Últimas Notícias -
Apresentação e Anais de Congressos
Seattle - Em Congresso
que está ocorrendo de 30 de maio a 3 de junho de 2001 são relatados pelo menos
19 trabalhos de um total de mais de 1150 abordando a terapia gênica com vetores
virais no tratamento da distrofia muscular. Esta linha de tratamento é
considerada internacionalmente como a mais promissora no tratamento das
distrofias musculares. O sistema imune é considerado um obstáculo ao sucesso
desta terapia. Em um estudo de Jeffrey Chamberlain e seu grupo da Universidade
de Washington em Seattle construiram um vetor sem as proteínas que podem
desencadear as respostas imunológicas denominado "gutted" adenovirus.
Usando camundongos portadores de distrofia muscular observaram que o vetor com o
gene da distrofina provoca uma mínima resposta imune; os genes são liberados
para os músculos e provocam melhora da força muscular. Em outro estudo os
autores desenvolveram micro-distrofinas com cerca de um terço do tamanho da
distrofina completa e observaram, em camundongos, melhora da força muscular
inclusive do músculo diafragma. Outro estudo independente da Universidade de
Pittsburgh realizado por Xiao-Xiao e colaboradores eles demonstraram, em
camundongos, que após sete meses da aplicação do minigene da distrofina
(a última notícia desta página) o músculo apresenta força significativa
maior do que o que não recebeu o tratamento com expressão da distrofina entre
20 e 50%. Leia os resumos
completos dos artigos relacionados a distrofia muscular
Today's
Findings From the ASGT Annual Meeting: New Cardiovascular Gene Therapy And New
Gene Therapy Research for Muscle and Connective Tissue Disease
http://www.mdausa.org/research/010602_progressgenetherapy.html
http://www.mdausa.org/research/010601_dmdminigene.html
Londres - nesta palestra,
Kay Davies da Universidade de Oxford aborda uma das formas possíveis de correção
das distrofias musculares com aumento da expressão do gene da utrofina, uma
protéina da membrana da célula que tem a capacidade de fazer as mesmas funções
da distrofina. O aumento da utrofina em 3 a 4 vezes nos estudos experimentais não
causou efeitos tóxicos às células. No momento a pesquisadora já está
testando moléculas que podem aumentar a expressão do gene da utrofina.
http://ex2.excerptamedica.com/01wcn/DetailSession.cfm?Abstract=11456
http://news.bmn.com/conferences/list/view?rp=2001-WCN-5-S4
Califórnia - mais de 40
trabalhos apresentados no Congresso abordam temas relacionados a diferentes
formas de distrofia; alguns trabalhos foram produzidos no Brasil. A maioria
deles tem interesse exclusivo para especialistas ou pesquisadores; de qualquer
modo para ler os resumos click aqui
http://www.faseb.org/genetics/ashg/meet-2001/2001meetmenu.htm
San Diego - este encontro,
que se realizará de 10 a 15 de novembro de 2001, disponibiliza online os
resumos a serem apresentados; alguns dos selecionados estão disponíveis aqui;
o mais significativo é sobre o transplante de medula óssea em um modelo
experimental de distrofia em camungongos realizado na China.
http://sfn.scholarone.com/
USA - de 8 a 12 de dezembro
de 2001 realizar-se-á em Washington está reunião de especialistas em pesquisa
experimental. Alguns trabalhos envolvem pesquisas com stem cells,
distrofia muscular, etc. Com 20 dias de antecedência disponibilizamos 22 resumos
do Congresso (em inglês).
Denver (USA) - no período de
14 a 20 de abril de 2002 realizar-se-á em Denver o Congresso
da Academia Americana de Neurologia, o mais conceituado encontro de
neurologistas dos USA. Dos 1541 artigos selecionei os que se relacionam a
distrofia muscular, sendo vários abordam a terapia gênica das
distrofias, alguns sobre o acompanhamento clínico (corticóides, função
pulmonar), genética, etc. Para ler os resumos em inglês clique aqui.
Strasbourg - Está sendo
realizado de 25 a 28 de maio de 2002 o Congresso da Sociedade Européia de Genética
Humana na cidade de Strasbourg . Os resumos do Congresso que se relacionam com
as distrofias musculares você pode ler aqui . Há
um resumo das pesquisas com transplantes de mioblastos na Rússia.
USA - Começa hoje em Boston,
USA, e irá até 9 de junho, o Encontro da Sociedade Americana de Terapia Gênica.
As mais recentes novidades em terapia gênica serão relatadas neste encontro.
Dos 1374 resumos apresentados, 20 se referem a distrofia muscular. Leia os
resumos aqui.
USA - neste Congresso
realizado em Baltimore de 15 a 19 de outubro de 2002 foram apresentados vários
trabalhos relacionados a distrofia muscular. Os mais importantes relacionados
com o tratamento da doença estão aqui. Em um deles
é descrito o uso de bifosfonato no tratamento da osteoporose de portadores de
distrofia muscular e outras doenças genéticas; nos outros três são descritas
formas de terapia gênica em modelos experimentais de distrofia muscular.
USA - este encontro anual será
realizado em São Francisco de 14 a 18/12/2002. Há poucos trabalhos
relacionados a distrofia muscular; os mais importantes estão relacionados
abaixo. No primeiro artigo é estudado o papel da TGF-beta no desenvolvimento da
fibrose muscular; existem estudo sendo realizados tentando inibir esta proteína
para retardar as alterações musculares da distrofia muscular. No segundo
trabalho os pesquisadores observaram que as células tronco são atraidas em
maior quantidade para o músculo na lesão aguda do que nas lesões crônicas.
O terceiro artigo fala do papel da inflamação no desenvolvimento das lesões
musculares da distrofia muscular experimental; os pesquisadores reiteram a opinião
que o tratamento desta inflamação no músculo pode retardar a evolução da
doença. No último trabalho os pesquisadores discutem as dificuldades em fazer
as células tronco se transformarem em células musculares em grande quantidade.
I) TGF-beta triggers the
differentiation of myogenic cells toward myofibroblasts: Implication for muscle
fibrosis
Y. Li, W. Foster, Y. Chan, B. M.
Deasy, T. Payne, T. Horaguchi, N. Badlani, J. Cummins, J. Huard; Dept. of
Orthopeadic Surgery, Children's Hospital of Pittsburgh, Pittsburgh, PA
Injured muscle can undergo
regeneration; however, the healing process is often inefficient and hindered by
progressive fibrosis, which is caused by stimulation of inflammatory factors.
Transforming Growth Factor-beta1 (TGF-b1) is considered to be a central factor
in fibrotic diseases of the liver, lung, heart, and kidney. Our previous
research has found that muscle-derived stem cells are capable of differentiating
into myofibroblasts following muscle laceration injury. We also have observed
TGF-b1 over-expression in injured skeletal muscle. In this study, we determined
that TGF-b1 stimulation can induce myoblasts (C2C12 cells) to express TGF-b1 in
an autocrine manner, down-regulate the expression of myogenic proteins, and
activate the production of fibrotic-related proteins in vitro. In addition,
direct injection of human recombinant TGF-b1 into skeletal muscle in vivo was
found to stimulate myogenic cells, including myofibers, to express TGF-b1 and
subsequently induce scar tissue formation within the injected area. These
results demonstrate that TGF-b1 is a key factor that can initiate the fibrotic
cascades in skeletal muscle and induce a population of myogenic cells, which
includes myofibers, to de-differentiate into myofibroblastic cells in injured
muscle. These observations shed new light on the process of scar tissue
formation, which commonly occurs in injured and diseased muscle.
II) Side population stem cells are
recruited to sites of acute but not chronic muscle degeneration
K. A. Lapidos, E. M. McNally;
Cardiology, University of Chicago, Chicago, IL
Muscular dystrophies are
degenerative disorders in which skeletal muscle undergoes chronic cycles of
degeneration and regeneration. Stem cells are a critical component of the
regenerative process. Recently, a pluripotent population of stem cells has been
isolated from bone marrow and muscle of adult mice, and these side population
(SP) cells were shown to regenerate hematopoietic lineages and skeletal muscle.
