|
PARKINSN Archives
|
PARKINSN@LISTSERV.UTORONTO.CA
|
|
|
|
Gene therapy, also known by the compounds and materials deposited in the
brain in search of a therapeutic response shows much promise. This paper =
is
by Dr. K.S. Bankiewicz, a scientist with Somatix Therapy Corp., Alameda, =
CA.
The Neurosciences Institute, which is associated with Good Samaritan=20
Hospital in Los Angeles, sponsored a symposium on Nov. 2-3, 1996
in Rancho Mirage, CA. This review was included in their syllabus.
I wish to thank Carole Hilton for making this available to the archive.
Application of Gene Therapy for Parkinson's Disease;=20
Non-Human Primate Experience
K.S. Bankiewicz
Somatix Therapy Corporation, 850 Marina Village Parkway,=20
Alameda, CA 94501
Introduction
Parkinson's disease (PD) is characterized by the=20
progressive loss of dopaminergic cells and a deficiency of=20
tyrosine hydroxylase (TH) which is necessary for the synthesis=20
of dopamine (DA). As the degeneration of nigral dopamine neurons=20
continues, intracellular biochemical responses occur in the=20
presynaptic dopamine neurons and the postsynaptic striatal=20
neurons that enable the nigrostriatal dopamine system to=20
compensate for great depletions of DA prior to the onset of
clinical symptoms. As the disease progresses, and up to 85%=20
of nigral dopaminergic neurons have degenerated, the synthesis=20
of TH and the release of DA from the presynaptic terminals is
increased in a compensatory manner.
Early in the disease, this TH deficiency can be offset=20
by oral administration of L-dopa. The exogenous L-dopa is taken=20
into the cell via active amino acid transport, where it is acted=20
upon by L-AADC - thus circumventing the rate limiting TH. Currently=20
it is thought that the exogenous L-dopa exerts its therapeutic effects=20
by converting to dopamine in the remaining striatonigral DA cells=20
which then release DA in a physiologically relevant manner.
Oral administration of L-dopa continues to be the mainstay=20
of current therapy for Parkinson's disease but is limited by:=20
(i) the inability to achieve site specific delivery, which results
in unwanted side-effects and limits the amount of drug which can=20
be given, (ii) the inability to maintain a sustained drug level within=20
the central nervous system (CNS), which is thought to contribute=20
to unpredictable "on-off" effects in the treatment, and (iii) the=20
inability to prevent the progressive degeneration of dopamine-
secreting nerve cells (1). Development of new therapeutic
approaches to Parkinson's disease must address these issues.
Local Delivery of Therapeutic Agents into CNS
Delivery of macromolecular therapeutic agents into the=20
CNS presents unique challenges. Systematically-administered=20
proteins do not enter the brain tissue due to exclusion by the=20
brain capillary endothelial cells. Since the blood brain barrier=20
(BBB) is permeable to lipid-soluble compounds, enhancement of=20
the lipid solubility of polypeptides has been attempted, but with
limited success. The permeability through brain capillaries can=20
also be increased with chemical modifications of proteins such as=20
cationization, Conjugation of peptides and proteins to anti-
transferin receptor antibodies also increases uptake into the brain,=20
but this has resulted in systemic toxicity. Drug-impregnated=20
polymer devices can provide sustained and local release into the CNS.
However, such delivery is only possible for a limited period of time,=20
and "empty" polymer capsules that have limited biodegradability=20
remain in the CNS after therapy. Regardless of the method used for=20
direct drug delivery, the ability of agents to distribute within the=20
brain following introduction at a local site is a critical issue in=20
determining drug efficacy and toxicity,
Direct administration of therapeutic agents into the=20
CNS eliminates the need to bypass the (BBB) and thereby, reducing=20
systemic side effects. Delivery of proteins into the cerebral=20
ventricles results in high protein concentration in the tissue=20
adjacent to the ventricle, but there is limited penetration into=20
surrounding regions and clearance into the peripheral circulation=20
is rapid, Direct injection into the brain parenchyma at the target=20
site has been shown to be a more effective means of achieving high=20
drug concentration at specific sites within the brain,=20
Intraparenchymal cannulae and infusion pumps are required for=20
chronic delivery, making this approach less desirable,
The short penetration distances of proteins pose an even=20
greater problem in the human brain than in the relatively small=20
rat brain, The treatment of many neurodegenerative disorders will
require protocols which allow for delivery of sufficient levels of=20
therapeutic agents over significant distances in brain parenchyma.
Cell-based delivery into CNS
Cellular implants can overcome the need for persistent=20
and local physical delivery of therapeutic agents into the CNS=20
Limited but striking successes have been achieved through the
implantation of DA-producing fetal cells in Parkinsonian patients,=20
The requirement of many embryos per patient is a major factor=20
restricting the wide scale adoption of this approach as a
possible treatment for PD.
Several alternative approaches utilizing genetic=20
engineering have been proposed by many investigators (2).=20
Primary or immortalized cells have been engineered to produce=20
a specific protein in culture and then implanted into the host=20
CNS either by direct cell transplantation or by encapsulating=20
cells into semipermeable membranes. Other approaches consist of=20
in vivo gene transfer based on direct introduction of genetic=20
material into the CNS using viral or synthetic vectors. Ex vivo=20
methods using primary fibroblasts transduced with retroviral=20
vectors and the in vivo method utilizing adeno-associated virus=20
(AAV) are described here as possible means of chronic delivery of=20
therapeutic agents into CNS. In order to develop a viable gene=20
therapy for any neurological disorder, four requirements must be=20
met: (i) demonstration of long-term expression; (ii) supply=20
of local and therapeutic levels of the desired protein (iii) lack=20
of toxicity of the procedure; and (vi) the ability to reverse the=20
treatment if adverse events occur.
