Dopaminergic neuron loss and up-regulation of chaperone protein mRNA induced by targeted over-expression of alpha-synuclein in mouse substantia nigra

Several transgenic mouse lines with altered alpha-synuclein expression have been developed that show a variety of Parkinson's disease-like symptoms without specific loss of dopaminergic neurons. Targeted over-expression of human alpha-synuclein using viral-vector mediated gene delivery into the substantia nigra of rats and non-human primates leads to dopaminergic cell loss and the formation of alpha-synuclein aggregates reminiscent of Lewy bodies. In the context of these recent findings, we used adeno-associated virus (AAV) to over-express wild type human alpha-synuclein in the substantia nigra of mice. We hypothesized that this over-expression would recapitulate pathological hallmarks of Parkinson's disease, creating a mouse model to further characterize the disease pathogenesis. Recombinant AAV expressing alpha-synuclein was stereotaxically injected into the substantia nigra of mice, leading to a 25% reduction of dopaminergic neurons after 24 weeks of transduction. Furthermore, examination of mRNA levels of stress-related proteins using laser capture microdissection and quantitative PCR revealed a positive correlation of Hsp27 expression with the extent of viral transduction at 4 weeks and a positive correlation of Hsp40, Hsp70 and caspase 9 with the extent of viral transduction at 24 weeks. Taken together, our findings suggest that targeted over-expression of alpha-synuclein can induce pathology at the gross anatomical and molecular level in the substantia nigra, providing a mouse model in which upstream changes in Parkinson's disease pathogenesis can be further elucidated.     

Modeling the mechanisms of acute hepatitis B virus infection

Mathematical models have been used to understand the factors that govern infectious disease progression in viral infections. Here we focus on hepatitis B virus (HBV) dynamics during the acute stages of the infection and analyze the immune mechanisms responsible for viral clearance. We start by presenting the basic model used to interpret HBV therapy studies conducted in chronically infected patients. We then introduce additional models to study acute infection where immune responses presumably play an important role in determining whether the infection will be cleared or become chronic. We add complexity incrementally and explain each step of the modeling process. Finally, we validate the model against experimental data to determine how well it represents the biological system and, consequently, how useful are its predictions. In particular, we find that a cell-mediated immune response plays an important role in controlling the virus after the peak in viral load.     

West Nile Virus Exposure in Black Bears of Northeastern Wisconsin

ABSTRACT Knowledge of the distribution and pathology of West Nile virus (WNV) in black bears is a necessary tool that allows wildlife managers to implement a management plan, set harvest quotas, and relocate nuisance bears. We studied the presence and significance of WNV titers in free-roaming black bears (Ursus americanus) in northeastern Wisconsin between February 2003 and March 2005. Serum neutralizing antibodies to WNV, with confirmation by plaque-reduction neutralization test to both WNV and Saint Louis encephalitis, identified exposure in 13 of 74 (17.6%) bears. This compares with a 6% infection rate in black bears in Virginia and 22% in European brown bears (Ursus arctos). Pathologic effects from exposure to WNV were not seen in any of the black bears studied.     

人源抗狂犬病毒单链二硫键稳定抗体的重组设计与表达

以人源抗狂犬病毒糖蛋白母本单链抗体ScFv为模板,利用PCR点突变分别在重链FR可变区VH(44)和轻链FR可变区VL(100)分别引入一个半胱氨酸,成功构建了重组单链二硫键稳定抗体基因。连接pET22b( )载体,转化入E.coli BL21(DE3)得到工程菌,IPTG诱导表达。体外复性并经Ni-NTA亲和层析对目的蛋白ScdsFv进行纯化;利用荧光抗体实验和ELISA检测抗体活性及稳定性。结果表明重组ScdsFv蛋白实现了原核高效表达,通过体外复性和Ni-NTA柱纯化获得纯度大于90%的ScdsFv蛋白。荧光抗体实验和ELISA结果表明ScdsFv具有特异的抗原结合活性,与母本ScFv比较,稳定性有明显提高。这种具有特异抗原结合活性的稳定ScdsFv蛋白的获得为其进一步的功能研究提供了材料。     

Seroprevalence study of Tick-borne encephalitis, Borrelia burgdorferi, Dengue and Toscana virus in Turin Province

Tick borne encephalitis virus (TBEV) is present in some European countries and it is transmitted by a tick bite. Ixodes ricinus is the main vector of the infection in Italy, where fortunately clinical neurological manifestations, typical of the more serious phase of the disease, are very rarely observed. This behaviour is different from other endemic Euroasiatic areas where numerous cases of encephalitis are described. However TBE transmission has not been widely investigated in Italy and available epidemiological data have been obtained only by studies performed in Central and Northern Regions of the country. In addition seroepidemiological researches were made prevalently on subjects at high risk of tick bite, such as hunters or forest guards from Trentin and Central Italy. No precise information about TBE virus diffusion was available in the Piedmont before our investigations. We found that hunters and wild boar breeders seem to be particularly exposed to the risk of TBE virus infection in Turin Province and in particular in the Susa valley, although no neurological involvement was observed in our population. In particular a seroprevalence of about 5% was detected by the use of purified antigens ELISA test, amongst the subjects at high risk of tick bite. Moreover low risk individuals showed a seroprevalence of below 2%. In addition a parallel seroepidemiological study was performed in Turin Province for Borrelia burgdorferi, the aetiological agent of Lyme disease, also transmitted by tick bite (e.g. Ixodes ricinus), for Dengue and Toscana (TOS) arboviruses, respectively transmitted by Aedes mosquitoes and phlebotomes. Data reported here demonstrate only a sporadic presence in our population of antibodies against Borrelia and Dengue infection. Moreover using an ELISA test performed with viral purified nucleoprotein, we reported a total percentage of about 3% of subjects positive for TOSV.     

