In JoVE (1)

Other Publications (11)

Articles by Gültekin Tamgüney in JoVE

Other articles by Gültekin Tamgüney on PubMed

Autocrine Stimulation of Rhadinovirus-transformed T Cells by the Chemokine CCL1/I-309

Oncogene. Nov, 2004  |  Pubmed ID: 15378023

The rhadinovirus herpesvirus saimiri transforms human T lymphocytes to stable growth in culture. Besides the viral oncogenes stpC and tip, little is understood about the transformation process at the cellular level. To identify cellular factors that might contribute to growth transformation, we compared cellular gene expression in pairs of herpesvirus saimiri-transformed and nontransformed human T-cell clones. Using cDNA arrays and suppressive subtractive hybridization, we were able to identify the chemokine CCL1/I-309 as one of the few cellular genes that are strongly overexpressed in T cells after growth transformation with herpesvirus saimiri. The transformed T cells expressed CCR8, the receptor for CCL1, which rapidly induced intracellular calcium ion levels. Neutralizing antibodies to CCL1 led to reduced secretion of interferon-gamma and tumor necrosis factor-alpha as well as to reduced proliferation rates in transformed T cells. Thus, we propose that growth transformation of human T cells with herpesvirus saimiri gives rise to an autocrine loop where the proliferation of transformed T cells is supported by the endogenous production of the chemokine CCL1.

Transmission of Elk and Deer Prions to Transgenic Mice

Journal of Virology. Sep, 2006  |  Pubmed ID: 16940522

Chronic wasting disease (CWD) is a fatal prion disease in deer and elk. Unique among the prion diseases, it is transmitted among captive and free-ranging animals. To facilitate studies of the biology of CWD prions, we generated five lines of transgenic (Tg) mice expressing prion protein (PrP) from Rocky Mountain elk (Cervus elaphus nelsoni), denoted Tg(ElkPrP), and two lines of Tg mice expressing PrP common to white-tailed deer (Odocoileus virginianus) and mule deer (Odocoileus hemionus), denoted Tg(DePrP). None of the Tg(ElkPrP) or Tg(DePrP) mice exhibited spontaneous neurologic dysfunction at more than 600 days of age. Brain samples from CWD-positive elk, white-tailed deer, and mule deer produced disease in Tg(ElkPrP) mice between 180 and 200 days after inoculation and in Tg(DePrP) mice between 300 and 400 days. One of eight cervid brain inocula transmitted disease to Tg(MoPrP)4053 mice overexpressing wild-type mouse PrP-A in approximately 540 days. Neuropathologic analysis revealed abundant PrP amyloid plaques in the brains of ill mice. Brain homogenates from symptomatic Tg(ElkPrP) mice produced disease in 120 to 190 days in Tg(ElkPrP) mice. In contrast to the Tg(ElkPrP) and Tg(DePrP) mice, Tg mice overexpressing human, bovine, or ovine PrP did not develop prion disease after inoculation with CWD prions from among nine different isolates after >500 days. These findings suggest that CWD prions from elk, mule deer, and white-tailed deer can be readily transmitted among these three cervid species.

Transmission and Detection of Prions in Feces

The Journal of Infectious Diseases. Jul, 2008  |  Pubmed ID: 18505383

In chronic wasting disease (CWD) in cervids and in scrapie in sheep, prions appear to be transmitted horizontally. Oral exposure to prion-tainted blood, urine, saliva, and feces has been suggested as the mode of transmission of CWD and scrapie among herbivores susceptible to these prion diseases. To explore the transmission of prions through feces, uninoculated Syrian hamsters (SHas) were cohabitated with or exposed to the bedding of SHas orally infected with Sc237 prions. Incubation times of 140 days and a rate of prion infection of 80%-100% among exposed animals suggested transmission by feces, probably via coprophagy. We measured the disease-causing isoform of the prion protein (PrP(Sc)) in feces by use of the conformation-dependent immunoassay, and we titrated the irradiated feces intracerebrally in transgenic mice that overexpressed SHa prion protein (SHaPrP). Fecal samples collected from infected SHas in the first 7 days after oral challenge harbored 60 ng/g PrP(Sc) and prion titers of approximately 10(6.6) ID(50)/g. Excretion of infectious prions continued at lower levels throughout the asymptomatic phase of the incubation period, most likely by the shedding of prions from infected Peyer patches. Our findings suggest that horizontal transmission of disease among herbivores may occur through the consumption of feces or foodstuff tainted with prions from feces of CWD-infected cervids and scrapie-infected sheep.