Ex-vivo Gene Therapy using Autologous Cells
One method for delivery of L-DOPA locally into the CNS=20
is to transplant cells that synthesize and secrete L-DOPA after=20
ex vivo genetic modification. In this scenario, a PD patient's=20
own cells would be genetically modified to express TH. Depending=20
on the cell type being used as a vehicle, it may be necessary to=20
express a second enzyme, GTP cyclohydrolase 1, to facilitate the=20
production of the cofactor for TH activity, tetrahydrobiopterin=20
(BH4). These cells then would be grafted into appropriate sites=20
in the striatum to provide a local supply of L-DOPA at the sites=20
in the brain that are normally innervated by dopaminergic neurons.=20
The L-DOPA secreted by these cells may then be taken up by=20
remaining neuronal and non-neuronal cells, converted to DA and=20
released either in a normally regulated or in a unregulated fashion to
ameliorate symptoms of the underlying Parkinsonism in these patients.
In-vivo Gene Transfer
Significant advances have recently been made regarding=20
the introduction of genetic material into the mammalian cells in
vivo. Several vehicles have been used for in vivo transfer of
cDNA, including herpes simplex viral vectors, adeno viral vectors,=20
direct plasmid DNA transfer and, most recently, retroviral=20
vectors (lentivirus) (2). An additional promising vector system
utilizes AAV, a nonpathogenic in human single stranded DNA=20
parvovirus (3). In addition to having no known pathology in man=20
the wild-type virus is incapable of replication without helper
functions provided by an adenovirus. Wild type AAV has been shown=20
to infect post-mitotic mammalian cells and to insert its DNA=20
into the host genome with some specificity for chromosome 19.=20
It should be stressed, however, that at the present time it is=20
difficult to predict which of these in vivo delivery systems will=20
prove to be the most optimal and clinically applicable.
Delivery of TH-AAV vector into monkey striatum
In addition to the difficulty of introducing therapeutic=20
agents into the CNS another significant challenge is presented=20
by PD. Since the disease affects mostly nigrostriatal and partially
mesolimbic systems, therapeutic agents must be spread over a=20
considerable area of the brain. This can be accomplished by=20
utilizing multiple delivery sites. However, with the increasing=20
number of penetration sites into the CNS, the risk of complication=20
increases as well. At the present time direct gene delivery into=20
the CNS can be accomplished only by introduction of viral vectors=20
into the brain parenchyma. Injection of the AAV vector results in=20
a limited and highly concentrated infection of the brain located=20
within close proximity to the injection cannulae. Such a limited=20
area of coverage is not desirable when large regions of the human=20
brain are to be targeted. In addition, due to the limited=20
diffusion of therapeutic agents in the brain, infections are=20
highly localized. An overproduction of a gene product in a limited=20
area can result in local toxicity, which give rise to unwanted=20
side effects.
Attempts to spread the infection into larger areas of=20
the brain using a slow infusion technique and fused silica=20
cannulae resulted in a higher number of infected cells and a less
concentrated area of infection. No signs of toxicity in the=20
striatum related to the volumes or techniques used were observed (4).
Gene expression at 4 months
in vivo approach
Gene expression was detected by immunostaining in monkey=20
striatum after l4-21 days and at 3 months survival for both=20
=DF-galactosidase (=DF-gal) and TH-AAV vectors. Gene expression after
3 months appeared less robust than at the earlier time points,=20
however, the level of gene expression over time is now under=20
investigation. Based on double labeling immunocytochemistry
using antibodies against the transgene and neurofilament or glial=20
fibrillary acidic protein (GFAP), it appears that the AAV vector=20
used infects almost exclusively neurons in the monkey brain.
Some glia-like cells were detected expressing =DF-gal. =20
However, they constituted a minority of AAV-infected cells as=20
they were estimated to be less than 5% of all the cells detected to
express =DF-gal. Furthermore, no GFAP-positive cell was observed to=20
express also =DF-gal.
The number of =DF-gal positive cells was the greatest when=20
the slow infusion methods were applied. In this case there was=20
estimated to be between 31,000 to 14,000 cells per single delivery
site (4).
ex-vivo approach
Expression of the TH gene was examined at four months=20
after the fibroblast implantation by in situ, hybridization,=20
immunostaining with a TH antibody and Southern PCR. The application
of the photographic emulsion combined with H&E counter staining=20
allows the cellular resolution of in-situ hybridization. The TH=20
mRNA-expressing cells were recognized by the accumulation of
silver grains over the cell bodies. The grains were highly=20
concentrated over the groups of elongated fibroblast inside the=20
implants. Positive TH staining was observed in the fibroblast
implants as well.
So far, we have examined the gene expression in=20
genetically modified fibroblasts for up to four months. We have=20
demonstrated persistent expression in the grafted cells for this=20
period. The addition of GTP-cyclohydrolase-l vectors, and therefore
the production of BH4, appears to facilitate TH immunostaining,=20
perhaps by lengthening the half-life of the TH enzyme in co-
infected fibroblasts. As has been shown in rat experiments, the=20
combination of expressing TH and GTP-cyclohydrolase- 1 cDNAs in=20
grafted cells also makes it possible to detect L-DOPA by
microdialysis.
The application of AAV-based vectors into the monkey=20
striatum results in robust gene expression which is limited mostly=20
to neuronal populations of cells in the stratum. The mechanism
for specific targeting of the AAV vector expression to neurons is=20
not clear at this time. However, we have observed this pattern=20
using both the cytomegalovirus (CMV) immediate-early promoter
and a Moloney Murine leukemia virus (MoMuLV) promoter driving=20
expression in the AAV vector. A critical question still remains=20
regarding the regulation of gene expression. The development=20
of regulatable promoters might help to overcome this in the=20
near future. Further technological advances are required to=20
optimize gene delivery, regulation of gene expression and
testing in appropriate functional models before gene therapy=20
can be used extensively.
References
1 Chase TN, Juncos J, Serrati C, Fabbrini G, Bruno G.=20
Fluctuations in response to chronic levodopa treatment therapy:=20
pathogenic and therapeutic considerations. In: Yahr MD, Bergmann
KJ (eds) Advances in Neurology: Parkinson's disease. New York:=20
Raven Press, 1986, 477-480.
2 Freese A, Stern M, Kaplitt MG, O'Connor W, Abbey M,=20
O'Connor MJ, During MJ, Prospects for gene therapy in=20
Parkinson's disease. Mov Dis 1996; 5:469-488
3 Muzyczka N, Use of adeno-associated virus as general=20
transduction vector for mammalian cells. Curr Top Microbiol=20
Imunol 1992; 158: 97-l29.
