A Targeted Survey for Scrapie in Jos Plateau State, Nigeria

Scrapie, a disease of sheep and goats with a progressive course and fatal outcome, has not been identified in Nigeria. Anecdotal scrapie reports by livestock workers abound. Livestock diseases like scrapie form huddles in livestock economics of countries. For 8 months we surveyed for scrapie targeting emergency/casualty slaughter sheep and goats in Jos, Nigeria. We clinically examined 510 sheep and 608 goats of local breeds, aged from 12 months to 5 years. In total 31 (5.10%) goats and no sheep were clinically suspicious for scrapie. Caudal brainstem tissues of suspect animals collected postmortem were analyzed for the disease specific form of the prion protein, PrPSc, using Bio-Rad’s TeSeE ELISA rapid test kit. No sample was positive for scrapie. Fluorescent antibody test for rabies and H&E staining on samples were carried out for differential diagnosis. These showed no pathological lesions indicative for neurological disease. While our findings do not exclude the presence of scrapie in Jos, we demonstrate that targeted sampling of small ruminants for neuroinfectious disease is feasible in developing countries, pointing to the possibility of implementing such a monitoring scheme in Nigeria to prevent economic losses in small ruminant livestock as scrapie caveats from endemic countries have shown.

Analysis of protein expression in zebrafish during gonad differentiation by targeted proteomics

The molecular mechanisms governing sex determination and differentiation in the zebrafish (Danio rerio) are not fully understood. To gain more insights into the function of specific genes in these complex processes, the expression of multiple candidates needs to be assessed, preferably on the protein level. Here, we developed a targeted proteomics method based on selected reaction monitoring (SRM) to study the candidate sex-related proteins in zebrafish which were selected based on a global proteomics analysis of adult gonads and representational difference analysis of male and female DNA, as well as on published information on zebrafish and other vertebrates. We employed the developed SRM protocols to acquire time-resolved protein expression profiles during the gonad differentiation period in vas::EGFP transgenic zebrafish. Evidence on protein expression was obtained for the first time for several candidate genes previously studied only on the mRNA level or suggested by bioinformatic predictions. Tuba1b (tubulin alpha 1b), initially included in the study as one of the potential housekeeping proteins, was found to be preferentially expressed in the adult testis with nearly absent expression in the ovary. The revealed changes in protein expression patterns associated with gonad differentiation suggest that several of the examined proteins, especially Ilf2 and Ilf3 (interleukin enhancer-binding factors 2 and 3), Raldh3 (retinaldehyde dehydrogenase type 3), Zgc:195027 (low density lipoprotein-related receptor protein 3) and Sept5a (septin 5a), may play a specific role in the sexual differentiation in zebrafish.

Molecular cloning and characterization of NcROP2Fam-1, a member of the ROP2 family of rhoptry proteins in Neospora caninum that is targeted by antibodies neutralizing host cell invasion in vitro.

Recent publications demonstrated that a fragment of a Neospora caninum ROP2 family member antigen represents a promising vaccine candidate. We here report on the cloning of the cDNA encoding this protein, N. caninum ROP2 family member 1 (NcROP2Fam-1), its molecular characterization and localization. The protein possesses the hallmarks of ROP2 family members and is apparently devoid of catalytic activity. NcROP2Fam-1 is synthesized as a pre-pro-protein that is matured to 2 proteins of 49 and 55 kDa that localize to rhoptry bulbs. Upon invasion the protein is associated with the nascent parasitophorous vacuole membrane (PVM), evacuoles surrounding the host cell nucleus and, in some instances, the surface of intracellular parasites. Staining was also observed within the cyst wall of 'cysts' produced in vitro. Interestingly, NcROP2Fam-1 was also detected on the surface of extracellular parasites entering the host cells and antibodies directed against NcROP2Fam-1-specific peptides partially neutralized invasion in vitro. We conclude that, in spite of the general belief that ROP2 family proteins are intracellular antigens, NcROP2Fam-1 can also be considered as an extracellular antigen, a property that should be taken into account in further experiments employing ROP2 family proteins as vaccines.