Genes Contributing to Prion Pathogenesis

The Journal of General Virology. Jul, 2008  |  Pubmed ID: 18559949

Prion diseases are caused by conversion of a normally folded, non-pathogenic isoform of the prion protein (PrP(C)) to a misfolded, pathogenic isoform (PrP(Sc)). Prion inoculation experiments in mice expressing homologous PrP(C) molecules on different genetic backgrounds displayed different incubation times, indicating that the conversion reaction may be influenced by other gene products. To identify genes that contribute to prion pathogenesis, we analysed incubation times of prions in mice in which the gene product was inactivated, knocked out or overexpressed. We tested 20 candidate genes, because their products either colocalize with PrP, are associated with Alzheimer's disease, are elevated during prion disease, or function in PrP-mediated signalling, PrP glycosylation, or protein maintenance. Whereas some of the candidates tested may have a role in the normal function of PrP(C), our data show that many genes previously implicated in prion replication have no discernible effect on the pathogenesis of prion disease. While most genes tested did not significantly affect survival times, ablation of the amyloid beta (A4) precursor protein (App) or interleukin-1 receptor, type I (Il1r1), and transgenic overexpression of human superoxide dismutase 1 (SOD1) prolonged incubation times by 13, 16 and 19 %, respectively.

Transmission of Scrapie and Sheep-passaged Bovine Spongiform Encephalopathy Prions to Transgenic Mice Expressing Elk Prion Protein

The Journal of General Virology. Apr, 2009  |  Pubmed ID: 19264659

Chronic wasting disease (CWD) is a transmissible, fatal prion disease of cervids and is largely confined to North America. The origin of CWD continues to pose a conundrum: does the disease arise spontaneously or result from some other naturally occurring reservoir? To address whether prions from sheep might be able to cause disease in cervids, we inoculated mice expressing the elk prion protein (PrP) transgene [Tg(ElkPrP) mice] with two scrapie prion isolates. The SSBP/1 scrapie isolate transmitted disease to Tg(ElkPrP) mice with a median incubation time of 270 days, but a second isolate failed to produce neurological dysfunction in these mice. Although prions from cattle with bovine spongiform encephalopathy (BSE) did not transmit to the Tg(ElkPrP) mice, they did transmit after being passaged through sheep. In Tg(ElkPrP) mice, the sheep-passaged BSE prions exhibited an incubation time of approximately 300 days. SSBP/1 prions produced abundant deposits of the disease-causing PrP isoform, denoted PrP(Sc), in the cerebellum and pons of Tg(ElkPrP) mice, whereas PrP(Sc) accumulation in Tg mice inoculated with sheep-passaged BSE prions was confined to the deep cerebellar nuclei, habenula and the brainstem. The susceptibility of 'cervidized' mice to 'ovinized' prions raises the question about why CWD has not been reported in other parts of the world where cervids and scrapie-infected sheep coexist.