4 Bankiewicz KS, Snyder R, Zhou SZ, Morton M, Conway J,=20
Nagy D. Adeno-associated (AAV) viral vector-mediated gene=20
delivery in non-human primates. Soc. Neurosci Abst
1996:22:768
John Cottingham To search the Parkinsn archive, send search requests
to [log in to unmask] with Archive Search as the subject.
LibraryH Searches of the Subject: line, From: line and Body are
possible. Look for "Revised Current Topics...." messag=
e
HomeBoy for Articles and Studies available by e-mail.
PARKINSONIANS WORLD-WIDE GIVE THANKS IN MEMORY OF ALAN=
=20
[log in to unmask] BONANDER.....WHERE EVER YOU ARE!
|
|
|
|
|
Archives |
March 2024, Week 4 January 2022, Week 4 November 2021, Week 4 February 2021, Week 2 December 2020, Week 2 October 2020, Week 4 June 2020, Week 4 May 2020, Week 2 May 2020, Week 1 April 2020, Week 5 April 2020, Week 1 March 2020, Week 5 March 2020, Week 4 March 2020, Week 2 March 2020, Week 1 February 2020, Week 4 February 2020, Week 3 February 2020, Week 1 January 2020, Week 5 January 2020, Week 2 October 2019, Week 1 September 2019, Week 5 September 2019, Week 3 July 2019, Week 1 June 2019, Week 5 June 2019, Week 4 June 2019, Week 3 June 2019, Week 1 April 2019, Week 5 April 2019, Week 4 April 2019, Week 2 March 2019, Week 5 March 2019, Week 3 March 2019, Week 2 March 2019, Week 1 February 2019, Week 4 January 2019, Week 3 January 2019, Week 2 January 2019, Week 1 December 2018, Week 5 December 2018, Week 4 December 2018, Week 2 November 2018, Week 3 November 2018, Week 2 November 2018, Week 1 October 2018, Week 4 October 2018, Week 3 October 2018, Week 1 September 2018, Week 4 September 2018, Week 3 August 2018, Week 4 August 2018, Week 3 August 2018, Week 1 July 2018, Week 4 July 2018, Week 3 July 2018, Week 2 July 2018, Week 1 June 2018, Week 5 June 2018, Week 3 June 2018, Week 1 May 2018, Week 5 May 2018, Week 4 May 2018, Week 3 May 2018, Week 2 May 2018, Week 1 April 2018, Week 4 April 2018, Week 3 April 2018, Week 2 February 2018, Week 3 January 2018, Week 5 January 2018, Week 2 January 2018, Week 1 December 2017, Week 4 December 2017, Week 3 December 2017, Week 1 November 2017, Week 5 November 2017, Week 4 November 2017, Week 3 November 2017, Week 2 November 2017, Week 1 October 2017, Week 4 October 2017, Week 2 October 2017, Week 1 September 2017, Week 5 September 2017, Week 4 September 2017, Week 3 September 2017, Week 2 September 2017, Week 1 August 2017, Week 4 August 2017, Week 2 August 2017, Week 1 July 2017, Week 5 July 2017, Week 4 July 2017, Week 3 July 2017, Week 2 July 2017, Week 1 June 2017, Week 5 June 2017, Week 4 June 2017, Week 3 June 2017, Week 2 June 2017, Week 1 May 2017, Week 5 May 2017, Week 4 May 2017, Week 3 May 2017, Week 2 May 2017, Week 1 April 2017, Week 3 April 2017, Week 2 April 2017, Week 1 March 2017, Week 4 March 2017, Week 3 March 2017, Week 2 March 2017, Week 1 February 2017, Week 3 February 2017, Week 2 February 2017, Week 1 January 2017, Week 4 January 2017, Week 2 January 2017, Week 1 December 2016, Week 5 December 2016, Week 4 December 2016, Week 2 December 2016, Week 1 November 2016, Week 4 November 2016, Week 3 November 2016, Week 2 November 2016, Week 1 October 2016, Week 4 October 2016, Week 3 October 2016, Week 1 September 2016, Week 3 September 2016, Week 2 September 2016, Week 1 August 2016, Week 4 July 2016, Week 5 July 2016, Week 4 July 2016, Week 3 July 2016, Week 2 July 2016, Week 1 June 2016, Week 5 June 2016, Week 3 June 2016, Week 2 June 2016, Week 1 May 2016, Week 5 May 2016, Week 4 May 2016, Week 3 May 2016, Week 2 May 2016, Week 1 April 2016, Week 5 April 2016, Week 4 April 2016, Week 3 April 2016, Week 2 April 2016, Week 1 March 2016, Week 5 March 2016, Week 4 March 2016, Week 3 March 2016, Week 2 March 2016, Week 1 February 2016, Week 5 February 2016, Week 4 February 2016, Week 3 February 2016, Week 2 February 2016, Week 1 January 2016, Week 5 January 2016, Week 4 January 2016, Week 3 January 2016, Week 2 January 2016, Week 1 December 2015, Week 5 December 2015, Week 4 December 2015, Week 3 December 2015, Week 2 December 2015, Week 1 November 2015, Week 5 November 2015, Week 3 November 2015, Week 2 November 2015, Week 1 October 2015, Week 5 October 2015, Week 4 October 2015, Week 3 October 2015, Week 2 October 2015, Week 1 September 2015, Week 5 September 2015, Week 4 September 2015, Week 3 September 2015, Week 2 September 2015, Week 1 August 2015, Week 5 August 2015, Week 4 August 2015, Week 3 August 2015, Week 2 August 2015, Week 1 July 2015, Week 5 July 2015, Week 4 July 2015, Week 3 July 2015, Week 2 July 2015, Week 1 June 2015, Week 5 June 2015, Week 4 June 2015, Week 3 June 2015, Week 2 June 2015, Week 1 May 2015, Week 5 May 2015, Week 4 May 2015, Week 3 May 2015, Week 2 May 2015, Week 1 April 2015, Week 4 April 2015, Week 3 April 2015, Week 2 April 2015, Week 1 March 2015, Week 5 March 2015, Week 4 March 2015, Week 3 March 2015, Week 2 March 2015, Week 1 February 2015, Week 4 February 2015, Week 3 February 2015, Week 2 February 2015, Week 1 