Based on their ability to differentiate into both cell types, we hypothesized
that SP cells comprise an in vivo, stable stem cell compartment available
for the regeneration of muscle tissue or the hematopoietic lineages. For example,
in muscular dystrophy, a high demand for stem cells in the muscle promotes
recruitment of SPs from the bone marrow. Over time, this may result in a
quantifiable decrease in SP cells from bone marrow and a quantifiable,
concomitant increase at the muscle. We tested this hypothesis by quantifying the
percentage of SP cells obtained from bone marrow or muscle in a chronic model of
muscular dystrophy, g-sarcoglycan null mice (gsg-/-). Whole
bone marrow and lower limb skeletal muscle were isolated from wild type and gsg-/-
mice at either 6 weeks or 6 months of age. Using the Hoechst dye exclusion
technique, SPs from bone marrow (HSPs) and muscle (MSPs) were quantified. We
found no statistically significant differences between the mutant and wild type
mice. We also studied an acute model of muscle degeneration elicited by
cardiotoxin injection of the crural muscles of normal mice. Three days
post-injection, SP cells showed a five to nine-fold increase in MSPs in
cardiotoxin-treated versus the control. In conclusion, we have shown that side
population cells increase at the site of acute, but not chronic, muscle
degeneration/regeneration.
III) Staging
Interactive Pathogenic Mechanisms in mdx Mouse Dystrophinopathy by
Temporal Gene Expression Signatures
J. D. Porter, A. P. Merriam, S.
Khanna; Ophthalmology, Case Western Reserve University, Cleveland, OH
Although Duchenne muscular dystrophy
(DMD) results from mutations of dystrophin, multiple interactive mechanisms
likely contribute toward pathogenesis. We used Affymetrix oligonucleotide arrays
to compare hindlimb muscle of the mdx mouse dystrophinopathy to wild type
mice at postnatal (P) ages 7, 14, 18, 23, 28, 56, and 112 days; 3 independent
replicates/age/strain. Extraocular muscle, which shows no signs of pathology in
dystrophinopathy, also was studied at most ages. Patterned alterations in
hindlimb gene expression between P7 and P14 (prenecrotic stage) included only 8
genes. Rapid accumulation of expression changes was observed thereafter, with a
20-fold increase in the number of up-regulated genes between P14 and P28, which
coincided with appearance of histologic changes in muscle. Assessed at the
expression level, disease peaked with 376 differentially regulated genes at P56,
but a persistent response was evident at P112 (~130 genes). Correlation analyses
provided insight into the interactive nature of inflammatory, fibrotic, and
muscle regenerative events, with proinflammatory mechanisms particularly evident.
By contrast, few changes were detected in extraocular muscle at any age.
Immunocytochemistry and quantitative PCR for chemotaxic molecules confirmed
their early role in recruitment of an inflammatory infiltrate to dystrophic
muscle, along with a corresponding absence in extraocular muscle. These findings
suggest that inflammation is a key contributor to, rather than a consequence of,
the pathogenesis of dystrophin deficiency. The absence of changes in extraocular
muscle suggest that this tissue does not adapt, but rather is constitutively
protected in muscular dystrophy. Taken together, genome-wide expression
profiling establishes an integrated profile of dystrophinopathy and the
resultant mdx temporal expression signature for hindlimb suggests that
blockade of inflammatory cell recruitment may be effective in ameliorating the
disease, an attractive prospect in the absence of effective therapy for the
primary mutation.
IV) Migration and Fusion of
Muscle SP Cells into Dystrophic Myofibers
R. L. Sohn,1 E. Gussoni2
; 1 Medicine/Cardiology, Brigham and Women's Hospital, Boston, MA, 2
Division of Genetics, Children's Hospital, Boston, MA
Cell therapy with muscle SP cells in
animal models is being explored as a potential treatment for muscular dystrophy.
However, though promising, stem cell therapy is plagued by its low efficiency:
only about 1-5% of these stem cells fuse into pre-existing myotubes. In fact,
how myoblasts, or even muscle SP cells, fuse to form multinucleate myotubes is a
fascinating and highly regulated process. Recent work in Drosophila has
elucidated some of the molecular components of this process. Our hope is to
improve the efficiency of cell therapy with muscle SP cells by utilizing the
tools and concepts of myoblast fusion found in Drosophila. As a first step, we
have looked for cell surface markers that promote the migration of SP cells from
the blood where they are injected to skeletal muscle where they then fuse into
myotubes. Chemokines are known to play major roles in leukocyte migration.
Therefore, we looked for the presence of chemokine receptors in skeletal muscle.
By immunohistochemistry on human fetal skeletal muscle, we have found cells
positive for the chemokine receptor CXCR-4 along the myofibers. These are not
satellite cells as they are not positive for the transcription factor Pax-7. We
have also found myotubes positive for CDO (CAM-regulated, down-regulated by
oncogenes), a cell surface protein of the immunoglobulin/fibronectin type III
repeat family that has been found to positively regulate myogenic
differentiation in vitro (Kang, et al. 1998, JCB 143: 403-13). Work is currently
underway to determine if these cell surface proteins are also present in muscle
SP cells. If so, it is possible that by increasing the expression of these
molecules, we will be able to also increase the efficiency of the SP cells to
migrate to muscle and to fuse into the myotubes.
USA - Esta semana foram
divulgados os mais de 1500 trabalhos que serão apresentados no Encontro Anual
da Academia Americana de Neurologia que realizar-se-á de 29 de março a 5 de
abril de 2003 no Hawaii. Destes artigos selecionamos 24 que se relacionam a
distrofia muscular e estão relacionados aqui. Os
principais são:
- o artigo número 1 que mostra que
os pacientes com Duchenne tratados com corticóides mantém a marcha por mais de
3 anos em relação aos que não usam e tem menor grau de escoliose. Os tratados
com corticóides apresentaram maior incidência de fraturas inclusive com
compressão vertebral, que pode ser explicada por maior atividade e maior incidência
de osteoporose.
- o artigo 2 descreve o uso da
ressonância nuclear magnética para avaliar o acometimento dos músculos que
será um exame muito utilizado num futuro próximo.
- o artigo 3 demonstra que o
ecocardiograma é o melhor exame para avaliar a função cardíaca em mulheres
que são portadoras do gene da distrofina e que a realização do
ecocardiograma ao esforço demonstrou anormalidades em 83% das portadoras
assintomáticas indicando que elas devam ser acompanhadas para o diagnóstico
(as mulheres estudadas estavam com idade entre 37+ 10 anos)
- o artigo 4 descreve um novo vetor
para levar o gene até o músculo com bons e prolongados resultados.
- o artigo 5 demonstra os bens
resultados da terapia gênica em camundongos utilizando corrente elétrica mas há
um declínio da expressão do gene ao longo do tempo que deverá ser estudada;
pelo resultado exposto a terapia gênica se bem sucedida deveria ser repetida
mais de uma vez.
- o artigo 6 estuda aspectos da lesão
muscular da distrofia e o papel dos receptores e do fator de crescimento plaquetário
nas lesões observadas.
- o artigo 14 e 15 descrevem alterações
no sistema nervoso central de portadores de distrofia miotônica; no artigo 14 são
analisadas alrterações na distrofia muscular miotônica congênita que
apresentam alterações compatíveis com retardo mental e atraso do
desenvolvimento. Não há adequada explicação para estas alterações.
USA - de 4 a 8 de junho de
2003 será realizado o Encontro Anual da Sociedade Americana em terapia gênica;
neste encontro os maiores pesquisadores em terapia gênica irão apresentar seus
trabalhos. Vinte e sete destes trabalhos se referem a pesquisas em terapia gênica
na distrofia muscular. Os resumos destes trabalhos em inglês estão aqui.
Todos eles demonstram avanços na terapia gênica experimental. Tentarei fazer
um resumo das principais pesquisas nos próximos dias. O conteúdo dos primeiros
resumos:
1) Sucesso com oligonucleotídeos para terapia gênica
2) Sucesso na correção da distrofia muscular
experimental intra-útero
3) Uso da gentamicina na mutação de ponto:
descrição de um teste que demonstra os prováveis pacientes que se beneficiarão
do tratamento.
4) Sucesso na terapia gênica com vetor viral
5) Sucesso da terapia gênica no músculo
diafragma utilizando vetor viral e gel.
6) Terapia gênica com sucesso em um modelo de
distrofia tipo cinturas com gene liberado por via intraarterial
7) Uso de terapia gênica utilizando o Igf-1
8) Terapia gênica com um indutor de formação
de vasos, o VEGF 165, induzindo a regeneração dos músculos.
9) Descrição de um novo modelo de distrofia
muscular em cães.
10) Sucesso com o uso do mini-gene da distrofina
em cães com distrofia muscular
11) Explora o uso do vetor lentiviral em modelos
de distrofia experimental; o vetor lentiviral é maior que o AAV e facilita a
colocação do gene da distrofina
12) Discute as alterações da células
musculares dos vasos dos músculos e a importância da introdução do gene da
distrofina também nestas células.
13) Demonstra os resultados positivos do minigene
da distrofina em modelos experimentais, com aumento da força muscular.