Measuring Prions by Bioluminescence Imaging

Proceedings of the National Academy of Sciences of the United States of America. Sep, 2009  |  Pubmed ID: 19706444

Prions are infectious proteins that cause fatal neurodegenerative diseases. Because astrocytic gliosis marked by the deposition of fibrils composed of GFAP is a prominent feature of prion disease, we asked whether GFAP might be used as a surrogate marker for prions. To interrogate this posit, we inoculated prions into transgenic (Tg) mice expressing luciferase (luc) under the GFAP gene (Gfap) promoter, denoted Tg(Gfap-luc) mice. Weekly noninvasive, bioluminescence imaging (BLI) detected an increase in light emitted from the brains of Tg(Gfap-luc) mice at approximately 55 d after inoculation and approximately 62 d before neurologic deficits appeared. To determine whether BLI could be used as a proxy bioassay for prion infectivity, we performed endpoint titrations of prions in Tg(Gfap-luc) mice. BLI bioassays were as or more sensitive than those determined by the onset of neurological dysfunction, and were completed in approximately half the time. Our studies argue that BLI is likely to be a suitable surrogate for measuring prion infectivity, and might be useful in the study of Tg mouse models for other neurodegenerative illnesses.

Asymptomatic Deer Excrete Infectious Prions in Faeces

Nature. Sep, 2009  |  Pubmed ID: 19741608

Infectious prion diseases-scrapie of sheep and chronic wasting disease (CWD) of several species in the deer family-are transmitted naturally within affected host populations. Although several possible sources of contagion have been identified in excretions and secretions from symptomatic animals, the biological importance of these sources in sustaining epidemics remains unclear. Here we show that asymptomatic CWD-infected mule deer (Odocoileus hemionus) excrete CWD prions in their faeces long before they develop clinical signs of prion disease. Intracerebral inoculation of irradiated deer faeces into transgenic mice overexpressing cervid prion protein (PrP) revealed infectivity in 14 of 15 faecal samples collected from five deer at 7-11 months before the onset of neurological disease. Although prion concentrations in deer faeces were considerably lower than in brain tissue from the same deer collected at the end of the disease, the estimated total infectious dose excreted in faeces by an infected deer over the disease course may approximate the total contained in a brain. Prolonged faecal prion excretion by infected deer provides a plausible natural mechanism that might explain the high incidence and efficient horizontal transmission of CWD within deer herds, as well as prion transmission among other susceptible cervids.

Salivary Prions in Sheep and Deer

Prion. Jan-Mar, 2012  |  Pubmed ID: 22453179

Scrapie of sheep and chronic wasting disease (CWD) of cervids are transmissible prion diseases. Milk and placenta have been identified as sources of scrapie prions but do not explain horizontal transmission. In contrast, CWD prions have been reported in saliva, urine and feces, which are thought to be responsible for horizontal transmission. While the titers of CWD prions have been measured in feces, levels in saliva or urine are unknown. Because sheep produce ~17 L/day of saliva, and scrapie prions are present in tongue and salivary glands of infected sheep, we asked if scrapie prions are shed in saliva. We inoculated transgenic (Tg) mice expressing ovine prion protein, Tg(OvPrP) mice, with saliva from seven Cheviot sheep with scrapie. Six of seven samples transmitted prions to Tg(OvPrP) mice with titers of -0.5 to 1.7 log ID₅₀ U/ml. Similarly, inoculation of saliva samples from two mule deer with CWD transmitted prions to Tg(ElkPrP) mice with titers of -1.1 to -0.4 log ID₅₀ U/ml. Assuming similar shedding kinetics for salivary prions as those for fecal prions of deer, we estimated the secreted salivary prion dose over a 10-mo period to be as high as 8.4 log ID₅₀ units for sheep and 7.0 log ID₅₀ units for deer. These estimates are similar to 7.9 log ID₅₀ units of fecal CWD prions for deer. Because saliva is mostly swallowed, salivary prions may reinfect tissues of the gastrointestinal tract and contribute to fecal prion shedding. Salivary prions shed into the environment provide an additional mechanism for horizontal prion transmission.