January 2015, Week 5 January 2015, Week 4 January 2015, Week 3 January 2015, Week 2 December 2014, Week 5 December 2014, Week 4 December 2014, Week 3 December 2014, Week 2 December 2014, Week 1 November 2014, Week 5 November 2014, Week 4 November 2014, Week 3 November 2014, Week 2 November 2014, Week 1 October 2014, Week 5 October 2014, Week 4 October 2014, Week 3 October 2014, Week 2 October 2014, Week 1 September 2014, Week 5 September 2014, Week 4 September 2014, Week 3 September 2014, Week 2 September 2014, Week 1 August 2014, Week 5 August 2014, Week 4 August 2014, Week 3 August 2014, Week 2 August 2014, Week 1 July 2014, Week 5 July 2014, Week 4 July 2014, Week 3 July 2014, Week 2 July 2014, Week 1 June 2014, Week 5 June 2014, Week 4 June 2014, Week 3 June 2014, Week 2 June 2014, Week 1 May 2014, Week 4 May 2014, Week 3 May 2014, Week 2 May 2014, Week 1 April 2014, Week 5 April 2014, Week 4 April 2014, Week 3 April 2014, Week 2 April 2014, Week 1 March 2014, Week 5 March 2014, Week 4 March 2014, Week 3 March 2014, Week 2 March 2014, Week 1 February 2014, Week 4 February 2014, Week 3 February 2014, Week 2 February 2014, Week 1 January 2014, Week 5 January 2014, Week 4 January 2014, Week 3 January 2014, Week 2 January 2014, Week 1 December 2013, Week 5 December 2013, Week 4 December 2013, Week 3 December 2013, Week 2 December 2013, Week 1 November 2013, Week 4 November 2013, Week 3 November 2013, Week 2 November 2013, Week 1 October 2013, Week 5 October 2013, Week 4 October 2013, Week 3 October 2013, Week 2 October 2013, Week 1 September 2013, Week 5 September 2013, Week 4 September 2013, Week 3 September 2013, Week 2 September 2013, Week 1 August 2013, Week 5 August 2013, Week 4 August 2013, Week 3 August 2013, Week 2 August 2013, Week 1 July 2013, Week 5 July 2013, Week 4 July 2013, Week 3 July 2013, Week 2 July 2013, Week 1 June 2013, Week 5 June 2013, Week 4 June 2013, Week 3 June 2013, Week 2 June 2013, Week 1 May 2013, Week 5 May 2013, Week 4 May 2013, Week 3 May 2013, Week 2 May 2013, Week 1 April 2013, Week 5 April 2013, Week 4 April 2013, Week 3 April 2013, Week 2 April 2013, Week 1 March 2013, Week 5 March 2013, Week 4 March 2013, Week 3 March 2013, Week 2 March 2013, Week 1 February 2013, Week 4 February 2013, Week 3 February 2013, Week 2 February 2013, Week 1 January 2013, Week 5 January 2013, Week 3 January 2013, Week 2 January 2013, Week 1 December 2012, Week 5 December 2012, Week 4 December 2012, Week 3 December 2012, Week 2 December 2012, Week 1 November 2012, Week 5 November 2012, Week 3 November 2012, Week 2 November 2012, Week 1 October 2012, Week 5 October 2012, Week 4 October 2012, Week 3 October 2012, Week 2 October 2012, Week 1 September 2012, Week 5 September 2012, Week 4 September 2012, Week 3 September 2012, Week 2 September 2012, Week 1 August 2012, Week 5 August 2012, Week 4 August 2012, Week 3 August 2012, Week 2 August 2012, Week 1 July 2012, Week 5 July 2012, Week 4 July 2012, Week 3 July 2012, Week 2 July 2012, Week 1 June 2012, Week 5 June 2012, Week 4 June 2012, Week 3 June 2012, Week 2 June 2012, Week 1 May 2012, Week 5 May 2012, Week 4 May 2012, Week 3 May 2012, Week 2 May 2012, Week 1 April 2012, Week 5 April 2012, Week 4 April 2012, Week 3 April 2012, Week 2 April 2012, Week 1 March 2012, Week 5 March 2012, Week 4 March 2012, Week 3 March 2012, Week 2 March 2012, Week 1 February 2012, Week 5 February 2012, Week 4 February 2012, Week 3 February 2012, Week 2 February 2012, Week 1 January 2012, Week 5 January 2012, Week 4 January 2012, Week 3 January 2012, Week 2 January 2012, Week 1 December 2011, Week 5 December 2011, Week 4 December 2011, Week 3 December 2011, Week 2 December 2011, Week 1 November 2011, Week 5 November 2011, Week 4 November 2011, Week 3 November 2011, Week 2 November 2011, Week 1 October 2011, Week 5 October 2011, Week 4 October 2011, Week 3 October 2011, Week 2 October 2011, Week 1 September 2011, Week 5 September 2011, Week 4 September 2011, Week 3 September 2011, Week 2 September 2011, Week 1 August 2011, Week 5 August 2011, Week 4 August 2011, Week 3 August 2011, Week 2 August 2011, Week 1 July 2011, Week 5 July 2011, Week 4 July 2011, Week 3 July 2011, Week 2 July 2011, Week 1 June 2011, Week 5 June 2011, Week 4 June 2011, Week 3 June 2011, Week 2 June 2011, Week 1 May 2011, Week 5 May 2011, Week 4 May 2011, Week 3 May 2011, Week 2 May 2011, Week 1 April 2011, Week 5 April 2011, Week 4 April 2011, Week 3 April 2011, Week 2 April 2011, Week 1 March 2011, Week 5 March 2011, Week 4 March 2011, Week 3 March 2011, Week 2 March 2011, Week 1 February 2011, Week 4 February 2011, Week 3 February 2011, Week 2 February 2011, Week 1 January 2011, Week 5 January 2011, Week 4 January 2011, Week 3 January 2011, Week 2 January 2011, Week 1 December 2010, Week 5 December 2010, Week 4 December 2010, Week 3 December 2010, Week 2 December 2010, Week 1 November 2010, Week 5 November 2010, Week 4 November 2010, Week 3 November 2010, Week 2 November 2010, Week 1 October 2010, Week 