Investigations of dengue-2 susceptibility and body size among Aedes aegypti populations

The mosquito Aedes aegypti (L.) (Diptera: Culicidae) is the primary global vector for dengue virus (DENV), yet considerable genetic variation exists among populations in terms of its competence to vector DENV. Variability in adult body size has also been observed among various mosquito populations and several studies have reported a relationship between body size and arbovirus dissemination, although most of these relied on artificially derived variation in body size. Here we examine the relationship between body size and disseminated DENV infection among 10 Ae. aegypti populations reared under optimum laboratory conditions. Body size variability was inferred from wing length measurements and DENV competence was evaluated as the proportion of individuals with disseminated infections following exposure to the dengue-2 JAM1409 strain. There were significant differences in mean wing lengths among populations (anova, F(9,22)= 7.10, P < 0.0001), ranging from 2.16 mm (Bangkok population) to 2.79 mm (MOYO-S [susceptible] population). We also observed significant differences among some populations in mean DENV infection rates (Waller-Duncan K-ratio t-test), ranging from 19.54% (MOYO-R [refractory] population) to 56.60% (MOYO-S population). However, we did not observe evidence for significant interactions between body size and DENV dissemination. We suggest that either the two traits are genetically independent or that our ability to detect interactions between them was limited by their respective inheritances as quantitative traits.     

Predominant Neuritic Pathology Induced by Viral Overexpression of <Emphasis Type="Bold">α</Emphasis>-Synuclein in Cell Culture

1. Alpha-synuclein is known to play an important role in the pathogenesis of Parkinson’s disease (PD). The pathogenicity of α-synuclein is related to its ability to form intraneuronal inclusions. The inclusions, which are found in brains of patients with PD and diffuse Lewy body disease consist partially of C-terminally truncated α-synuclein. This α-synuclein species has an increased ability to form aggregates compared to full length α-synuclein. 2. We have used an adeno-associated virus (AAV) vector system to overexpress either C-terminally truncated or full length α-synuclein containing the A53T mutation, which have both been identified in brains of familial PD patients and transgenic mouse models. Dissociated mesencephalic neurons, cerebellar granule neurons, and organotypic midbrain slice cultures were infected with AAV containing the transgene under the control of the cytomegalovirus promoter. 3. We demonstrate that viral overexpression of α-synuclein(A53T) leads to the formation of distorted neurites, intraneuritic swellings, and granular perikaryal deposits in cultured neurons. Our results indicate that these cell culture models may represent an early phase of PD reflecting pathologic neuritic alterations before significant neuronal cell loss occurs.     

Overexpression of proline oxidase induces proline-dependent and mitochondria-mediated apoptosis

The neuropathogenesis of influenza-associated encephalopathy in children and Reye's syndrome remains unclear. A surveillance effort conducted during 2000-2003 in South-West Japan reveals that almost all fatal and handicapped influenza-associated encephalopathy patients exhibit a disorder of mitochondrial β-oxidation with elevated serum acylcarnitine ratios (C_16:0+C_18:1)/C₂. Here we show invasion by a non-neurotropic epidemic influenza A H3N2 virus in cerebral capillaries with progressive brain edema after intranasal infection of mice having impaired mitochondrial β-oxidation congenitally or posteriorly in the newborn/ suckling periods. Mice genetically lacking of carnitine transporter OCTN2, resulting in carnitine deficiency and impaired β-oxidation, exhibited significant higher virus-genome numbers in the brain, accumulation of virus antigen exclusively in the cerebral capillaries and increased brain vascular permeability compared to in wild type mice. Mini-plasmin, which proteolytically potentiates influenza virus multiplication in vivo and destroys the blood-brain barrier, accumulated with virus antigen in the brain capillaries of OCTN2-deficient mice but only a little in wild-type mice. These results suggest that the impaired mitochondrial β-oxidation changes the susceptibility to a non-neurotropic influenza A virus as to multiplication in the brain capillaries and to cause brain edema. These pathological findings in the brain of mice having impaired mitochondrial β-oxidation after influenza virus infection may have implications for human influenza-associated encephalopathy.     

Costs versus benefits: best possible and best practical treatment regimens for HIV

Current HIV therapy, although highly effective, may cause very serious side effects, making adherence to the prescribed regimen difficult. Mathematical modeling may be used to evaluate alternative treatment regimens by weighing the positive results of treatment, such as higher levels of helper T cells, against the negative consequences, such as side effects and the possibility of resistance mutations. Although estimating the weights assigned to these factors is difficult, current clinical practice offers insight by defining situations in which therapy is considered “worthwhile”. We therefore use clinical practice, along with the probability that a drug-resistant mutation is present at the start of therapy, to suggest methods of rationally estimating these weights. In our underlying model, we use ordinary differential equations to describe the time course of in-host HIV infection, and include populations of both activated CD4⁺ T cells and CD8⁺ T cells. We then determine the best possible treatment regimen, assuming that the effectiveness of the drug can be continually adjusted, and the best practical treatment regimen, evaluating all patterns of a block of days “on” therapy followed by a block of days “off” therapy. We find that when the tolerance for drug-resistant mutations is low, high drug concentrations which maintain low infected cell populations are optimal. In contrast, if the tolerance for drug-resistant mutations is fairly high, the optimal treatment involves periods of reduced drug exposure which consequently boost the immune response through increased antigen exposure. We elucidate the dependence of the optimal treatment regimen on the pharmacokinetic parameters of specific antiviral agents.