Chimeric Elk/mouse Prion Proteins in Transgenic Mice

The Journal of General Virology. Feb, 2013  |  Pubmed ID: 23100369

Chronic wasting disease (CWD) of deer and elk is a highly communicable neurodegenerative disorder caused by prions. Investigations of CWD are hampered by slow bioassays in transgenic (Tg) mice. Towards the development of Tg mice that will be more susceptible to CWD prions, we created a series of chimeric elk/mouse transgenes that encode the N terminus of elk PrP (ElkPrP) up to residue Y168 and the C terminus of mouse PrP (MoPrP) beyond residue 169 (mouse numbering), designated Elk3M(SNIVVK). Between codons 169 and 219, six residues distinguish ElkPrP from MoPrP: N169S, T173N, V183I, I202V, I214V and R219K. Using chimeric elk/mouse PrP constructs, we generated 12 Tg mouse lines and determined incubation times after intracerebral inoculation with the mouse-passaged RML scrapie or Elk1P CWD prions. Unexpectedly, one Tg mouse line expressing Elk3M(SNIVVK) exhibited incubation times of <70 days when inoculated with RML prions; a second line had incubation times of <90 days. In contrast, mice expressing full-length ElkPrP had incubation periods of >250 days for RML prions. Tg(Elk3M,SNIVVK) mice were less susceptible to CWD prions than Tg(ElkPrP) mice. Changing three C-terminal mouse residues (202, 214 and 219) to those of elk doubled the incubation time for mouse RML prions and rendered the mice resistant to Elk1P CWD prions. Mutating an additional two residues from mouse to elk at codons 169 and 173 increased the incubation times for mouse prions to >300 days, but made the mice susceptible to CWD prions. Our findings highlight the role of C-terminal residues in PrP that control the susceptibility and replication of prions.

Prion-like Propagation of Human Brain-derived Alpha-synuclein in Transgenic Mice Expressing Human Wild-type Alpha-synuclein

Acta Neuropathologica Communications. Nov, 2015  |  Pubmed ID: 26612754

Parkinson's disease (PD) and multiple system atrophy (MSA) are neurodegenerative diseases that are characterized by the intracellular accumulation of alpha-synuclein containing aggregates. Recent increasing evidence suggests that Parkinson's disease and MSA pathology spread throughout the nervous system in a spatiotemporal fashion, possibly by prion-like propagation of alpha-synuclein positive aggregates between synaptically connected areas. Concurrently, intracerebral injection of pathological alpha-synuclein into transgenic mice overexpressing human wild-type alpha-synuclein, or human alpha-synuclein with the familial A53T mutation, or into wild-type mice causes spreading of alpha-synuclein pathology in the CNS. Considering that wild-type mice naturally also express a threonine at codon 53 of alpha-synuclein, it has remained unclear whether human wild-type alpha-synuclein alone, in the absence of endogenously expressed mouse alpha-synuclein, would support a similar propagation of alpha-synuclein pathology in vivo.

Neuroinvasion of α-Synuclein Prionoids After Intraperitoneal and Intraglossal Inoculation

Journal of Virology. Oct, 2016  |  Pubmed ID: 27489279

α-Synuclein is a soluble, cellular protein that in a number of neurodegenerative diseases, including Parkinson's disease and multiple system atrophy, forms pathological deposits of protein aggregates. Because misfolded α-synuclein has some characteristics that resemble those of prions, we investigated its potential to induce disease after intraperitoneal or intraglossal challenge injection into bigenic Tg(M83(+/-):Gfap-luc(+/-)) mice, which express the A53T mutant of human α-synuclein and firefly luciferase. After a single intraperitoneal injection with α-synuclein fibrils, four of five mice developed paralysis and α-synuclein pathology in the central nervous system, with a median incubation time of 229 ± 17 days. Diseased mice accumulated aggregates of Sarkosyl-insoluble and phosphorylated α-synuclein in the brain and spinal cord, which colocalized with ubiquitin and p62 and were accompanied by gliosis. In contrast, only one of five mice developed α-synuclein pathology in the central nervous system after intraglossal injection with α-synuclein fibrils, after 285 days. These findings are novel and important because they show that, similar to prions, α-synuclein prionoids can neuroinvade the central nervous system after intraperitoneal or intraglossal injection and can cause neuropathology and disease.

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