5 October 2010, Week 4 October 2010, Week 3 October 2010, Week 2 October 2010, Week 1 September 2010, Week 5 September 2010, Week 4 September 2010, Week 3 September 2010, Week 2 September 2010, Week 1 August 2010, Week 5 August 2010, Week 4 August 2010, Week 3 August 2010, Week 2 August 2010, Week 1 July 2010, Week 5 July 2010, Week 4 July 2010, Week 3 July 2010, Week 2 July 2010, Week 1 June 2010, Week 5 June 2010, Week 4 June 2010, Week 3 June 2010, Week 2 June 2010, Week 1 May 2010, Week 5 May 2010, Week 4 May 2010, Week 3 May 2010, Week 2 May 2010, Week 1 April 2010, Week 5 April 2010, Week 4 April 2010, Week 3 April 2010, Week 2 April 2010, Week 1 March 2010, Week 5 March 2010, Week 4 March 2010, Week 3 March 2010, Week 2 March 2010, Week 1 February 2010, Week 4 February 2010, Week 3 February 2010, Week 2 February 2010, Week 1 January 2010, Week 5 January 2010, Week 4 January 2010, Week 3 January 2010, Week 2 January 2010, Week 1 December 2009, Week 5 December 2009, Week 4 December 2009, Week 3 December 2009, Week 2 December 2009, Week 1 November 2009, Week 5 November 2009, Week 4 November 2009, Week 3 November 2009, Week 2 November 2009, Week 1 October 2009, Week 5 October 2009, Week 4 October 2009, Week 3 October 2009, Week 2 October 2009, Week 1 September 2009, Week 5 September 2009, Week 4 September 2009, Week 3 September 2009, Week 2 September 2009, Week 1 August 2009, Week 5 August 2009, Week 4 August 2009, Week 3 August 2009, Week 2 August 2009, Week 1 July 2009, Week 5 July 2009, Week 4 July 2009, Week 3 July 2009, Week 2 July 2009, Week 1 June 2009, Week 5 June 2009, Week 4 June 2009, Week 3 June 2009, Week 2 June 2009, Week 1 May 2009, Week 5 May 2009, Week 4 May 2009, Week 3 May 2009, Week 2 May 2009, Week 1 April 2009, Week 5 April 2009, Week 4 April 2009, Week 3 April 2009, Week 2 April 2009, Week 1 March 2009, Week 5 March 2009, Week 4 March 2009, Week 3 March 2009, Week 2 March 2009, Week 1 February 2009, Week 4 February 2009, Week 3 February 2009, Week 2 February 2009, Week 1 January 2009, Week 5 January 2009, Week 4 January 2009, Week 3 January 2009, Week 2 January 2009, Week 1 December 2008, Week 5 December 2008, Week 4 December 2008, Week 3 December 2008, Week 2 December 2008, Week 1 November 2008, Week 5 November 2008, Week 4 November 2008, Week 3 November 2008, Week 2 November 2008, Week 1 October 2008, Week 5 October 2008, Week 4 October 2008, Week 3 October 2008, Week 2 October 2008, Week 1 September 2008, Week 5 September 2008, Week 4 September 2008, Week 3 September 2008, Week 2 September 2008, Week 1 August 2008, Week 5 August 2008, Week 4 August 2008, Week 3 August 2008, Week 2 August 2008, Week 1 July 2008, Week 5 July 2008, Week 4 July 2008, Week 3 July 2008, Week 2 July 2008, Week 1 June 2008, Week 5 June 2008, Week 4 June 2008, Week 3 June 2008, Week 2 June 2008, Week 1 May 2008, Week 5 May 2008, Week 4 May 2008, Week 3 May 2008, Week 2 May 2008, Week 1 April 2008, Week 5 April 2008, Week 4 April 2008, Week 3 April 2008, Week 2 April 2008, Week 1 March 2008, Week 5 March 2008, Week 4 March 2008, Week 3 March 2008, Week 2 March 2008, Week 1 February 2008, Week 5 February 2008, Week 4 February 2008, Week 3 February 2008, Week 2 February 2008, Week 1 January 2008, Week 5 January 2008, Week 4 January 2008, Week 3 January 2008, Week 2 January 2008, Week 1 December 2007, Week 5 December 2007, Week 4 December 2007, Week 3 December 2007, Week 2 December 2007, Week 1 November 2007, Week 5 November 2007, Week 4 November 2007, Week 3 November 2007, Week 2 November 2007, Week 1 October 2007, Week 5 October 2007, Week 4 October 2007, Week 3 October 2007, Week 2 October 2007, Week 1 September 2007, Week 5 September 2007, Week 4 September 2007, Week 3 September 2007, Week 2 September 2007, Week 1 August 2007, Week 5 August 2007, Week 4 August 2007, Week 3 August 2007, Week 2 August 2007, Week 1 July 2007, Week 5 July 2007, Week 4 July 2007, Week 3 July 2007, Week 2 July 2007, Week 1 June 2007, Week 5 June 2007, Week 4 June 2007, Week 3 June 2007, Week 2 June 2007, Week 1 May 2007, Week 5 May 2007, Week 4 May 2007, Week 3 May 2007, Week 2 May 2007, Week 1 April 2007, Week 5 April 2007, Week 4 April 2007, Week 3 April 2007, Week 2 April 2007, Week 1 March 2007, Week 5 March 2007, Week 4 March 2007, Week 3 March 2007, Week 2 March 2007, Week 1 February 2007, Week 4 February 2007, Week 3 February 2007, Week 2 February 2007, Week 1 January 2007, Week 5 January 2007, Week 4 January 2007, Week 3 January 2007, Week 2 January 2007, Week 1 December 2006, Week 5 December 2006, Week 4 December 2006, Week 3 December 2006, Week 2 December 2006, Week 1 November 2006, Week 5 November 2006, Week 4 November 2006, Week 3 November 2006, Week 2 November 2006, Week 1 October 2006, Week 5 October 2006, Week 4 October 2006, Week 3 October 2006, Week 2 October 2006, Week 1 September 2006, Week 5 September 2006, Week 4 September 2006, Week 3 September 2006, Week 2 September 2006, Week 1 August 2006, Week 5 August 2006, Week 