14) Estuda a relação entre o aumento da
utrofina e a redução da calpaína em camundongos que receberam o gene da
distrofina; a utrofina pode exercer o mesmo papel da distrofina.
15) Estudo com o uso da corrente elétrica para
melhorar a passagem do gene para as células musculares.
16) Obteve maior sobrevida dos mioblastos com a
utilização da interleucina 10 por terapia gênica em camundongos.
17) Apresenta um estudo sobre as diferentes
formas de célula tronco.
18) Demonstra que a superexpressão da
matrilisina aumenta a migração dos mioblastos e reduz a necessidade de tantas
injeções.
19) Estuda maneiras de modificar os mioblastos
antes do transplante para evitar rejeição.
20) Analisa as possiblidades de terapia gênica
na cardiomiopatia das distrofias.
21) Demonstra os bons resultados da terapia gênica
intra-útero (antes do nascimento)
22) Estudo o uso de oligonucleotídeos na
distrofia miotônica (estuodo in vitro,em laboratório)
23) Observou que a injeção de células tronco
por via intramuscular aumenta a expressão da distrofina e que o transplante de
medula óssea não produziu efeito.
24) Estuda a manipulação prévia (em laboratório)
das células tronco para terapia gênica
25) Utiliza na terapia gênica vetores com
distrofina e proteínas imunossupressoras e obtem a expressão do gene por mais
tempo.
26) Demonstra os efeitos do minigene da proteína
agrin em camundongos com uma forma de distrofia muscular congênita
27) Estuda a terapia gênica nas células satélites
dos músculos responsavéis pela regeneração muscular.
USA - O Encontro Anual da
Sociedade de Neurociências será realizado de 8 a 12 de novembro de 2003, em
New Orleans. Deste encontro selecionei três artigos relacionados com a
distropia muscular: o mais importante é relacionado com a terapia gênica e foi
realizado na China. Neste estudo os camundongos com distrofia muscular foram
submetidos a transplante de células tronco da medula óssea. Os animais foram
submetidos a radioterapia antes do tratamento. Após três meses do tratamento
10% dos animais apresentavam expressão da distrofina. Houve melhora da força
muscular nos animais tratados e aumento da sobrevida em relação aos animais do
grupo controle. No segundo artigo os autores demonstraram o aumento da expressão
do receptor da transferrina em fibras de regeneração em pacientes com
distrofia muscular. Este receptor esta relacionado a entrada do ferro e pode
indicar um papel deste elemento no retardo da deterioração muscular. O
terceiro artigo estuda o efeito dos corticóides na distrofia muscular
experimental, tentando entender como esta droga atua.
1) C.
Zhang, S. Chen, Y. Xie, W. Huang, S. Li, W. Zhang, Q. Li, X. Liu. THERAPEUTIC
EFFECT OF DUCHENNE MUSCULAR DYSTROPHY MICE WITH BONE MARROW STEM CELLS
TRANSPLANTATION. Program No. 413.9. 2003
Objective:
To investigate the locomotive function, electronic physiology changes, and
expression of dystrophin in Duchenne muscular dystrophy mice(mdx and dko) with
bone marrow stem cells transplantation.
Methods: The bone marrow stem cells of C57BL/6
4-to-5
weeks age
were
isolated and cultured in vitro for 3 days, then the stem cells about 5.0X106
injected intravenously into the mdx mice and dko mic
7-to-8
weeks age
,
respectively. Before injecting, 10 mdx mice and 10 dko mice were preconditioned
with 7 Gy gamma ray to decrease the immune reaction. The clinical features of
the graft verse host disease (GVHD). in the transplanted mice were assessed and
investigated after bone marrow stem cell transplatation..12 weeks after being
transplanted, the locomotion function, muscle electrophysiologic features(EMG)
and dystrophin expression of the mdx mice and dko mice were investigated..
Results: Three month after bone marrow stem cells transplanting, we find that
(1) there were more than 10% of muscle cells expression dystrophin protein in
each transplanted mice(mdx and dko); (2) the electromyography showed almost
normal waves in the transplantation mdx mice and dko mice; (3)the locomotive
function improved obviously in transplantation dko mice; (4)the life span of the
transplantation dko mice were more than 200 days( the life span of control of
dko mice were less than 140 days).
Conclusions: Allogenic bone marrow stem cells transplantation can improve the
electromyographic items, dystrophin expression in muscles of mdx and dko mice.
Further more, the stem cell transplantation can improve the locomotive function
and prolong the life span of dko mice.
2)
Y. Zhu, B. Chen, H. Gong, J. Pan, W. Zhang. EXPRESSION OF TRANSFERRIN RECEPTOR
IN DUCHENNE MUSCULAR DYSTROPHY TISSUES. Program No. 80.2. 2003
Duchenne
muscular dystrophy (DMD) is an X-linked, fatal disease caused by mutations of
the gene encoding the cytoskeletal protein dystrophin. Skeletal muscle of DMD
patients is characterized by an ongoing process of degeneration and
regeneration. Transferrin receptor (TfR, CD71), an integral membrane
glycoprotein, mediates cellular uptake of iron. In most tissues, TfR expression
is correlated positively with proliferation and regulated at the
post-transcriptional level. To determine if TfR is involved in muscular
regeneration in DMD patients, we examined the expression of TfR in muscle
samples of 42 patients with DMD, and 10 from normal volunteers.
Immunohistochemical staining of TfR was positive with variable intensity in
normal control and DMD patients. In normal control, TfR was faintly expressed on
the surface of muscle fibers. In DMD patients, strong TfR reaction was detected.
In some muscular fibers from DMD patients, TfR was found strongly expressed
inside the fibers. Further detection with Enzyme histochemistry and acridine
orange (AO) fluorescence techniques showed that these fibers with strong TfR
expression were regenerative IIc type fibers. The increase of TfR expression in
regenerative fibers suggests that the high uptake of iron may promote the
capacity of muscle regeneration and thus may delay the deterioration of DMD
patients.
3)
C.G. Carlson. STEADY STATE CALCIUM AND RESTING CALCIUM INFLUX IN NONDYSTROPHIC
AND MDX MYOTUBES EXPOSED TO PREDNISONE OR PREDNISOLONE. Program No. 413.10. 2003
Certain
glucorticoids, such as prednisone, prednisolone and deflazacort, increase muscle
strength and slow the progress of Duchenne muscular dystrophy (Fenichel et al.,
Neurology, 41, 1874, 1991) by one or more mechanisms that have not yet been
fully characterized. Evidence from 45calcium uptake studies in the C2C12 muscle
cell line suggests that one mechanism may involve decreases in calcium influx
following one or more days of exposure to these agents beginning at the period
of myoblast fusion (Passaquin et al., Brit. J. of Pharmacol., 1998). Recent
evidence from this laboratory obtained in FURA-loaded cultured lens epithelial
cells indicates that some steroids, however, may induce very rapid (seconds to
minutes) effects on resting calcium influx measured by the manganese quench
technique (Samadi et al., Pfluegers Arch. Europ. J. Physiol.,444, 700-709,
2002). To determine whether glucorticoids exert a unique action on resting
calcium influx in dystrophic muscle, studies have begun to characterize any
acute, nongenomic effects of prednisone or prednisolone on steady state calcium
levels and resting calcium influx in cultured nondystrophic and mdx myotubes.
Initial results indicate that neither prednisone (10 microM, 100 microM) nor
prednisolone (100 microM) has immediate effects on steady state calcium levels
in nondystrophic myotubes, but that prednisolone may induce a slight decrease in
manganese quench rate in mdx myotubes at relatively early stages in culture
(culture day 12-14). These results further demonstrate the utility of the
manganese quench technique in assessing whether short or long term exposure to
glucorticoids induces a beneficial reduction in resting calcium influx in
dystrophic skeletal muscle.
USA - em dezembro, de 13 a
17, será realizado na Califórnia o Encontro Anual da Sociedade Americana de
Biologia Celular. Neste encontro serão apresentadas inúmeras pesquisas, das
quais eu selecionei as duas que se relacionam com terapia na distrofia muscular.
No primeiro artigo os pesquisadores utilizaram a negamicina, um antibiótico da
mesma classe da gentamicina para tratamento da mutação de ponto na distrofia
muscular de camundongos. Os resultados obtidos com o estudo das células dos
camundongos demonstrou que a negamicina tem a mesma capacidade que a gentamicina
em corrigir a mutação de ponto e é menos tóxica. No segundo artigo os
pesquisadores criaram um camundongo com distrofia muscular e expressava a
calcineurina. A calcineurina promoveu aumento da utrofina A, que pode substituir
as funções da distrofina. Drogas que aumentem a expressão da calcineurina
podem se constituir em uma alternativa para a terapia medicamentosa da distrofia
muscular.
