HIV-hepatitis B co-infected patients with additional hepatitis C and D virus.

Virological interactions of hepatitis B (HBV), hepatitis C (HCV) and hepatitis D (HDV) viruses in HIV-infected patients have been poorly characterized especially under treatment influences. Undetection rates of hepatitis viruses were longitudinally analyzed in a 3-year cohort of 308 HIV-HBV co-infected patients and compared using Generalized Estimating Equation models adjusted for age, HIV-RNA, CD4 cell-count and antiviral treatment. Chronic hepatitis co-infection in HIV-infected patients (age years, SD) was: 265 HBV (40.7, 8.2); 19 HBV-HCV (39.7, 4.1); 12 HBV-HDV (35.2, 9.9); 12 HBV-HCV-HDV (39.2, 5.2). At inclusion, treatment with lamivudine/tenofovir was not significantly different between co-infection groups. HBV suppression was significantly associated with HDV (aOR = 3.85, 95%CI 1.13-13.10, P = 0.03) and HCV tri-infection (aOR = 2.65, 95%CI 1.03-6.81, P = 0.04), but marginally associated with HIV-HBV-HCV-HDV (aOR = 2.32, 95%CI 0.94-5.74, P = 0.07). In quad-infection, lower HDV-undetectability (vs HIV-HBV-HDV, P = 0.2) and higher HCV-undetectability (vs HIV-HBV-HCV, P = 0.1) were demonstrated. The degree of HBV suppression varied between visits and co-infection groups [range of aOR during follow-up (vs HIV-HBV co-infection): HIV-HBV-HCV = 2.23-5.67, HIV-HBV-HDV = 1.53-15.17]. In treated co-infected patients, HDV expressed continuous suppression over HCV- and HBV-replications. Peaks and rebounds from undetectable hepatitis B, C and/or D viremia warrant closer follow-up in this patient population. HDV-replication was uncontrolled even with antiviral treatment.

Bovine papillomavirus type 1 DNA and E5 oncoprotein expression in water buffalo fibropapillomas.

Papillomas and fibropapillomas may occur in the skin and in different organs in animals. Ten different genotypes of bovine papillomavirus (BPV) have been identified. BPV-1 through BPV-10 are all strictly species-specific, but BPV-1/2 may also infect other species such as equids, inducing fibroblastic tumors. BPV-1 and BPV-2 are associated with fibropapillomas in cattle; these tumors are formed by excessive proliferation of virus-infected dermal fibroblasts and epidermal keratinocytes. Nine water buffalo (Bubalus bubalis) were examined for the presence of multiple cutaneous and perivulvar tumors. Cutaneous and perivulvar fibropapillomatosis were confirmed histologically. Negative-stain transmission electron microscopic examination revealed papillomavirus-like particles in the fibropapillomas, and papillomaviral DNA was also detected by the polymerase chain reaction. The amplified long control region (LCR) DNA sequence was identical to that of BPV-1. The BPV-1 E5 oncoprotein was strongly expressed in the tumor cells thus confirming a causal role of the virus. This article represents the first report of cutaneous, perivulvar, and vulvar fibropapilloma associated with BPV-1 infection in the water buffalo and describes another example of cross-species infection by BPV-1.

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.

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.

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.

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.

Optimising management in Turner syndrome: from infancy to adult transfer.

Turner syndrome can be defined as loss or abnormality of the second X chromosome in at least one cell line in a phenotypic female. The condition occurs in approximately 1 in every 2000 live female births,(1) so that in the UK the prevalence for any year of life is in the region of 200 girls. The condition is much more common in utero, it being estimated that 1-2% of all conceptuses are affected, of whom only 1% will survive to term.

The in vitro effects of piroxicam and meloxicam on canine cell lines.

OBJECTIVES: To analyse the direct antiproliferative effects of both piroxicam and meloxicam at a variety of concentrations on a series of canine cancer cell lines and the mechanism of cell death. METHODS: The in vitro effects of piroxicam and meloxicam at various concentrations on canine cell cultures (Madin-Darby canine kidney cells, osteosarcoma, mammary carcinoma, and lymphoma) were assessed with respect to proliferation inhibition and apoptosis induction. Western blot analysis of cyclooxygenase-1 and cyclooxygenase-2 expression was performed on all cell lines. RESULTS: All cell lines used in this study were cyclooxygenase-1 and cyclooxygenase-2 positive apart from Madin-Darby canine kidney cells which were negative for both cyclooxygenase-1 and cyclooxygenase-2. Both meloxicam and piroxicam were able to inhibit proliferation in cell lines in a dose-dependent manner. However, the drug concentration required for a given effect was cell line dependent. CLINICAL SIGNIFICANCE: The results suggest that significant inhibition of proliferation and induction of apoptosis would only occur when drug concentrations were in excess of those that can be achieved in vivo following maximum recommended dose rates. It is possible, however, that local or topical treatment or altered dosing regimens may offer alternative approaches to the use of these drugs as antineoplastic agents.