4 August 2006, Week 3 August 2006, Week 2 August 2006, Week 1 July 2006, Week 5 July 2006, Week 4 July 2006, Week 3 July 2006, Week 2 July 2006, Week 1 June 2006, Week 5 June 2006, Week 4 June 2006, Week 3 June 2006, Week 2 June 2006, Week 1 May 2006, Week 5 May 2006, Week 4 May 2006, Week 3 May 2006, Week 2 May 2006, Week 1 April 2006, Week 5 April 2006, Week 4 April 2006, Week 3 April 2006, Week 2 April 2006, Week 1 March 2006, Week 5 March 2006, Week 4 March 2006, Week 3 March 2006, Week 2 March 2006, Week 1 February 2006, Week 4 February 2006, Week 3 February 2006, Week 2 February 2006, Week 1 January 2006, Week 5 January 2006, Week 4 January 2006, Week 3 January 2006, Week 2 January 2006, Week 1 December 2005, Week 5 December 2005, Week 4 December 2005, Week 3 December 2005, Week 2 December 2005, Week 1 November 2005, Week 5 November 2005, Week 4 November 2005, Week 3 November 2005, Week 2 November 2005, Week 1 October 2005, Week 5 October 2005, Week 4 October 2005, Week 3 October 2005, Week 2 October 2005, Week 1 September 2005, Week 5 September 2005, Week 4 September 2005, Week 3 September 2005, Week 2 September 2005, Week 1 August 2005, Week 5 August 2005, Week 4 August 2005, Week 3 August 2005, Week 2 August 2005, Week 1 July 2005, Week 5 July 2005, Week 4 July 2005, Week 3 July 2005, Week 2 July 2005, Week 1 June 2005, Week 5 June 2005, Week 4 June 2005, Week 3 June 2005, Week 2 June 2005, Week 1 May 2005, Week 5 May 2005, Week 4 May 2005, Week 3 May 2005, Week 2 May 2005, Week 1 April 2005, Week 5 April 2005, Week 4 April 2005, Week 3 April 2005, Week 2 April 2005, Week 1 March 2005, Week 5 March 2005, Week 4 March 2005, Week 3 March 2005, Week 2 March 2005, Week 1 February 2005, Week 4 February 2005, Week 3 February 2005, Week 2 February 2005, Week 1 January 2005, Week 5 January 2005, Week 4 January 2005, Week 3 January 2005, Week 2 January 2005, Week 1 December 2004, Week 5 December 2004, Week 4 December 2004, Week 3 December 2004, Week 2 December 2004, Week 1 November 2004, Week 5 November 2004, Week 4 November 2004, Week 3 November 2004, Week 2 November 2004, Week 1 October 2004, Week 5 October 2004, Week 4 October 2004, Week 3 October 2004, Week 2 October 2004, Week 1 September 2004, Week 5 September 2004, Week 4 September 2004, Week 3 September 2004, Week 2 September 2004, Week 1 August 2004, Week 5 August 2004, Week 4 August 2004, Week 3 August 2004, Week 2 August 2004, Week 1 July 2004, Week 5 July 2004, Week 4 July 2004, Week 3 July 2004, Week 2 July 2004, Week 1 June 2004, Week 5 June 2004, Week 4 June 2004, Week 3 June 2004, Week 2 June 2004, Week 1 May 2004, Week 5 May 2004, Week 4 May 2004, Week 3 May 2004, Week 2 May 2004, Week 1 April 2004, Week 5 April 2004, Week 4 April 2004, Week 3 April 2004, Week 2 April 2004, Week 1 March 2004, Week 5 March 2004, Week 4 March 2004, Week 3 March 2004, Week 2 March 2004, Week 1 February 2004, Week 5 February 2004, Week 4 February 2004, Week 3 February 2004, Week 2 February 2004, Week 1 January 2004, Week 5 January 2004, Week 4 January 2004, Week 3 January 2004, Week 2 January 2004, Week 1 December 2003, Week 5 December 2003, Week 4 December 2003, Week 3 December 2003, Week 2 December 2003, Week 1 November 2003, Week 5 November 2003, Week 4 November 2003, Week 3 November 2003, Week 2 November 2003, Week 1 October 2003, Week 5 October 2003, Week 4 October 2003, Week 3 October 2003, Week 2 October 2003, Week 1 September 2003, Week 5 September 2003, Week 4 September 2003, Week 3 September 2003, Week 2 September 2003, Week 1 August 2003, Week 5 August 2003, Week 4 August 2003, Week 3 August 2003, Week 2 August 2003, Week 1 July 2003, Week 5 July 2003, Week 4 July 2003, Week 3 July 2003, Week 2 July 2003, Week 1 June 2003, Week 5 June 2003, Week 4 June 2003, Week 3 June 2003, Week 2 June 2003, Week 1 May 2003, Week 5 May 2003, Week 4 May 2003, Week 3 May 2003, Week 2 May 2003, Week 1 April 2003, Week 5 April 2003, Week 4 April 2003, Week 3 April 2003, Week 2 April 2003, Week 1 March 2003, Week 5 March 2003, Week 4 March 2003, Week 3 March 2003, Week 2 March 2003, Week 1 February 2003, Week 4 February 2003, Week 3 February 2003, Week 2 February 2003, Week 1 January 2003, Week 5 January 2003, Week 4 January 2003, Week 3 January 2003, Week 2 January 2003, Week 1 December 2002, Week 5 December 2002, Week 4 December 2002, Week 3 December 2002, Week 2 December 2002, Week 1 November 2002, Week 5 November 2002, Week 4 November 2002, Week 3 November 2002, Week 2 November 2002, Week 1 October 2002, Week 5 October 2002, Week 4 October 2002, Week 3 October 2002, Week 2 October 2002, Week 1 September 2002, Week 5 September 2002, Week 4 September 2002, Week 3 September 2002, Week 2 September 2002, Week 1 August 2002, Week 5 August 2002, Week 4 August 2002, Week 3 August 2002, Week 2 August 2002, Week 1 July 2002, Week 5 July 2002, Week 4 July 2002, Week 3 July 2002, Week 2 July 2002, Week 1 June 2002, Week 5 June 2002, Week 4 June 2002, Week 3 June 2002, Week 2 June 2002, Week 