Abstracts from 43rd
Annual Meeting The American Society for Cell Biology - California,
13-17 december, 2003
Negamycin
restores dystrophin expression in skeletal and cardiac muscles of mdx mice
M. SHIOZUKA,1 M. ARAKAWA,1 Y. NAKAYAMA,2 T.
HARA,2 M. HAMADA,3 D. IKEDA,3 Y. TAKAHASHI,3
R. SAWA,3 Y. NONOMURA,3 K. SHEYKHOLESLAMI,4 K.
KONDO,4 K. KAGA,4 S. TAKEDA,5 R. MATSUDA1
; 1 Dept. of Life Sciences, The University of Tokyo, Tokyo, Japan, 2
Dept. of Tumor Biochemistry, Tokyo Metropolitan Institute of Medical Sciences,
Tokyo, Japan, 3 The Institute of Microbial Chemistry, Tokyo, Japan, 4
Dept. of Otolaryngology, The University of Tokyo, Tokyo, Japan, 5
Dept. of Molecular Therapy, National Institute of Neuroscience, Tokyo, Japan
The
ability of aminoglycoside antibiotics to promote read-through of nonsense
mutations has attracted interests in these drugs as potential therapeutic agents
in genetic diseases. However, strong toxicity of aminoglycoside antibiotics may
cause severe side effects during long-term treatment. In this study, we report
that negamycin, a dipeptide antibiotic, also restores dystrophin expression in
skeletal and cardiac muscles of mdx mouse; an animal model of Duchenne muscular
dystrophy (DMD) with a nonsense mutation in dystrophin gene, and in cultured mdx
myotubes. Dystrophin expression was confirmed by immunohistochemistry and
immunoblotting. We also compared the toxicity of negamycin and gentamicin, and
found negamycin to be less toxic. Furthermore, we demonstrated that negamycin
bound to a partial sequence decording the eukaryotic rRNA A-site. We conclude
that negamycin is a promising new candidate for chemotherapy for DMD and other
genetic diseases caused by nonsense mutations.
Calcineurin-Induced
Upregulation of Utrophin Attenuates the Dystrophic Pathology in mdx Mouse Muscle
J. V. Chakkalakal,1 M. Harrison,2 E. R. Chin,3
R. N. Michel,2 B. J. Jasmin1 ; 1 Cellular &
Molecular Medicine, University of Ottawa, Ottawa, ON, Canada, 2
Neuromuscular Lab, Laurentian University, Sudbury, ON, Canada, 3
Cardiovascular and Metabolic Diseases, Pfizer Global Research and Development,
Groton, CT
We
recently showed that mice expressing a constitutively active form of calcineurin
(CnA*) have elevated levels of utrophin A in their muscles (Proc. Natl. Acad.
Sci. USA, 100: 7791-7796, 2003). In the present study, we crossed these
transgenic animals with mdx mice to determine whether mdx/CnA* mice would be
less affected by the dystrophic process. Since expression of CnA* has been shown
previously to stimulate the slow/oxidative myofiber program, we examined the
expression of myosin heavy chain isoforms in mdx/CnA* and mdx mice. By
immunofluorecence and RT-PCR assays, we observed in mdx/CnA* a shift in myosin
heavy chain profile towards a slower, more oxidative phenotype compared to mdx
muscles. In addition, we determined that expression of utrophin A and its
transcript were increased by ~ 2 fold in mdx/CnA* mouse muscles. Immunodetection
of IgM inside myofibers, used in this case as an index of sarcolemmal disruption,
showed that the number of IgM-positive fibers was significantly reduced in
muscles from mdx/CnA* mice. Consistent with these findings, we also observed
that muscles from mdx/CnA* mice showed less variability in fiber size and
contained fewer central nuclei. Together, these results demonstrate that
enhanced calcineurin activity can have important beneficial effects on the
dystrophic phenotype by stimulating the expression of utrophin A. Furthermore,
these findings provide specific targets for which pharmacological strategies may
be designed to enhance utrophin levels in muscles from patients with Duchenne
muscular dystrophy. Funded by the MDA and CIHR.
USA -
No encontro dos cardiologistas foram
apresentados poucos artigos sobre o coração na distrofia muscular. Selecionei
os dois mais importantes: no primeiro os pesquisadores franceses utilizaram o
peridropril em portadores de Duchenne com função cardíaca adequada. O
peridropril é uma droga inibidora da enzima de conversão da angiotensina. Os
resultados demonstraram que o medicamento foi bem tolerada pelos pacientes
e retardaram o aparecimento da disfunção cardíaca nestes pacientes. No
segundo artigo os autores ingleses apresentaram os resultados de um novo método
de diagnóstico da doença cardíaca em portadores de distrofia muscular de
Duchenne. Este método se demonstrou mais eficiente e permitiu o diagnóstico
mais precoce que o ecocardiograma.
Perindopril
Prevents the Onset and Progression of Left Ventricular Dysfunction in Children
with Duchenne Muscular Dystrophy
Henri-Marc
Becana, Pitie-Salpetriere Hospital, Paris, France; Christophe Meune, Cochin
Hospital, Paris, France; Guy Lerebours, Pitie-Salpetriere Hospital, Paris,
France; Jean-Yves Devaux, Denis Duboc, Cochin Hospital, Paris, France; The
French Working Group on Heart Involvement in Myopathies
Background:
Duchenne Muscular disease (DMD) is an inherited X-linked disease due to the
absence of dystrophin and clinically characterized by progressive muscle
weakness and constant myocardial involvement responsible for sudden death or
end-stage heart failure in 40% of patients between the age of 15 and 30. The aim
of this study was to evaluate the preventive effect of early ACEI treatment on
left ventricular (LV) dysfunction in young patients with DMD and normal LVEF at
inclusion. Methods: In phase I, children with genetically proven
DMD and radionuclide ejection fraction (EF) superior than 55% were enrolled in a
multicentric, controlled, randomized, double blind trial of perindopril 2-4
mg/day versus placebo for 3 years. In phase II, all patients then received
open-label perindopril for 24 more months. Radionuclide LVEF was performed at 0,
36 and 60 months. Student's t-tests and chi-square analysis were used for
comparisons. Results: 60 children asymptomatic for heart disease
were included in the phase I study (10.6±1.2 years, EF 65.0±5.4%)(31 in the
perindropil group and 29 in the placebo group) and 46 in the phase II study
(n=23 in both groups). No adverse effect related to treatment was documented. At
the end of phase I, LVEF remained stable in both groups (from 64.8±5.3% to
59.6±8.5% in the perindopril group and 65.5±5.4% to 64.5±9.9% in the placebo
group, p=0.114). However, at 60 months, 6 patients in the control group (26%)
had a LVEF of less than 45, versus one in the perindopril group (4%)(p=0.0319). Conclusions:
Early treatment with ACEI perindopril is well tolerated in these young patients
and delay onset of LVEF deterioration. This preventive efficacy of ACEI has to
be evaluated in other groups of patients genetically exposed to develop LV
dysfunction.
Impaired Strain Rate Measurements
in Patients With Duchenne Muscular Dystrophy
Nickolaos Giatrakos, Cardiology
dept, Hammersmith Hospital, NHLI, ICSM, London, United Kingdom; Maria Kinali,
Dubowitz Neuromuscular Centre, Department of Paediatrics, Hammersmith Hospital,
ICSM, London, UK, London, United Kingdom; George Koutroulis, Cardiology dept,
Hammersmith Hospital, NHLI, ICSM, London, United Kingdom; Francesco Muntoni,
Dubowitz Neuromuscular Centre, Department of Paediatrics, Hammersmith Hospital,
ICSM, London, UK, London, United Kingdom; Petros Nihoyannopoulos; Cardiology
dept, Hammersmith Hospital, NHLI, ICSM, London, United Kingdom
Background: Patients with Duchenne
muscular dystrophy (DMD) develop dilated cardiomyopathy at the later stages of
the disease. Strain rate (SR) has been used to study myocardial function in
ischeamia and cardiomyopathies. The aim of this study was to investigate the
usefulness of SR for the early detection of cardiac involvement in young,
asymptomatic patients with DMD.
Methods: We studied 53 patients with genetically confirmed diagnosis of DMD
(mean age 8.7±2.8 years) without clinical symptoms from the cardiovascular
system and normal conventional echocardiographic studies, and compared with 22
normal controls matched for age (mean age 8.5±2.5 years). We used the HDI 5000
(Philips Medical Systems) to acquire from the parasternal long axis the colour
M-mode tissue Doppler (TDI) of the posterior wall of the LV. Images were
digitally stored for offline analysis with dedicated software HDI-lab (Philips
Medical Systems). We calculated the SR using the formula SR=Ua-Ub/d where U the
velocities of the endocardium a and epicardium b, and d the distance of a and b
at each time point.