Targeted radiotherapy of prostate cancer with a gastrin-releasing peptide receptor antagonist is effective as monotherapy and in combination with rapamycin

UNLABELLED The gastrin-releasing peptide receptor (GRPr) is overexpressed in prostate cancer and is an attractive target for radionuclide therapy. In addition, inhibition of the protein kinase mammalian target of rapamycin (mTOR) has been shown to sensitize various cancer cells to the effects of radiotherapy. METHODS To determine the effect of treatment with rapamycin and radiotherapy with a novel (177)Lu-labeled GRPr antagonist ((177)Lu-RM2, BAY 1017858) alone and in combination, in vitro and in vivo studies were performed using the human PC-3 prostate cancer cell line. PC-3 cell proliferation and (177)Lu-RM2 uptake after treatment with rapamycin were assessed in vitro. To determine the influence of rapamycin on (177)Lu-RM2 tumor uptake, in vivo small-animal PET studies with (68)Ga-RM2 were performed after treatment with rapamycin. To study the efficacy of (177)Lu-RM2 in vivo, mice with subcutaneous PC-3 tumors were treated with (177)Lu-RM2 alone or after pretreatment with rapamycin. RESULTS Stable expression of GRPr was maintained after rapamycin treatment with doses up to 4 mg/kg in vivo. Monotherapy with (177)Lu-RM2 at higher doses (72 and 144 MBq) was effective in inducing complete tumor remission in 60% of treated mice. Treatment with 37 MBq of (177)Lu-RM2 and rapamycin in combination led to significantly longer survival than with either agent alone. No treatment-related toxicity was observed. CONCLUSION Radiotherapy using a (177)Lu-labeled GRPr antagonist alone or in combination with rapamycin was efficacious in inhibiting in vivo tumor growth and may be a promising strategy for treatment of prostate cancer.

CHARACTERIZING AND TREATING THE NEUROPATHOLOGY OF TUBEROUS SCLEROSIS COMPLEX IN THE MOUSE

Tuberous sclerosis complex (TSC) is a multisystem, autosomal dominant disorder affecting approximately 1 in 6000 births. Developmental brain abnormalities cause substantial morbidity and mortality and often lead to neurological disease including epilepsy, cognitive disabilities, and autism. TSC is caused by inactivating mutations in either TSC1 or TSC2, whose protein products are known inhibitors of mTORC1, an important kinase regulating translation and cell growth. Nonetheless, neither the pathophysiology of the neurological manifestations of TSC nor the extent of mTORC1 involvement in the development of these lesions is known. Murine models would greatly advance the study of this debilitating disorder. This thesis will describe the generation and characterization of a novel brain-specific mouse model of TSC, Tsc2flox/ko;hGFAP-Cre. In this model, the Tsc2 gene has been removed from most neurons and glia of the cortex and hippocampus by targeted Cre-mediated deletion in radial glial neuroprogenitor cells. The Tsc2flox/ko;hGFAP-Cre mice fail to thrive beginning postnatal day 8 and die from seizures around 23 days. Further characterization of these mice demonstrated megalencephaly, enlarged neurons, abnormal neuronal migration, altered progenitor pools, hypomyelination, and an astrogliosis. The similarity of these defects to those of TSC patients establishes this mouse as an excellent model for the study of the neuropathology of TSC and testing novel therapies. We further describe the use of this mouse model to assess the therapeutic potential of the macrolide rapamycin, an inhibitor of mTORC1. We demonstrate that rapamycin administered from postnatal day 10 can extend the life of the mutant animals 5 fold. Since TSC is a neurodevelopmental disorder, we also assessed in utero and/or immediate postnatal treatment of the animals with rapamycin. Amazingly, combined in utero and postnatal rapamycin effected a histologic rescue that was almost indistinguishable from control animals, indicating that dysregulation of mTORC1 plays a large role in TSC neuropathology. In spite of the almost complete histologic rescue, behavioral studies demonstrated that combined treatment resulted in poorer learning and memory than postnatal treatment alone. Postnatally-treated animals behaved similarly to treated controls, suggesting that immediate human treatment in the newborn period might provide the most opportune developmental timepoint for rapamycin administration.