1 May 2002, Week 5 May 2002, Week 4 May 2002, Week 3 May 2002, Week 2 May 2002, Week 1 April 2002, Week 5 April 2002, Week 4 April 2002, Week 3 April 2002, Week 2 April 2002, Week 1 March 2002, Week 5 March 2002, Week 4 March 2002, Week 3 March 2002, Week 2 March 2002, Week 1 February 2002, Week 4 February 2002, Week 3 February 2002, Week 2 February 2002, Week 1 January 2002, Week 5 January 2002, Week 4 January 2002, Week 3 January 2002, Week 2 January 2002, Week 1 December 2001, Week 5 December 2001, Week 4 December 2001, Week 3 December 2001, Week 2 December 2001, Week 1 November 2001, Week 5 November 2001, Week 4 November 2001, Week 3 November 2001, Week 2 November 2001, Week 1 October 2001, Week 5 October 2001, Week 4 October 2001, Week 3 October 2001, Week 2 October 2001, Week 1 September 2001, Week 5 September 2001, Week 4 September 2001, Week 3 September 2001, Week 2 September 2001, Week 1 August 2001, Week 5 August 2001, Week 4 August 2001, Week 3 August 2001, Week 2 August 2001, Week 1 July 2001, Week 5 July 2001, Week 4 July 2001, Week 3 July 2001, Week 2 July 2001, Week 1 June 2001, Week 5 June 2001, Week 4 June 2001, Week 3 June 2001, Week 2 June 2001, Week 1 May 2001, Week 5 May 2001, Week 4 May 2001, Week 3 May 2001, Week 2 May 2001, Week 1 April 2001, Week 5 April 2001, Week 4 April 2001, Week 3 April 2001, Week 2 April 2001, Week 1 March 2001, Week 5 March 2001, Week 4 March 2001, Week 3 March 2001, Week 2 March 2001, Week 1 February 2001, Week 4 February 2001, Week 3 February 2001, Week 2 February 2001, Week 1 January 2001, Week 5 January 2001, Week 4 January 2001, Week 3 January 2001, Week 2 January 2001, Week 1 December 2000, Week 5 December 2000, Week 4 December 2000, Week 3 December 2000, Week 2 December 2000, Week 1 November 2000, Week 5 November 2000, Week 4 November 2000, Week 3 November 2000, Week 2 November 2000, Week 1 October 2000, Week 5 October 2000, Week 4 October 2000, Week 3 October 2000, Week 2 October 2000, Week 1 September 2000, Week 5 September 2000, Week 4 September 2000, Week 3 September 2000, Week 2 September 2000, Week 1 August 2000, Week 5 August 2000, Week 4 August 2000, Week 3 August 2000, Week 2 August 2000, Week 1 July 2000, Week 5 July 2000, Week 4 July 2000, Week 3 July 2000, Week 2 July 2000, Week 1 June 2000, Week 5 June 2000, Week 4 June 2000, Week 3 June 2000, Week 2 June 2000, Week 1 May 2000, Week 5 May 2000, Week 4 May 2000, Week 3 May 2000, Week 2 May 2000, Week 1 April 2000, Week 5 April 2000, Week 4 April 2000, Week 3 April 2000, Week 2 April 2000, Week 1 March 2000, Week 5 March 2000, Week 4 March 2000, Week 3 March 2000, Week 2 March 2000, Week 1 February 2000, Week 5 February 2000, Week 4 February 2000, Week 3 February 2000, Week 2 February 2000, Week 1 January 2000, Week 5 January 2000, Week 4 January 2000, Week 3 January 2000, Week 2 January 2000, Week 1 December 1999, Week 5 December 1999, Week 4 December 1999, Week 3 December 1999, Week 2 December 1999, Week 1 November 1999, Week 5 November 1999, Week 4 November 1999, Week 3 November 1999, Week 2 November 1999, Week 1 October 1999, Week 5 October 1999, Week 4 October 1999, Week 3 October 1999, Week 2 October 1999, Week 1 September 1999, Week 5 September 1999, Week 4 September 1999, Week 3 September 1999, Week 2 September 1999, Week 1 August 1999, Week 5 August 1999, Week 4 August 1999, Week 3 August 1999, Week 2 August 1999, Week 1 July 1999, Week 5 July 1999, Week 4 July 1999, Week 3 July 1999, Week 2 July 1999, Week 1 June 1999, Week 5 June 1999, Week 4 June 1999, Week 3 June 1999, Week 2 June 1999, Week 1 May 1999, Week 5 May 1999, Week 4 May 1999, Week 3 May 1999, Week 2 May 1999, Week 1 April 1999, Week 5 April 1999, Week 4 April 1999, Week 3 April 1999, Week 2 April 1999, Week 1 March 1999, Week 5 March 1999, Week 4 March 1999, Week 3 March 1999, Week 2 March 1999, Week 1 February 1999, Week 4 February 1999, Week 3 February 1999, Week 2 February 1999, Week 1 January 1999, Week 5 January 1999, Week 4 January 1999, Week 3 January 1999, Week 2 January 1999, Week 1 December 1998, Week 5 December 1998, Week 4 December 1998, Week 3 December 1998, Week 2 December 1998, Week 1 November 1998, Week 5 November 1998, Week 4 November 1998, Week 3 November 1998, Week 2 November 1998, Week 1 October 1998, Week 5 October 1998, Week 4 October 1998, Week 3 October 1998, Week 2 October 1998, Week 1 September 1998, Week 5 September 1998, Week 4 September 1998, Week 3 September 1998, Week 2 September 1998, Week 1 August 1998, Week 5 August 1998, Week 4 August 1998, Week 3 August 1998, Week 2 August 1998, Week 1 July 1998, Week 5 July 1998, Week 4 July 1998, Week 3 July 1998, Week 2 July 1998, Week 1 June 1998, Week 5 June 1998, Week 4 June 1998, Week 3 June 1998, Week 2 June 1998, Week 1 May 1998, Week 5 May 1998, Week 4 May 1998, Week 3 May 1998, Week 2 May 1998, Week 1 April 1998, Week 5 April 1998, Week 4 April 1998, Week 3 April 1998, Week 2 April 1998, Week 1 March 1998, Week 5 March 1998, Week 4 March 1998, Week 