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.

Disease- and cell-type-specific transcriptional targeting of vectors for osteoarthritis gene therapy: further development of a clinical canine model.

OBJECTIVES: The potential for undesirable systemic effects related to constitutive expression of certain therapeutic transgenes may be limited through the development of transcriptionally targeted disease- and cell-type-specific vectors. The objective of this study was to analyse the canine matrix metalloproteinase-9 (MMP-9) promoter and deletion constructs for its ability to drive expression in response to pro-inflammatory cytokines (interleukin-1beta and tumour necrosis factor-alpha). METHODS: Initial analysis of MMP-9 deletion constructs was made using a luciferase reporter system. The promoter was subsequently engineered to incorporate multiple NF-kappaB sites. In parallel experiments we used the mouse collagen type XI promoter to study cell-type-specific promoter activity in chondrocyte-specific cells (SW1353) and undifferentiated chondroprogenitor cells (ATDC5). RESULTS: Incorporation of multiple NF-kappaB sites into the MMP-9 promoter enhanced activity while maintaining disease specificity. Further, manipulation of the mouse collagen type XI (mColXI) promoter by the incorporation of SOX9 enhancer sites downstream of a reporter gene, increased gene activity while maintaining cell type specificity. CONCLUSIONS: Manipulation of promoter and enhancer regions can improve transcriptionally targeted genes. A combination of these systems, in the context of the canine model, has the potential to improve the safety of osteoarthritis gene therapy vectors.

Quantitative analysis of HCMV DNA load in whole blood of renal transplant patients using real-time PCR assay.

BACKGROUND: Preemptive antiviral treatment of Human Cytomegalovirus (HCMV) disease is a major goal in the management of organ transplant patients. It requires sensitive diagnostic methods. Automated real-time PCR systems have been recently proposed to monitor HCMV infection in such patients. OBJECTIVE: Objectives of this study was to compare a real-time quantitative PCR on whole blood with the HCMV pp65 antigenemia assay in renal transplant recipients, and also to evaluate two different DNA extraction methods. STUDY DESIGN: A total of 248 specimens from 21 patients were tested by quantitative pp65 antigenemia and quantitative real-time PCR. DNA was extracted from whole blood samples using two different methods: a conventional column manual assay and an automated system. RESULTS: Quantification of HCMV DNA using the two extraction methods showed highly similar results (Spearman rank test, r=0.863). We found a significant correlation between DNA quantification by real-time PCR in whole blood and pp65 antigenemia test (Spearman rank test, r=0.767). This correlation was not modified when the HCMV DNA results were normalized by quantification of the albumin cellular gene. In eight patients, HCMV infection was detected earlier with quantitative PCR than with the antigenemia test (mean delay of 11.25 days). HCMV DNA load equivalent of 50 pp65 positive cells/200,000 polymorphonuclear leukocytes (PMNLs) is log4.095 copies per ml of blood. CONCLUSIONS: Real-time PCR in whole blood is a sensitive method for estimating the HCMV genome load in renal transplant patients, and is more rapid and practicable than using PMNLs for pp65 antigenemia tests.

Feline epitheliotrophic T-cell lymphoma with paraneoplastic eosinophilia - immunochemotherapy with vinblastine and human recombinant interferon alpha(2b).

A cat with epitheliotrophic T-cell lymphoma with paraneoplastic eosinophilia is described. Initial attempts to control the disease with conventional therapies failed. The addition of recombinant human interferon alpha(2b) (rhINFalpha(2b)) resulted in a clinical, haematogenous and sonographic improvement for 49 days. The overall survival time from initial diagnosis was 100 days. Relapse was correlated with the development of serum antibodies directed against rhINFalpha(2b). To our knowledge, this is the first report describing the clinical use of IFNalpha in the treatment of neoplasia in the cat.

Evaluation of telomerase-targeted therapies in canine cancer cell lines.

Despite advances in conventional therapeutics, cancer remains an invariably fatal disease, the major challenge being to develop tumour-specific cancer treatment strategies. Current treatments such as chemotherapy and radiotherapy rely on a crude distinction between cancer cells and normal cells. However, with an increased understanding of the molecular events in the development of cancer, it is possible that far more innovative and targeted approaches can be developed. From studies on humans and dogs, the enzyme telomerase has emerged as a central unifying mechanism underlying the immortal phenotype of cancer and has thus become a candidate for differentiating between normal and cancer cells. The level and frequency of telomerase activity and component gene expression in cancers reinforces this as a potential target for cancer therapies. This article describes two approaches to target cancer by capitalizing on the expression of this enzyme. In the first approach, we target the enzyme itself, the goal being to cause cancer cell death. In the second approach, we utilize the respective gene promoters for telomerase component enzymes to drive expression of a reporter gene in cancer cell lines. The results demonstrated that targeted gene expression using promoter elements can be achieved specifically in telomerase-positive cell lines. However, targeting the enzyme itself proved less successful and warrants investigations into alternative approaches.