Results: There was no significant difference for the parameters from
conventional echocardiographic studies between the two groups. The velocities
derived from the TDI, mean velocity at systole (26,99±7,12mm/sec vs.
33,4±7,3mm/sec, p<0,000), at early diastole (-45,79±13,93mm/sec
vs.-60,46±7,58mm/sec, p<0,000) and late diastole (-10,93±3,41mm/sec
vs.-13,32±6,4mm/sec, p<0,02) were significantly different in patients with
DMD when compared with controls. SR was found to be significantly lower at
systole (1,78±0,75s-1 vs. 2,82±0,5s-1, p<0,000) and
early diastole (-5,17±1,98s-1 vs.-9,02±1,25s-1,
p<0,000) but not at late diastole (-1,52±0,84s-1vs.-1.6±0,46s-1,
p<0,568) in patients with DMD.
Conclusions: Estimation of SR of the posterior wall of the LV showed systolic
and diastolic dysfunction at early stages in asymptomatic patients with DMD and
when the conventional echocardiography is still normal. Estimation of SR could
be a sensitive method to investigate the pathophysiology of the disease and
identify early impairment of the cardiac function.
USA - este congresso será
realizado em São Francisco a partir de 24 de abril de 2004 e é considerado um
dos maiores encontros de neurologistas do mundo. No entanto o número de artigos
falando sobre distrofia é muito baixo, muito inferior ao de outras doenças como
a esclerose múltipla e a doença de Parkinson. Além disso poucos destes resumos
abordam aspectos da terapia da doença. A maioria descreve algumas formas de
distrofia, características genéticas, etc. Isto demonstra um desinteresse por
este grupo de doentes, em especial com relação aos aspectos importantes da
doença como tratamento medicamentoso, fisioterapia, cirurgia, etc. Os resumos
que falam sobre distrofia (todas as formas) podem ser lidos em inglês
aqui.
USA - este é o congresso
mundial mais importante na área de doenças pulmonares. Neste ano o congresso
ocorrerá em Orlando, em maio e lá serão apresentados 8 trabalhos relacionados à
distrofia. Um dos artigos (o número 1) fala dos resultados positivos com terapia
genética para o músculo diafragma de camundongos com distrofia. O artigo sete
fala de uma nova forma de ventilação mecânica em pacientes com distrofia
muscular. O número 8 descreve os efeitos da associação entre corticóides e
creatina em ratos normais. Os resumos em inglês podem ser lidos
aqui. Encontrei também no site do congresso 22 artigos
apresentados nos últimos anos (2001-2003) nestes encontros. Há importantes
artigos falando na melhora da função pulmonar com o uso dos corticóides, nos
benefícios da ventilação não invasiva (que já é usada no exterior há 10 anos),
alterações do sono na distrofia, equipamentos de estimulação da tosse,
experimentos com terapia gênica em animais entre outros. Os resumos em inglês
podem ser lidos aqui.
USA - no Encontro que reunirá
os principais pesquisadores na área de terapia gênica serão apresentados 20
artigos que falam sobre esta forma de tratamento na distrofia muscular; a
maioria é experimental, em camundongos ou cães, e não trazem nenhum avanço
significativo. O único trabalho em humanos (artigo 7) fala do uso de mioblastos
na distrofia muscular, trabalho realizado no Canadá, e que já foi divulgado aqui
mais de uma vez (estão ainda com poucos crianças com distrofia muscular de
Duchenne e o tratamento está sendo realizado em apenas um músculo). Os resumos
em inglês dos artigos pode ser lido
aqui.
São Paulo - em 2003 eu
decidi realizar pesquisas com camundongos com distrofia muscular na Faculdade de
Medicina da Fundação do ABC, além de apoiar as pesquisas que estão sendo
realizadas em Ribeirão Preto, no Centro de Pesquisas em Distrofia Muscular da
AADM. O meu primeiro estudo experimental foi aprovado para apresentação no 44o
Encontro Anual da Sociedade Americana em Biologia Celular. Neste trabalho eu
estudei a droga tamoxifeno, que é utilizada para tratamento de mulheres com
câncer de mama. Eu decidi estudar esta droga porque ela atua sobre as TGF beta,
um grupo de proteínas que está alterado na distrofia muscular de Duchenne. Para
este estudo eu utilizei a droga nos camundongos mdx, que tem uma mutação do gene
da distrofina. Os animais que receberam a droga apresentaram aumento da força
muscular após a quinta semana que se manteve até o final do experimento na 12a
semana. Biópsias musculares foram realizadas e estão ainda sob análise (no
Centro de Estudos do Genoma Humano). As primeiras análises demonstram que os
animais tratados com tamoxifeno tem menor grau de degeneração muscular do que os
não tratados. Há ainda outros estudos bioquímicos que estão sendo realizados nos
músculos dos animais cujos resultados serão conhecidos em breve. Neste mês
estamos iniciando o estudo de uma segunda droga no mesmo modelo experimental. O
resumo do meu trabalho que será apresentado em Washington, USA, em dezembro de
2004 é o seguinte:
Tamoxifen increases muscular strength of the mdx dystrophic mice
NEW
(Abstract from 44th Annual
Meeting - American Society for Cell Biology (December, 4-8, 2004, Washington)
A. Cavalsan,1 R. F. Vasquez,1 R. C.
Lin,1 S. B. Zyngier,1 F. J. Velloso,2 B. H.
Santos,2 L. L. Fogaca,2 M. Vainzof,2
D. Feder1
; 1 Pharmacology, ABC Faculty of Medicine, Santo Andre, Brazil,
2 Human Genome Research Center - IB USP, Sao Paulo, Brazil
The mdx mice is a well-known model of Xp21 dystrophin-deficient muscular
dystrophy. Although a good genetic and biochemical model, the mdx shows no
muscle weakness, but under physical exercise, a loss of muscular strength can be
detected. Here we have tested the possible therapeutic beneficial effect of
Tamoxifen in the degenerative process of the dystrophic muscle, analyzing muscle
strength of tamoxifen treated mdx mice, under intensive physical exercise. A
total of 22 mices aged 4 weeks were divided into 3 groups: control (n= 8)
treated with 0.5 mL saline ip, Tamoxifen 5 mg/Kg body weight ip (n= 8) daily and
Tamoxifen 10 mg/Kg ip (n=6) daily. The exercise protocol was done in a wheel
revolving at 18 cm/s, for 10 minutes, twice a day, 5 days/week, up to 12 weeks.
Whole-body strength was measured weekly using a force transducer coupled to a
computer. Mice tails were attached via a non-flexibile nylon cord to the
transducer, the animals were electricly stimulated to run, and the force to pull
the cord was registered continuously. Results were analyzed by the Kruskal-Wallis
test. The mice treated either with 5 mg/Kg (6.26+ 1.44 dynes/g body
weight) or 10 mg/Kg (6.46+ 2.52) showed a significant increase of
muscular strength (p<0.05) compared to the control group (3.66+ 0.77)
starting in the 5th week, and maintaining significance up to the end
of the experiment. Histological and histochemical analysis of the complex of the
gastrocnemius muscle are under analysis, but preliminary results suggest a less
degree of degeneration in the Tamoxifen treated groups.
Tamoxifen is an anti tumoral drug and act on TGF-beta. Its possible therapeutic
effect in the degenerative process of the dystrophic muscle could ameliorates
the clinical course of dystrophic patients.
FAPESP-CEPID, CNPq, PRONEX, NEPAS-FMABC.
USA - neste Congresso que se
realizará em dezembro em Washington, além da minha pesquisa sobre tamoxifeno
outros estudos serão apresentados:
1) um estudo fala dos resultados
positivos com o uso do gene da distrofina retiniana humana utilizado em
camundongos sem distrofina e utrofina que melhoram os sintomas musculares; de
uma forma severa de distrofia eles passaram a apresentar poucos sintomas
musculares;
2) o segundo estudo fala da relação
entre calcificação ectópica de musculos com distrofia muscular pode ser um sinal
de regeneração e auxiliar na avaliação do tratamento com medicamentos;
3) o terceiro estudo trata dos níveis de
protoglicans em distrofia muscular de Duchenne em relação às outras formas da
doença; há redução dos níveis de protoglicans em Duchenne pode estar relacionado
aos altos níveis de TGF beta e TNF
4) O quarto estudo demonstra que as
células tronco originadas de músculos de camundongos fêmeas com distrofia
muscular são mais eficientes para a terapia gênica do que as células dos machos.