3 March 1998, Week 2 March 1998, Week 1 February 1998, Week 5 February 1998, Week 4 February 1998, Week 3 February 1998, Week 2 February 1998, Week 1 January 1998, Week 5 January 1998, Week 4 January 1998, Week 3 January 1998, Week 2 January 1998, Week 1 December 1997, Week 5 December 1997, Week 4 December 1997, Week 3 December 1997, Week 2 December 1997, Week 1 November 1997, Week 5 November 1997, Week 4 November 1997, Week 3 November 1997, Week 2 November 1997, Week 1 October 1997, Week 5 October 1997, Week 4 October 1997, Week 3 October 1997, Week 2 October 1997, Week 1 September 1997, Week 5 September 1997, Week 4 September 1997, Week 3 September 1997, Week 2 September 1997, Week 1 August 1997, Week 5 August 1997, Week 4 August 1997, Week 3 August 1997, Week 2 August 1997, Week 1 July 1997, Week 5 July 1997, Week 4 July 1997, Week 3 July 1997, Week 2 July 1997, Week 1 June 1997, Week 5 June 1997, Week 4 June 1997, Week 3 June 1997, Week 2 June 1997, Week 1 May 1997, Week 5 May 1997, Week 4 May 1997, Week 3 May 1997, Week 2 May 1997, Week 1 April 1997, Week 5 April 1997, Week 4 April 1997, Week 3 April 1997, Week 2 April 1997, Week 1 March 1997, Week 5 March 1997, Week 4 March 1997, Week 3 March 1997, Week 2 March 1997, Week 1 February 1997, Week 5 February 1997, Week 4 February 1997, Week 3 February 1997, Week 2 February 1997, Week 1 January 1997, Week 5 January 1997, Week 4 January 1997, Week 3 January 1997, Week 2 January 1997, Week 1 December 1996, Week 5 December 1996, Week 4 December 1996, Week 3 December 1996, Week 2 December 1996, Week 1 November 1996, Week 5 November 1996, Week 4 November 1996, Week 3 November 1996, Week 2 November 1996, Week 1 October 1996, Week 5 October 1996, Week 4 October 1996, Week 3 October 1996, Week 2 October 1996, Week 1 September 1996, Week 5 September 1996, Week 4 September 1996, Week 3 September 1996, Week 2 September 1996, Week 1 August 1996, Week 5 August 1996, Week 4 August 1996, Week 3 August 1996, Week 2 August 1996, Week 1 July 1996, Week 5 July 1996, Week 4 July 1996, Week 3 July 1996, Week 2 July 1996, Week 1 June 1996, Week 5 June 1996, Week 4 June 1996, Week 3 June 1996, Week 2 June 1996, Week 1 May 1996, Week 5 May 1996, Week 4 May 1996, Week 3 May 1996, Week 2 May 1996, Week 1 April 1996, Week 5 April 1996, Week 4 April 1996, Week 3 April 1996, Week 2 April 1996, Week 1 March 1996, Week 5 March 1996, Week 4 March 1996, Week 3 March 1996, Week 2 March 1996, Week 1 February 1996, Week 5 February 1996, Week 4 February 1996, Week 3 February 1996, Week 2 February 1996, Week 1 January 1996, Week 5 January 1996, Week 4 January 1996, Week 3 January 1996, Week 2 January 1996, Week 1 December 1995, Week 5 December 1995, Week 4 December 1995, Week 3 December 1995, Week 2 December 1995, Week 1 November 1995, Week 5 November 1995, Week 4 November 1995, Week 3 November 1995, Week 2 November 1995, Week 1 October 1995, Week 5 October 1995, Week 4 October 1995, Week 3 October 1995, Week 2 October 1995, Week 1 September 1995, Week 5 September 1995, Week 4 September 1995, Week 3 September 1995, Week 2 September 1995, Week 1 August 1995, Week 5 August 1995, Week 4 August 1995, Week 3 August 1995, Week 2 August 1995, Week 1 July 1995, Week 5 July 1995, Week 4 July 1995, Week 3 July 1995, Week 2 July 1995, Week 1 June 1995, Week 5 June 1995, Week 4 June 1995, Week 3 June 1995, Week 2 June 1995, Week 1 May 1995, Week 5 May 1995, Week 4 May 1995, Week 3 May 1995, Week 2 May 1995, Week 1 April 1995, Week 5 April 1995, Week 4 April 1995, Week 3 April 1995, Week 2 April 1995, Week 1 March 1995, Week 5 March 1995, Week 4 March 1995, Week 3 March 1995, Week 2 March 1995, Week 1 February 1995, Week 4 February 1995, Week 3 February 1995, Week 2 February 1995, Week 1 January 1995, Week 5 January 1995, Week 4 January 1995, Week 3 January 1995, Week 2 January 1995, Week 1 December 1994, Week 5 December 1994, Week 4 December 1994, Week 3 December 1994, Week 2 December 1994, Week 1 November 1994, Week 5 November 1994, Week 4 November 1994, Week 3 November 1994, Week 2 November 1994, Week 1 October 1994, Week 5 October 1994, Week 4 October 1994, Week 3 October 1994, Week 2 October 1994, Week 1 September 1994, Week 5 September 1994, Week 4 September 1994, Week 3 September 1994, Week 2 September 1994, Week 1 August 1994, Week 5 August 1994, Week 4 August 1994, Week 3 August 1994, Week 2 August 1994, Week 1 July 1994, Week 5 July 1994, Week 4 July 1994, Week 3 July 1994, Week 2 July 1994, Week 1 June 1994, Week 5 June 1994, Week 4 June 1994, Week 3 June 1994, Week 2 June 1994, Week 1 May 1994, Week 5 May 1994, Week 4 May 1994, Week 3 May 1994, Week 2 May 1994, Week 1 April 1994, Week 5 April 1994, Week 4 April 1994, Week 3 April 1994, Week 2 April 1994, Week 1 March 1994, Week 5 March 1994, Week 4 March 1994, Week 3 March 1994, Week 2 March 1994, Week 1 February 1994, Week 4 February 1994, Week 3 February 1994, Week 2 February 1994, Week 1 February 1994 January 1994 December 1993 November 1993
|
|
|
|