Potentiation of in vivo neuroprotection by BclX(L) and GDNF co-expression depends on post-lesion time in deafferentiated CNS neurons.

To elucidate effective and long-lasting neuroprotective strategies, we analysed a combination of mitochondrial protection and neurotrophic support in two well-defined animal models of neurodegeneration, traumatic lesion of optic nerve and complete 6-hydroxydopamine (6-OHDA) lesion of nigrostriatal pathway. Neuroprotection by BclX(L), Glial cell line-derived neurotrophic factor (GDNF) or BclX(L) plus GDNF co-expression were studied at 2 weeks and at 6-8 weeks after lesions. In both lesion paradigms, the efficacy of this combination approach significantly differed depending on post-lesion time. We show that BclX(L) expression is more important for neuronal survival in the early phase after lesions, whereas GDNF-mediated neuroprotection becomes more prominent in the advanced state of neurodegeneration. BclX(L) expression was not sufficient to finally inhibit degeneration of deafferentiated central nervous system neurons. Long-lasting GDNF-mediated neuroprotection depended on BclX(L) co-expression in the traumatic lesion paradigm, but was independent of BclX(L) in the 6-OHDA lesion model. The results demonstrate that neuroprotection studies in animal models of neurodegenerative diseases should generally be performed over extended periods of time in order to reveal the actual potency of a therapeutic approach.

Evaluation of epitope tags for protein detection after in vivo CNS gene transfer.

Functional characterization of disease-related proteins, their splice variants and dominant negative mutants in the context of complex CNS tissues such as brain and retina is frequently assessed by in vivo gene transfer. For correct interpretation of results it is imperative that the protein under investigation is unambiguously detected in the transduced cell types and can be distinguished from any endogenously expressed physiological variants. Therefore the first systematic evaluation of epitope tags used to trace ectopically expressed proteins in the central nervous system is presented here. Substantial differences in the performances of various epitope tag-antibody combinations with respect to sensitivity, specificity and influence of the epitope tag on the fusion protein are elucidated. Epitope tags already established for protein detection in vitro and to some extent in vivo (c-Myc, HA and FLAG tags) were immunohistochemically detected with high sensitivity. However, detection of these tags revealed problems with background staining and we also document structural and functional influence of the tags on the fusion protein. In order to prevent such unwanted side-effects, epitope tags which have not yet been used for in vivo applications (IRS, EE and AU1 tags) were characterized in brain, retina and cultured neurons. While use of the IRS and EE tags was hindered by low sensitivity or specificity, optimal results were obtained with the AU1 epitope, which may develop into a standard tool for detection of ectopic protein expression in the central nervous system.

Long-term in vivo inhibition of CNS neurodegeneration by Bcl-XL gene transfer.

The inherently low regenerative capacity of the CNS demands effective strategies to inhibit neurodegeneration in acute lesions but also in slowly progressive neurological disorders. Therefore, therapeutic targets that can interact with the degeneration cascade to block, not just postpone, neuronal degeneration need to be defined. Bcl-X(L), a protein protecting the integrity of the mitochondrial membrane potential, was investigated for its neuroprotective properties in a long-term in vivo model of neuronal cell death. An AAV-2-based vector was used to express both Bcl-X(L) and EGFP in retinal ganglion cells (RGCs) of the adult rat retina. Transection of the optic nerve results in degeneration of RGCs in control retinae, while Bcl-X(L)-overexpressing ganglion cells were protected from degeneration. At 2 weeks after axotomy, 94% of the transduced RGCs survived the lesion (15% in controls). For the first time, we investigated RGC survival up to 8 weeks after axotomy and detected that 46% of the Bcl-X(L)-overexpressing RGCs still survived, representing significantly increased neuroprotection compared to neurotrophin-based approaches. We could also show that the axons of AAV-Bcl-X(L)-transduced RGCs remained morphologically intact after the lesion, thus providing the basis for regeneration-inducing attempts.

Promoters and serotypes: targeting of adeno-associated virus vectors for gene transfer in the rat central nervous system in vitro and in vivo.