Os resumos em inglês destes estudos
estão a seguir:
Human Retinal
Dystrophin Transgene Converts Lethal Muscular Dystrophy into Viable Mild
Myopathy in Dystrophin-Utrophin Null Mice
R. Gaedigk,1,2 D. J. Law,3 K. M. Fitzgerald-Gustafson,4,2
S. G. McNulty,1 N. N. Nsumu,1 A. C. Modrcin,5
R. J. Rinaldi,5 D. Pinson,6 S. C. Fowler,7 M.
Bilgen,4,8 J. Burns,9 S. D. Hauschka,10 R. A.
White1,2 ; 1 Medical Research, Children's Mercy Hospitals
& Clinics, Kansas City, MO, 2 Pediatrics, UMKC School of Medicine,
Kansas City, MO, 3 School of Biological Sciences, Univ. of
Missouri-Kansas City, Kansas City, MO, 4 Hoglund Brain Imaging
Center, Univ. of Kansas Medical Center, Kansas City, KS, 5
Rehabilitation Medicine, Children's Mercy Hospitals & Clinics, Kansas City, MO,
6 Pathology & Laboratory Medicine, Univ. of Kansas Medical Center,
Kansas City, KS, 7 Pharmacology & Toxicology, Univ. of Kansas,
Lawrence, KS, 8 Molecular & Integrative Physiology, Univ. of Kansas
Medical Center, Kansas City, KS, 9 Veterinary Imaging Services,
Topeka, KS, 10 Biochemistry, Univ. of Washington, Seattle, WA
Duchenne muscular dystrophy (DMD) is a progressive muscle disease caused by
severe dystrophin gene mutations that often result in death by the third decade.
The mdx mouse is the most commonly used DMD model. Although they lack
dystrophin and have underlying muscle disease, mdx mice appear physically
normal. This may be partially due to compensatory expression of the dystrophin-related
protein, utrophin. In contrast, double mutant mice (DM), deficient for both
dystrophin and utrophin (mdx/+, utrn -/-), die by 3 months of age
and suffer from severe muscle weakness, pronounced growth retardation, kyphosis,
weight loss, slack posture, and immobility (Deconinck et al. (1997) Cell 90:
717-727; Grady et al. (1997) Cell 90: 729-738). These features make them an
excellent physiological model for DMD research. The capacity of a naturally
occurring isoform, human retinal dystrophin (Dp260), to compensate for the
missing muscle isoform Dp427 was tested in DM mice. The expression of this
transgene prevents premature death and reduces the severe muscular dystrophy
phenotype to a mild myopathy. Electromyography (EMG), histology, radiography,
magnetic resonance imaging, and behavior studies show that DM transgenic mice
grow normally, have normal spinal curvature and locomotion, and have reduced
muscle pathology. EMG and histologic data from transgenic DM mice are typical of
mild myopathy, while the DM mice exhibit severe abnormalities commonly seen in
human dystrophinopathies. The expression of human Dp260 in DM mice converts a
severe and lethal muscular dystrophy into a non-lethal mild myopathy.
Muscle-specific expression of Dp260 could have several advantages over other
treatments of DMD
Visualization of
Ectopic Calcification in mdx Mouse Skeletal Muscle
N. Kikkawa, T. Ohno, M. Shiozuka, R. Matsuda; The Graduate School of Arts and
Sciences, The University of Tokyo, Tokyo, Japan
It has been demonstrated that osteogenic differentiation of skeletal muscle
cells are induced by osteogenic factors, such as bone morphogenetic proteins,
both in vitro and in vivo. Spontaneous ectopic bone formation in vivo has also
been reported in, for instance, fibrodysplasia ossificans progressiva,
which is a rare bone disorder. Another example of self-generating ectopic
calcification has been found in skeletal muscle of mdx mouse, a model of
Duchenne muscular dystrophy (DMD).
We observed the ectopic calcification in mdx mouse thigh muscle by using
x-ray micro CT-scanner SkyScan-1074, which gives resolution of 22um. The x-ray
images were reconstructed into three dimentional visions. Calcifications were
found as spicular structures running parallel to the muscle fiber. Paraffin
sections of the regions found by X-ray microtomography were Von Kossa stained to
confirm calcium deposits. We also detected ectopic calcifications of living mice
with mouse-whole-body x-ray CT-scanner of Aloka. No ectopic calcification was
observed in skeletal muscle of B10 mouse, which possesses normal dystrophin gene
and was used as a negative control.
Calcified regions of mdx mouse thigh did not overlap with the Evans
blue-positive areas but corresponded to some of the regenerating areas. Thus,
ectopic calcification can be a diagnostic marker of muscle regeneration in
mdx mice, and be available to determine the effects of drug or gene
therapies.
Muscle
Proteoglycan Levels in Duchenne Muscular Dystrophy Differ from Other Muscular
Dystrophies
S. Zanotti, E. Canioni, F. Cornelio, L. Morandi, M. Mora; Neuromuscular Diseases
and Neuroimmunology, National Neurological Institute C. Besta, Milano, Italy
Biglycan and decorin are small leucine-rich extracellular proteoglycans that
interact with several matrix proteins particularly collagens, and also with
cytokines whose activity they may modulate. To better understand the role of
these proteoglycans in muscle fibrosis in muscular dystrophies, we investigated
the expression of their transcripts and proteins in several forms of muscular
dystrophy compared to age-matched controls. mRNAs of both proteoglycans were
significantly downregulated in Duchenne muscular dystrophy and significantly
upregulated in Becker muscular dystrophy, sarcoglycanopathies and
dysferlinopathy. By immunohistochemistry, decorin and biglycan were mainly
localized in connective tissue and apparently increased with age. Their presence
increased in relation to increased fibrosis in all types of dystrophic muscle.
The increase in biglycan and decorin in most muscle diseases indicates a role of
these proteoglycans in the extracellular matrix organization. The significant
decrease of decorin and biglycan mRNA in DMD distinguishes this disease from the
other studied and may be related to the high levels of TGF-beta1 and TNF-alpha
found in this disease.
Sex Differences in
Muscle Stem Cell Transplantation Efficiency
B. Deasy,1 A. Lu,1 K. Urish,1 J. Tebbets,1
B. Gharaibeh,1 R. Rubin,2 J. Huard1 ; 1
University of Pittsburgh, Pittsburgh, PA, 2 Allegheny General
Hospital, Pittsburgh, PA
We previously demonstrated that muscle-derived stem cells (MDSCs) efficiently
regenerate skeletal muscle tissue after transplantation into dystrophic mdx
mice, which model muscular dystrophy. Here we show that transplantation of
female MDSC (F-MDSC) rather than male MDSCs (M-MDSC) significantly improves
skeletal muscle regeneration.
We transplanted F-MDSCs into the gastrocnemius muscles of female mdx mice and
the same number of M-MDSCs into the gastrocnemius muscles of male mdx mice. We
used a previously described protocol to measure the cells’ regeneration index (RI)_the
number of dystrophin-positive fibers in the host muscle per 105 donor
cells_two weeks after transplantation. The F-MDSCs elicited significantly
superior muscle regeneration equivalent to a 6-fold increase in efficiency (F-MDSC
RI=686±120 versus M-MDSCs RI=105±25, P< 0.01). Sex-crossed
transplantation also demonstrated the same trend for the donor cells. We
hypothesize that this difference may be related to reduced ability of the M-MDSC
to self-renew or to tolerate hypoxic stress. F-MDSCs maintained CD34 expression
(>80% of total cells) for up to 200 population doublings, whereas M-MDSCs
exhibited a rapid drop in CD34 expression (from >80% to <30% of total cells)
after only 45 population doublings. To support the notion that the F-MDSC are
more tolerant to stress, we also found that proliferation of F-MDSC was less
affected by low O2 conditions as compared with M-MDSC.
Our discovery of sex-related differences in muscle stem cell biology reveals
clear limitations to using male stem and progenitor cells for cell-tracking
purposes. This finding also warrants careful consideration by researchers
working to identify optimal populations of stem and progenitor cells for use in
cell therapy and tissue engineering, clinicians who perform bone marrow stem
cell transplantation, and basic scientists investigating cell and developmental
biology.
Canadá - neste Congresso realizado em
Toronto de 26 a 30 de outubro de 2004 foram apresentados apenas dois trabalhos
relatando experimentos em terapia das distrofias musculares.
No primeiro trabalho realizado nos Estados
Unidos os pesquisadores apresentam uma nova forma de terapia gênica em
camundongos; eles associaram ao vetor viral uma proteína (fator de crescimento
do endotélio vascular); com esta terapia os camundongos passaram a expressar
distrofina em quantidade suficiente para reduzir os sintomas da doença. O vetor
viral foi injetado em uma veia, que é um passo importante para poder tratar
todos os músculos; a forma de administração do gene da distrofia muscular é um
dos problemas ainda não resolvido na terapia gênica da doença.