Investigating the Effects of Silencing EphA2 in Metastatic Breast Cancer Cells

EphA2, also known as ECK (epithelial cell kinase), is a transmembrane receptor tyrosine kinase that is commonly over-expressed in cancers such as those of the prostate, colon, lung, and breast. For breast cancers, EphA2 overexpression is most prominent in the ER-negative subtype, and is associated with a higher rate of lung metastasis. Studies conducted to demonstrate the role of EphA2 in a non-cancerous environment have shown that it is very important in developmental processes, but not in normal adult tissues. These results make EphA2 a prospective therapeutic target since new therapies are needed for the more aggressive ER-negative breast cancers. A panel of breast cancer cell lines was screened for expression of EphA2 by immunoblotting. Several of the overexpressing cell lines, including BT549, MDA-MB-231, and HCC 1954 were selected for experiments utilizing siRNA for transient knockdown and shRNA for stable knockdown. Targeted knockdown of EphA2 was measured using RT-PCR and immunoblotting techniques. Here, the functions of EphA2 in the process of metastasis have been elucidated using in vitro assays that indicate cancer cell metastatic potential and in vivo studies that reveal the effect of EphA2 on mammary fat pad tumor growth, vessel formation, and the effect of using EphA2-targeting siRNA on pre-established mammary fat pad tumors. A decrease in EphA2 expression both in vitro and in vivo correlated with reduced migration and experimental metastasis of breast cancer cells. Current work is being done to investigate the mechanism behind EphA2’s participation in some of these processes. These studies are important because they have contributed to understanding the role that EphA2 plays in the progression of breast cancers to a metastatic state.

Knowledge and Perception of the Role of Targeted Ultrasound in Detecting Down Syndrome Among a High Risk Population

The purpose of this study was to determine the perception and knowledge of targeted ultrasound in women who screen positive for Down syndrome in the first or second trimester, and to assess the perceived detection rate of Down syndrome by targeted ultrasound in this population. While several studies have reported patient perceptions’ of routine ultrasound, no study has specifically examined knowledge regarding the targeted ultrasound and its role in detecting Down syndrome. A targeted ultrasound is a special ultrasound during the second trimester offered to women who may be at a higher-than-average risk of having a baby with some type of birth defect or complication. The purpose of the ultrasound is to evaluate the overall growth and development of the baby as well as screen for birth defects and genetic conditions. Women under the age of 35 referred for an abnormal first or second trimester maternal serum screen to several Houston area clinics were asked to complete a questionnaire to obtain demographic and ultrasound knowledge information as well as assess perceived detection rate of Down syndrome by ultrasound. Seventy-seven women completed the questionnaire and participated in the study. Our findings revealed that women have limited background knowledge about the targeted ultrasound and its role in detecting Down syndrome. These findings are consistent with other studies that have reported a lack of understanding about the purpose of ultrasound examinations. One factor that seems to increase background knowledge about the targeted ultrasound is individuals having a higher level of education. However, most participants regardless of race, education, income, and exposure to targeted ultrasound information did not know the capabilities of a targeted ultrasound. This study confirmed women lack background knowledge about the targeted ultrasound and do not know enough about the technology to form a perception regarding its ability to detect Down syndrome. Additional studies to identify appropriate education techniques are necessary to determine how to best inform our patient population about targeted ultrasound.

Simulator Network project report: a tool for improvement of teaching materials and targeted resource usage in Skills Labs

During the last decade, medical education in the German-speaking world has been striving to become more practice-oriented. This is currently being achieved in many schools through the implementation of simulation-based instruction in Skills Labs. Simulators are thus an essential part of this type of medical training, and their acquisition and operation by a Skills Lab require a large outlay of resources. Therefore, the Practical Skills Committee of the Medical Education Society (GMA) introduced a new project, which aims to improve the flow of information between the Skills Labs and enable a transparent assessment of the simulators via an online database (the Simulator Network).

Activation of heat shock and antioxidant responses by the natural product celastrol: transcriptional signatures of a thiol-targeted molecule.

Stress response pathways allow cells to sense and respond to environmental changes and adverse pathophysiological states. Pharmacological modulation of cellular stress pathways has implications in the treatment of human diseases, including neurodegenerative disorders, cardiovascular disease, and cancer. The quinone methide triterpene celastrol, derived from a traditional Chinese medicinal herb, has numerous pharmacological properties, and it is a potent activator of the mammalian heat shock transcription factor HSF1. However, its mode of action and spectrum of cellular targets are poorly understood. We show here that celastrol activates Hsf1 in Saccharomyces cerevisiae at a similar effective concentration seen in mammalian cells. Transcriptional profiling revealed that celastrol treatment induces a battery of oxidant defense genes in addition to heat shock genes. Celastrol activated the yeast Yap1 oxidant defense transcription factor via the carboxy-terminal redox center that responds to electrophilic compounds. Antioxidant response genes were likewise induced in mammalian cells, demonstrating that the activation of two major cell stress pathways by celastrol is conserved. We report that celastrol's biological effects, including inhibition of glucocorticoid receptor activity, can be blocked by the addition of excess free thiol, suggesting a chemical mechanism for biological activity based on modification of key reactive thiols by this natural product.

Thoracic aortic disease in tuberous sclerosis complex: molecular pathogenesis and potential therapies in Tsc2+/- mice.