The brain parenchyma consists of several different cell types, such as neurones, astrocytes, microglia, oligodendroglia and epithelial cells, which are morphologically and functionally intermingled in highly complex three-dimensional structures. These different cell types are also present in cultures of brain cells prepared to serve as model systems of CNS physiology. Gene transfer, either in a therapeutic attempt or in basic research, is a fascinating and promising tool to manipulate both the complex physiology of the brain and that of isolated neuronal cells. Viral vectors based on the parvovirus, adeno-associated virus (AAV), have emerged as powerful transgene delivery vehicles. Here we describe highly efficient targeting of AAV vectors to either neurones or astrocytes in cultured primary brain cell cultures. We also show that transcriptional targeting can be achieved by the use of small promoters, significantly boosting the transgene capacity of the recombinant viral genome. However, we also demonstrate that successful targeting of a vector in vitro does not necessarily imply that the same targeting works in the adult brain. Cross-packaging the AAV-2 genome in capsids of other serotypes adds additional benefits to this vector system. In the brain, the serotype-5 capsid allows for drastically increased spread of the recombinant vector as compared to the serotype-2 capsid. Finally, we emphasize the optimal targeting approach, in which the natural tropism of a vector for a specific cell type is employed. Taken together, these data demonstrate the flexibility which AAV-based vector systems offer in physiological research.

[An experimental study of immunity to hepatitis A in monkeys]. / Eksperimental'noe izuchenie immuniteta k gepatitu A na obez'ianakh.

In this work the experimental model of hepatitis A on monkeys, adequate to human hepatitis A, was used. Ten monkeys (6 Macaca mulatta and 4 Cercopithecus aethiops) were reinfected with different doses of hepatitis A virus (HAV) a year after recovery from spontaneous and experimental hepatitis A. The monkeys were completely resistant to the inoculation of the virus in moderate doses (10(3) ID50). The inoculation of HAV in large doses (10(4)-10(5) ID50) induced a mild form of this infection in the animals with a transient rise in the level of serum alanin aminotransferase and HAV shedding in feces, but in the absence of morphological changes in the liver. It should be specially pointed out that after the reinfection of monkeys virus shedding in feces was observed, which may be of great epidemiological importance. After reinfection the absence of IgM and a pronounced rise in the titers of IgC antibodies were observed.

[The properties of simian coronavirus]. / Izuchenie svoistv koronavirusa obez'ian.

Properties of 20 coronavirus (CV) isolates obtained from spontaneously infected Papio hamadryas and rhesus monkeys were investigated. Two of them were selected as simian CV prototype strains-CVRM 281 (rhesus monkey) and CVP 250 (Papio hamadryas), and can be offered as candidates to the Coronaviridae family. They are closely related to each other, but differ in some biological properties and polypeptides. These strains belong to the 2nd antigen group of mammalian CV with the prototype strain HCV OC 43 but differ from the latter. The strain CVRM 281 induces experimental CV infection which can be used as a model for investigations of some obscure aspects of human infection. The properties of these viruses suggest their usefulness for diagnostic purposes.

[Spontaneous and experimental hepatitis A in Papio hamadryas]. / Spontannyi i éksperimental'nyi gepatit A u pavianov gamadrilov.

Data on high susceptibility of Papio hamadryas to HAV are presented. For the first time, P. hamadryas were shown to be able to respond to both natural and experimental infection developing the features typical of hepatitis A: increased aminotransferase activity, virus shedding in feces, production of anti-HAV IgG and IgM, histological liver lesions. An infection lingering for 3-4 months was observed, as well as a case of chronic experimental hepatitis A with relapse in 7 months of the disease. Virological evidence of HAV infection was obtained in both lingering and chronic disease. HAV-PH strain was isolated for the first time and is described at length. It was isolated from a baboon with spontaneous infection which did not differ from that in man by antigenic and morphological features. The virus replicated in continuous African green monkey kidney cell line (AGMK) and was pathogenic for P. hamadryas. The HAV-PH isolate can be used for modelling hepatitis A in P. hamadryas.

[Virus persistence in hepatitis A in monkeys]. / Persistentsiia virusa pri gepatite A obez'ian.

A long-term complex observation of 16 cynomolgus monkeys (Macaca fascicularis) and 8 African green monkeys (Cercopithecus aethiops) with spontaneous and experimental hepatitis A revealed two forms of the illness: acute and chronic. Some monkeys developed undulating chronic course of the disease consisting of 2-6 waves. Others developed relapses (1 to 3) which occurred within 2-4 or 6-11.5 months of the infection. The morphological changes in the liver persisted for 7-28 months. Alaninaminotransferase elevations in the blood and HAV shedding in feces were observed periodically for 7-20 months. HAV persistence was documented by radioimmunoassay, enzyme immunoassay, immune electron microscopy and molecular hybridization. Persisting HAV was shown to remain pathogenic for monkeys. Virological evidence of the etiological association of HAV with chronic infection and late relapses has been obtained for the first time.