No segundo trabalho os pesquisadores
holandeses estudam os oligonucleotídeos para "consertar" o gene da distrofina.
Estudando células musculares humanas em laboratório ou camundongos com distrofia
muscular eles tem conseguido demonstrar que esta forma de tratamento é viável.
Estes oligonucleotideos tem que ser construidos para cada forma de mutação do
gene e eles tem desenvolvido vários destes oligonucleotideos para utilização em
seres humanos.
Os resumos destes dois trabalhos em inglês:
1) Body-wide delivery of a microdystrophin
gene via intravascular administration of rAAV6 vectors for treatment of muscular
dystrophy. P. Gregorevic, M. Blankinship, J. Allen, L. Meuse, S. Abmayr,
J. Han, J. Chamberlain. Senator Paul D. Wellstone Muscular Dystrophy
Cooperative Research Center, Dept. of Neurology, University of Washington,
Seattle, WA.
Severe neuromuscular disorders, such as Duchenne muscular dystrophy (DMD),
lead to reduced quality of life and premature mortality. Historically, genetic
interventions for these diseases have been limited by an inability to achieve
widespread gene transfer to the affected tissues. Here we demonstrate a method
that, for the first time, enables transduction of the vast majority of both the
cardiac and skeletal musculature of adult mammals via a single intravenous
administration of pseudotype 6 recombinant adeno-associated virus (rAAV6)
vectors. As a means to enhance gene transfer, we have determined that IV
coadministration of vascular endothelium growth factor (VEGF) with rAAV6 vectors
can significantly increase vector accumulation and transgene expression in
striated muscles. Having established in reporter gene studies that this
technique enables tolerable, high-level transgene expression, we sought to
evaluate the potential of this technique for delivering a therapeutic transgene
to the musculature of the mdx dystrophic mouse, a model of DMD. Using this
technique, we have observed that treated mdx mice exhibit expression of
microdystrophin throughout the striated musculature in a manner that is
sufficient to reduce pathological features of the dystrophic phenotype. Compared
with untreated mice, the muscles of treated animals exhibit reduced
susceptibility to contraction-induced injury, and reduced serum creatine kinase
levels, reflecting a global reduction in muscle degeneration. These data are the
first to demonstrate body-wide amelioration of symptoms associated with
dystrophin deficiency in an adult animal following a genetic intervention. In
subsequent studies we have established that these techniques can be adapted to
express alternate transgenes for the treatment of other muscle diseases. We are
currently undertaking studies to determine whether the existing techniques are
effective in animals more similar in size and immunology to humans, and present
relevant data summarizing our progress
2) Therapeutic antisense-induced exon
skipping for Duchenne muscular dystrophy. A. Aartsma-Rus1, A.
Janson1, G. Platenburg2, M. Bremmer-Bout1, J.T.
Den Dunnen1, J.C.T. Van Deutekom1, G-J.B. Van Ommen1.
1) LUMC, Leiden, the Netherlands; 2) Prosensa, Leiden, the Netherlands.
The severe Duchenne muscular dystrophy (DMD) is mostly caused by frame
disrupting mutations in the dystrophin gene, which result in non-functional
proteins. Mutations that keep the reading frame intact give rise to internally
deleted, semi-functional dystrophins and result in the milder Becker muscular
dystrophy (BMD). Antisense oligonucleotides (AONs) have the potential to
modulate the pre-mRNA splicing such that a specific exon is skipped. As a result,
the reading frame can be restored, which allows the synthesis of a BMD-like
dystrophin.
We have recently demonstrated the skipping of 20 different human DMD exons
using 2'-O-methyl RNA AONs with a full-length phosphorothioate backbone
(2OMePS), and confirmed the therapeutic applicability of this strategy in
cultures from 10 different DMD patients. We now compare the efficacy and
efficiency of 2OMePS AONs to morpholino, locked nucleic acid (LNA) and peptide
nucleic acid (PNA) AONs. While LNA was most efficient in inducing exon skipping,
we find that it acts in a less sequence-specific manner, probably due to its
extremely high RNA affinity. Awaiting further improvements in oligochemistry, we
therefore consider 2OMePS AONs currently the most favourable compounds.
To facilitate clinical application, multiple in vivo studies in animal
models are ongoing to develop safe and efficient AON-delivery methods. However,
as exon skipping is a sequence-specific therapy, it is desirable to directly
target the human DMD gene. We have therefore set up sequence-specific human exon
skipping in vivo in transgenic mice carrying the an intact copy of the
full-length 2.5 Mb human gene (hDMD). We injected 2OMePS AONs targeting
human exons 44, 46 and 49 into the m. gastrocnemicus of hDMD mice,
and showed skipping of the human (but not the murine) exons, which persisted for
at least 28 days. Based on pre-clinical data obtained by our group and others,
we are currently setting up a clinical trial aiming at local dystrophin
restoration following intramuscular injections of exon 46 and 51 specific AONs.
Espanha - realizou-se em junho de 2004
esta reunião de neurologistas europeus na qual foram apresentadas 4 pesquisas em
distrofia muscular. Na primeira os autores descrevem a melhora da força e da
fadiga muscular em camundongos com distrofia muscular utilizando duas drogas: a
IRFI 042, antioxidante, e a PDTC, um inibidor da NF-kB, uma proteína que causa
degeneração muscular. Na segunda os pesquisadores encontraram um pequeno
resultado com o transplante de células da medula óssea em camundongos com
distrofia muscular (baixa expressão da distrofina nos músculos). Na terceira
pesquisa os autores encontraram déficits de linguagem em portadores de distrofia
muscular de Duchenne (déficit em compressão de sintaxe e gramática, sem
dificuldade de leitura). Na quarta pesquisa os autores discutem as causas de
insuficiência respiratória na distrofia miotônica (apnéia do sono, fraqueza
muscular). Os trabalhos como foram apresentados no Congresso podem ser lidos
aqui:
1) Effect of treatment with PDTC and IRFI 042 on
strength and fatigue in MDX mice
S. Messina, P. Seminara, M. Aguennouz, M.C. Monici, H.
Marini, F. Squadrito, G. Vita (Messina, I)
Previous studies provided evidences that generation of
reactive oxygen species and activation of transcription factor NF-kB may play
important roles in the pathogenesis of Duchenne muscular dystrophy. We tested
whether IRFI 042, a vitamin E-like antioxidant, and PDTC, a NF-kB inhibitor,
could have an effect on muscle weakness in mdx mice. We treated 48 5/6-week old
mdx and wild type mice with intraperitoneal injections of PDTC (50 mg/kg), IRFI
042 (20 mg/kg), or vehicle, three times a week for five weeks. Data regarding
weight, survival and forelimb strength and fatigue were collected. Motor
performance measurements were carried out using a grip meter attached to a force
transducer which measures peak force generated. Mdx mice treated with IRFI 042
or PDTC showed at the end of treatment a significantly higher forelimb strength
than vehicle controls (IRFI 042: +53.6%, p<0.001; PDTC: +53.1%, p<0.05) as well
as higher strength normalised to weight (IRFI 042: +57.8%, p<0.001; PDTC: +54%,
p<0.05). Furthermore PDTC-treated mdx mice had significantly less fatigue than
vehicle animals (-120%, p<0.004).
Our results suggest that PDTC and IRFI 042 might have a
beneficial effect on weakness and fatigue in mdx mice. Further studies are
needed to investigate the morphological and biochemical substrates of such
encouraging preliminary results.
2) Bone marrow transplantation poorly restores
dystrophin expression in MDX mice: comparison of GFP and dystrophin expression
F. Chretien, P.A. Dreyfus, P. Caramelle, B. Chazaud, R.K.
Gherardi (Créteil, F)
We have previously developed a murine model of bone marrow
(BM) transplantation from B6-TgGFP transgenic mice to normal irradiated B6 mice,
the cytoplasmic green fluorescent protein (GFP) being used as an unambiguous
marker of donor-derived cells in host muscle. Using this model we were able to
demonstrate that stem cell marker-expressing cells found in connective tissue
and myogenic precursor cells (satellite cells) may be derived from bone-marrow
in adulthood. To investigate the therapeutic potential of BM transplantation in
muscle diseases such as dystrophinopathies, we compared the results obtained in
C57Bl6 (B6) mice and mdx mice, a model for Duchenne muscular dystrophy in which
muscle regeneration is dramatically increased. In mdx muscle compared to B6
muscle, we observed numerous GFP+ mononucleated cells corresponding to
macrophages and numerous necrotic fibers filled by GFP+ macrophages and
myoblasts. The number of GFP+ satellite cells number was similar in mdx and B6
mice 6 months post transplantation. However, GFP+ muscle fibers were more
numerous in mdx mice. They were from month 1 post transplantation, a time point
showing no GFP+ fiber in B6 mice. Six months post transplantation, 4 fold more
GFP+ muscle fibers were found in mdx mice compared to B6 mice.