Tuberous sclerosis complex (TSC) is a genetic disorder with pleiotropic manifestations caused by heterozygous mutations in either TSC1 or TSC2. One of the less investigated complications of TSC is the formation of aneurysms of the descending aorta, which are characterized on pathologic examination by smooth muscle cell (SMC) proliferation in the aortic media. SMCs were explanted from Tsc2(+/-) mice to investigate the pathogenesis of aortic aneurysms caused by TSC2 mutations. Tsc2(+/-) SMCs demonstrated increased phosphorylation of mammalian target of rapamycin (mTOR), S6 and p70S6K and increased proliferation rates compared with wild-type (WT) SMCs. Tsc2(+/-) SMCs also had reduced expression of SMC contractile proteins compared with WT SMCs. An inhibitor of mTOR signaling, rapamycin, decreased SMC proliferation and increased contractile protein expression in the Tsc2(+/-) SMCs to levels similar to WT SMCs. Exposure to alpha-elastin fragments also decreased proliferation of Tsc2(+/-) SMCs and increased levels of p27(kip1), but failed to increase expression of contractile proteins. In response to artery injury using a carotid artery ligation model, Tsc2(+/-) mice significantly increased neointima formation compared with the control mice, and the neointima formation was inhibited by treatment with rapamycin. These results demonstrate that Tsc2 haploinsufficiency in SMCs increases proliferation and decreases contractile protein expression and suggest that the increased proliferative potential of the mutant cells may be suppressed in vivo by interaction with elastin. These findings provide insights into the molecular pathogenesis of aortic disease in TSC patients and identify a potential therapeutic target for treatment of this complication of the disease.

Progenitor cell therapies for traumatic brain injury: barriers and opportunities in translation.

Traumatic brain injury (TBI) directly affects nearly 1.5 million new patients per year in the USA, adding to the almost 6 million cases in patients who are permanently affected by the irreversible physical, cognitive and psychosocial deficits from a prior injury. Adult stem cell therapy has shown preliminary promise as an option for treatment, much of which is limited currently to supportive care. Preclinical research focused on cell therapy has grown significantly over the last decade. One of the challenges in the translation of this burgeoning field is interpretation of the promising experimental results obtained from a variety of cell types, injury models and techniques. Although these variables can become barriers to a collective understanding and to evidence-based translation, they provide crucial information that, when correctly placed, offers the opportunity for discovery. Here, we review the preclinical evidence that is currently guiding the translation of adult stem cell therapy for TBI.

Targeted Multistage Delivery of Nanoparticles to the Bone Marrow

Bone marrow is a target organ site involved in multiple diseases including myeloproliferative disorders and hematologic malignancies and metastases from breast and prostate. Most of these diseases are characterized with poor quality of life, and the treatment options are only palliative due to lack of delivery mechanisms for systemically injected drugs which results in dose limitation to protect the healthy hematopoietic cells. Therefore, there is a critical need to develop effective therapeutic strategies that allow for selective delivery of therapeutic payload to the bone marrow. Nanotechnology-based drug delivery systems provide the opportunity to deliver drugs to the target tissue while decreasing exposure to normal tissues. E-selectin is constitutively expressed on the bone marrow vasculature, but almost absent in normal vessels, and therefore, E-selectin targeted drug delivery presents an ideal strategy for the delivery of therapeutic nanoparticles to the bone marrow. The objective of this study was to develop a novel bone marrow targeted multistage vector (MSV) via E-selectin for delivery of therapeutics and imaging agents. To achieve this goal, Firstly, an E-selectin thioaptamer (ESTA) ligand was identified through a two-step screening from a combinatorial thioaptamer library. Next, ESTA-conjugated MSV (ESTA-MSV) were developed and evaluated for their stability and binding to E-selectin expressing endothelial cells. Different types of nanoparticles including liposomes, quantum dots, and iron oxide nanoparticles were loaded into the porous structure of ESTA-MSV. In vivo targeting experiments demonstrated 8-fold higher accumulation of ESTA-MSV in the mouse bone marrow as compared to non-targeted MSV Furthermore, intravenous injection of liposomes loaded ESTA-MSV resulted in a significantly higher accumulation of liposome in the bone marrow space as compared to injection of non-targeted MSV or liposomes alone. Overall this study provides first evidence that E-selectin targeted multistage vector preferentially targets to bone marrow vasculature and delivers larger amounts of nanoparticles. This delivery strategy holds potential for the selective delivery of large amounts of therapeutic payload to the vascular niches in the bone marrow for the treatment of bone marrow associated diseases.