[The chronic course of spontaneous and experimental hepatitis A in rhesus monkeys with viral persistence]. / Khronicheskoe techenie spontannogo i éksperimental'nogo gepatita A u makak rezusov s persistentsiei virusa.

The prolonged (up to 2 years) complex observation of 11 rhesus macaques (Macaca mulatta) with spontaneous hepatitis A and 14 rhesus macaques with experimental hepatitis A developing after their intravenous and/or oral infection with human hepatitis A virus (HAV). Both natural and experimental infection took a chronic course (15-18 months). In 13 monkeys showing morphological changes in the liver during the whole period of the disease elevated enzyme levels in the blood and virus shedding in feces were periodically observed. Only one monkey had acute hepatitis A which lasted 1.5 months. In 11 monkeys the disease took an undulating course with 1-2 relapses when virological, biochemical and morphological signs of the disease could be detected. Seroconversion was observed in all monkeys. Anti-HAV IgM antibodies were retained for not more than 6-7 months and total anti-HAV antibodies, during the whole period of observation. Relapses were found to induce no antibody formation. Evidence on the prolonged (up to 12-16 months) persistence of HAV in primates was obtained for the first time.

[The modelling of hepatitis A in macaques]. / Modelirovanie gepatita A na makakakh.

Characteristics of experimental hepatitis A in Macaca fascicularis and M. mulatta produced with HAV, strain MP, isolated from M. mulatta in an outbreak of spontaneous hepatitis are presented. The HAV-MP strain induced the disease in all the animals used in the experiment. The infection was manifest, with all virological, serological, biochemical and morphological features typical of hepatitis A. In M. mulatta, the process was protracted, with virus persistence in feces for at least 4 months. Modeling of hepatitis A in Macaca monkeys using HAV-MP strain may be used for the study of the pathogenesis and trials of immune preparations.

[The sensitivity of rhesus and cynomolgus macaques to the human hepatitis A virus]. / Chuvstvitel'nost' makak rezusov i iavanskikh makak k virusu gepatita A cheloveka.

Hepatitis A infection characterized by virus excretion in feces, synthesis of specific IgM antibody, increased activity of alanine aminotransferase in the blood serum, and a complex of morphological lesions in the liver typical of acute hepatitis was reproduced in M. fascicularis (M. f.) and Macaca rhesus (M. r.) using 2 strains of hepatitis A virus (HAV) isolated from human patients. The incubation period varying from 9 to 23 (mean 16) days in M. f. and from 12 to 35 (mean 18) days in M. r. in primary infection shortened to 1-12 (mean 10) and 3-6 (mean 5) days in the process of virus passage from monkey to monkey. The disease was observed to run both manifest forms (except jaundice) typical of human HA and an inapparent form in which the level of enzymes remained within normal limits but HAV could be detected in feces, anti-HAV-IgM in the blood serum, and morphologically acute hepatitis in the liver. Immune electron microscopy of both the initial material and in monkey feces at the levels of all three passages revealed complexes consisting of spherical viral particles 27-29 nm in size coated with antibodies. The immune complexes formed upon addition to the fecal extracts under study of IgG isolated both from human convalescent sera and from sera of experimentally infected monkeys collected in the acute stage of the illness.

Transforming activity and antigenicity of an Epstein-Barr-like virus from lymphoblastoid cell lines of baboons with lymphoid disease.

Two lymphoblastoid cell lines were established from baboons with lymphoid disease. Cells of these lines were positive for complement and Fc receptors but lacked sheep cell receptors, theraby indicating B-cell origin. The cells contained antigens which cross-reacted with Epstein-Barr virus (EBV), viral capsid antigen (VCA), early antigen (EA) and membrane antigen (MA). Both lines released virus with in vitro transforming activity for lymphocytes of several primate species including humans. Cells of the original lines and transformed cells showed no staining for EB nuclear antigen (EBNA). The virus was neutralized by anti-MA positive baboon and human sera. Baboon virus and EBV had different but overlapping in vitro host-cell ranges.