In contrast, dystrophin expression was only mildly increased (0,65% at month 6)
and colocalizations between GFP and dystrophin was rarely found on serial cross
sections. We conclude that the nuclear domain of dystrophin is probably much
shorter than that of GFP.
3) Language disorders in Duchenne's muscular
dystrophy
F. Civati, M. Guglieri, M.G. D' Angelo, A. Tavano, F.
Fabbro, M.L. Lorusso, M. Sironi, A.C. Turconi, G.P. Comi, N. Bresolin (Bosisio
Parini, San Vito, Milan, I)
Background: Duchenne Muscular Dystrophy (DMD) is a fatal
recessive x-linked muscular disease caused by the absence of dystrophin.
Dystrophin isoforms are also expressed in the cerebral neocortex and in the
cerebellum. Cognitive impairment is described in 1/3 of DMD patients,
particularly in patients carrying deletions in the distal part of the gene.
Difficulties in verbal skills and reading abilities are more frequently
described in English speaking patients. According to the literature children
with DMD show linguistic deficits already in the early phases of language
development which mainly consist of poor expressive verbal abilities and
deficits in short-term memory. These observations suggest that in some cases
dystrophinopathy may be associated to language disorders. Preliminary results in
our group of Italian speaking children showed deficit in Grammatical and Syntax
Comprehension.
Aims of the study are:
1) to describe the language and reading ability in a group of italian DMD
children
2) to clarify the nature of the language and reading deficits
Patients: 13 children with DMD (mean age 8,3 years; standard deviation 1,7) were
diagnosed according to international standard criteria. Full Intelligence
Quotient(assessed using Wechsler Intelligence Scale) was >70.
Methods: Language and Reading abilities were determined through: “Test dello
sviluppo Morfosintattico”, Battery of standardized tests, Battery to evaluate
Dyslexia and Dysortography and Tasks of correctness and rapidity. To exclude
additional cognitive deficits we evaluated attention/executive functions domain
and memory and learning domain thought a Developmental Neuropsychological
Assessment (NEPSY particularly: Auditory attention and response set - Visual
attention - List learning - Memory for names).
Results: 8 patients out of 13 showed deficit in Syntax Comprehension. 7 patients
out of 13 manifested deficits in Grammatical Comprehension. No DMD patients
(except one) presented disabilities in reading. Most of the patients showed mild
attention and memory deficits.
Discussion: An early identification of the language and eventually
attention/memory difficulties, would be important for an early treatment, to
support DMD children in their learning course.
4) Sleep apnoea does not simply attribute to
respiratory failure in myotonic dystrophy: evaluation by somnography
H. Takada, S. Kon (Namioka, JP)
Sleep apnoea and hypopnoea have been reported in myotonic dystrophy (MD), but it
is unclear whether those are simply attributable to respiratory impairment as
well as respiratory muscle weakness. The aim of this study was to assess the
correlation of the disrupted nocturnal sleep and awake respiratory function, and
to deduce a mechanism of respiratory failure in MD. The study population
comprised eighteen patients with MD and seventeen patients with other types of
muscular dystrophy for a control group. Patients in the control group were
chosen to represent a similar range of the results of routine spirometry
examination, particularly the expected/observed forced vital capacity (%VC).
Arterial blood gases were also examined early in the morning. Sleep apnoea and
hypopnoea were evaluated in all patients using somnography (Sleep Tester LT-200,
Fukuda Denshi Co., Japan). The number of apnoea and hyponoea during night (NA)
was counted and types of apnoea were judged; the percentage of central type
apnoea (%CSA) and obstructive apnoea (%OSA) among total apnoea. Transcutaneous
recording of oxygen saturation during night were simultaneously performed. The
mean nadir and minimal value of oxygen saturation were obtained. The apnoea
index (AI) and the nocturnal hypoxia index (NHI) were calculated. The mean age
of the MD group was significantly younger than that of the control group (42.9
+/- 11.7 vs. 51.6 +/- 12.8 years). There was no significant difference in the
%VC between the two groups (63.0 +/- 15.4 vs. 65.7 +/- 23.4). The mean PaO2 in
MD was significantly lower and the mean PaCO2 in MD was higher than those in the
control (78.8 +/- 13.2 vs. 94.1 +/- 10.9 mmHg, 49.1 +/- 3.9 vs. 40.3 +/- 4.9
mmHg). The mean nadir (94.7 +/- 1.6 vs. 96.9 +/- 0.7 mm Hg) and minimal value
(73.0 +/- 15.9 vs. 85.6 +/- 7.5 mm Hg) of oxygen saturation in MD were
significantly lower than those in the control. The mean NHI in MD was
significantly higher than that in the control (96.1 +/- 45.3 vs. 36.0 +/- 16.7).
On the other hand, there were not significant differences in the NA, the %CSA,
the %OSA, or the AI between the two groups. Moreover, all the parameters for
apnoea did not correlate with the results of spirometry or arterial blood gases,
although the AI correlated with the NHI or the %CSA in MD. Our results suggest
that respiratory failure in MD is not simply owing to sleep apnoea or
respiratory muscle weakness, but might be due to impaired chemosensitivity or
ventiratory output.
USA - este congresso se realizará em
Miami, em abril de 2005. É o mais importante encontro de neurologistas. Eu
selecionei todos os resumos com temas em distrofia muscular. Os mais importantes
estão relacionados com a droga PTC 124, que começou a ser testada em seres
humanos para tratamento da mutação de ponto da distrofia muscular de Duchenne.
Outro artigo inovador fala do uso de corticóides em mulheres portadoras do gene
da distrofia muscular, com resultados positivos na melhora da fraqueza muscular
progressiva. Outro artigo fala do efeito positivo do uso da mexelitina para
controle da miotonia na distrofia miotônica e o outro relata que a droga
modafinil não melhora a sonolência diurna nesta forma de distrofia. Os resumos
em inglês de todos os trabalhos pode ser lido aqui.
USA - neste encontro que
realizar-se-á em julho, de 1 a 5, serão apresentados 29 artigos sobre terapia
gênica em distrofia muscular. A maioria das pesquisas é americana (12), seguida
do Canadá (7) e França e Japão (4 cada). A grande maioria é experimental (cães e
camundongos) e algumas são realizadas em células musculares humanas ou estudam a
terapia gênica em um único músculo. Apesar de promissoras não há nenhuma
pesquisa em andamento que permita a sua utilização.
Brasil - este Congresso reune
importantes pesquisadores na área da distrofia muscular, tanto na parte clínica
quanto experimental; inúmeros trabalhos serão apresentados neste encontro e eu
selecionei os principais, que se relacionam com a parte de tratamento das
doenças. Há muitos artgios falando dos benefícios dos corticóides em distrofia
muscular de Duchenne. Há também interessantes pesquisas na área de tratamento
das distrofias, inclusive com experimentos com células tronco e terapia gênica.
Os resumos são escritos em inglês e pelo volume de informação não é possível
traduzí-los na integra. Esta é a versão mais completa dos resumos do que a
colocada na página News in DMD. Os resumos podem ser lidos clickando abaixo:
Estudos clínicos
Estudos experimentais
USA - neste encontro serão
apresentados poucos artigos importantes relacionados com distrofia muscular. Os
principais resumos em inglês podem ser lidos aqui.
Os mais importantes são dois artigos descrevendo mediadores da fibrose muscular
em camundongos com distrofia muscular cuja inibição poderia retardar a evolução
da doença; outro artigo interessante é a pesquisa das células tronco que tem
maior capacidade de se diferenciar em células musculares.
USA - neste encontro de especialistas
em terapia gênica serão apresentados 30 pesquisas sobre o uso da terapia gênica
em diversas formas de distrofia muscular experimental. São estudos importantes
mas ainda sob pesquisa, não havendo nenhum estudo de aplicação imediata.
Bélgica - este congresso que ocorrerá
em outubro de 2006 é o maior encontro de especialistas em doenças
neuromusculares. Dos trabalhos que serão apresentados eu selecionei os que se
relacionam às distrofias musculares em especial com aspectos de tratamento (são
29 trabalhos que eu selecionei). Há várias pesquisas com terapia gênica,
corticóide, osteoporose mas muitas poucas pesquisas com drogas para uso
imediato. A maioria dos trabalhos é experimental mas há vários sobre Duchenne.
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