NOVEL AMINO ACID TRANSPORTER-TARGETED RADIOTRACERS FOR BREAST CANCER IMAGING

Breast cancer is the most common malignancy among women in the world. Its 5-year survival rate ranges from 23.4% in patients with stage IV to 98% in stage I disease, highlighting the importance of early detection and diagnosis. 18F-2-Fluoro-2-deoxy-glucose (18F-FDG), using positron emission tomography (PET), is the most common functional imaging tool for breast cancer diagnosis currently. Unfortunately, 18F-FDG-PET has several limitations such as poorly differentiating tumor tissues from inflammatory and normal brain tissues. Therefore, 18F-labeled amino acid-based radiotracers have been reported as an alternative, which is based on the fact that tumor cells uptake and consume more amino acids to sustain their uncontrolled growth. Among those radiotracers, 18F-labeled tyrosine and its derivatives have shown high tumor uptake and great ability to differentiate tumor tissue from inflammatory sites in brain tumors and squamous cell carcinoma. They enter the tumor cells via L-type amino acid transporters (LAT), which were reported to be highly expressed in many cancer cell lines and correlate positively with tumor growth. Nevertheless, the low radiosynthesis yield and demand of an on-site cyclotron limit the use of 18F-labeled tyrosine analogues. In this study, four Technetium-99m (99mTc) labeled tyrosine/ AMT (α-methyl tyrosine)-based radiotracers were successfully synthesized and evaluated for their potentials in breast cancer imaging. In order to radiolabel tyrosine and AMT, the chelators N,N’-ethylene-di-L-cysteine (EC) and 1,4,8,11-tetra-azacyclotetradecane (N4 cyclam) were selected to coordinate 99mTc. These chelators have been reported to provide stable chelation ability with 99mTc. By using the chelator technology, the same target ligand could be labeled with different radioisotopes for various imaging modalities for tumor diagnosis, or for internal radionuclide therapy in future. Based on the in vitro and in vivo evaluation using the rat mammary tumor models, 99mTc-EC-AMT is considered as the most suitable radiotracer for breast cancer imaging overall, however, 99mTc-EC-Tyrosine will be more preferred for differential diagnosis of tumor from inflammation.

APPLICATIONS OF EPHB4 RECEPTOR SPECIFIC PEPTIDES IN TARGETED CANCER IMAGING AND THERAPY

EphB4 receptors, a member of the largest family of receptor tyrosine kinases, are found over-expressed in a variety of tumors cells including glioma cells as well as angiogenic blood vessels. Noninvasive imaging of EphB4 could potentially increase early detection rates, monitor response to therapy directed against EphB4, and improve patient outcomes. Targeted delivery of EphB4 receptor specific peptide conjugated hollow gold nanoshells (HAuNS) into tumors has great potential in cancer imaging and photothermal therapy. In this study, we developed an EphB4 specific peptide named TNYL-RAW and labeled with radioisotope 64Cu and Cy5.5 dye. We also conjugate this specific peptide with hollow gold nanoshells (HAuNS) to evaluate targeted photothermal therapy of cancers. In vitro, 64Cu-DOTA-TNYL- RAW specifically bind to CT26 and PC-3M cells but not to A549 cells. In vivo, Small-animal PET/CT clearly showed the significant uptake of 64Cu-DOTA-TNYL-RAW in CT26 and PC-3M tumors but not in A549 tumors. Furthermore, µPET/CT and near-infrared optical imaging clearly showed the uptake of the dual labeled TNYL-RAW peptide in both U251 and U87 tumors in the brains of nude mice. In U251 tumors, Cy5.5-labeled peptide can bind to EphB4-expressing tumor blood vessels and tumors cells. But in U87 models, dual labeled peptide only could bind to tumor associated blood vessels. Also, Irradiation of PC-3M and CT-26 cell treated with TNYL-PEG-HAuNS nanopatilces with near-infrared (NIR) laser resulted in selective destruction of these cells in vitro. EphB4 targeted TNYL-PEG-HAuNS showed more photothermal killing effect on CT26 tumor model than PEG-HAuNS did. In summary, tumors with overexpression of EphB4 receptors can be noninvasively visualized by micro PET/CT with 64Cu labeled or dual labeled TNYL-RAW peptide. Targeted delivery of TNYL-RAW conjugated HAuNS into tumors can greatly improve the treatment effect of photothermal therapy. The information acquired with this study should be advantageous in improving diagnostics and future applications in photothermal ablation therapy in clinical.