sábado, 14 de maio de 2011

Reprogrammed cells trigger immune reactions in mice

Medical applications of induced pluripotent stem cells called into question.
Erika Check Hayden
iPS cellsAn infiltration of T cells, shown by dark brown color, can be seen in the tissues formed by induced pluripotent stem cells.Yang Xu, UC San Diego
Cells that have been reprogrammed to grow into different types of tissue might be rejected by the body — even when they are transplanted into the individual from whom they are made, researchers report in a study published today in Nature1.
The study was led by Yang Xu, a molecular biologist at the University of California, San Diego. It will shake up the regenerative-medicine field, because until now, most scientists have assumed that reprogrammed cells made from an individual's own tissue could be safely transplanted back into the same person.
"This is a surprise; it's going to put a spanner in the works for the whole field," says Paul Fairchild, an immunologist and stem-cell biologist at the University of Oxford, UK.
The latest study looked at mouse embryonic stem cells and mouse induced pluripotent stem (iPS) cells. Both types of cell are pluripotent, meaning that they can grow into many other cell varieties.

Potential for change

Xu's team transplanted the cells into mice with the same genetic make-up as those that had donated the source cells. This mimics transplantation of cells from one human individual back into the same individual.
When transplanted, the embryonic stem cells gave rise to teratomas — tumours containing a chaotic jumble of cell types, which are used as a signifier of a cell's pluripotency. Most of the iPS cells, by contrast, were not able to form teratomas, or made teratomas that were attacked or rejected by the immune systems of the host mice.
"We expected that iPS cells generated from patients would be able to be transplanted back into patients," says Nissim Benvenisty, a stem-cell biologist at the Hebrew University of Jerusalem. "This paper indicates that that may not be the case."
The team found that certain genes were expressed at much higher levels in the teratomas formed by iPS cells than in those formed by embryonic stem cells. Two of the genes — Zg16 and Hormad1 — were specifically targeted in immune attacks. Xu suggests that these genes are normally turned off by the time a fetus begins the process of developing immune tolerance to its own tissues, so they are not recognized as 'self' by the host's body; the iPS reprogramming procedure might alter the normal expression of these genes.

Facing rejection

But Xu's study is not necessarily the dire news for the iPS field that it might seem.
Researchers working with iPS-derived cells that have matured to an adult fate — for instance, neurons or heart cells — have been able to transplant them into mice without rejection, but these experiments have mostly looked at mice without functional immune systems. And scientists designing therapies are mostly proposing to transplant only one type of differentiated cell at a time made from patients' own skin cells back into their bodies, rather than the jumble of differentiated cells found in a teratoma.
Xu and other researchers don't yet know whether purified differentiated iPS-derived cells would be rejected, or whether the problem is specific to undifferentiated cells.
Xu's study "doesn't really mirror the clinical situation at all", says Fairchild.
Fairchild points out that the iPS cells in Xu's study were derived from embryonic skin cells, rather than from adult skin cells, as would be the case for human patients. Perhaps these immature skin cells are more likely than adult cells to trigger an immune reaction. And he adds that it is not clear which cells in the teratomas triggered the immune rejection, or whether human cells would behave in the same way. Until these questions are resolved, Fairchild says, Xu's paper "might cast a shadow over the whole field of regenerative medicine unnecessarily".
Joseph Wu, a transplant biologist at Stanford University in Palo Alto, California, agrees that the paper "may trigger additional regulatory concerns" for iPS-cell derived patient-specific transplants.
The finding comes on top of the publication of a spate of studies suggesting that iPS cells might contain more genetic abnormalities than embryonic stem cells2. The US Food and Drug Administration heard concerns about genetic mutations in iPS cells at a meeting in Bethesda, Maryland, in March.

Fine-tuned therapies

Wu does not think that companies would be interested in developing patient-specific iPS-derived therapies. They are more likely to focus on cell-derived therapies that can be used in many people, which would require suppression of the immune system anyway.
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"The overall significance of the finding is that more research [needs] to be done to examine whether iPS cells are immunoprivileged or not," says Wu.
Xu agrees. His group's next steps will be to examine which specific cells in the teratomas trigger immune rejection, and under what conditions. The team used two different methods to make the iPS cells, and they showed slightly different propensities to trigger immune rejection, so it may be that reprogramming methods can be fine-tuned to avoid the problem altogether.
"We propose that the technology to generate iPS cells needs to be improved in order to minimize the difference between iPS and embryonic stem cells, so that iPS cells can be more useful in human therapies," says Xu. 

  • References

    1. Zhao, T. , Zhang, Z.-N. , Rong, Z. & Xu, Y. Naturedoi:10.1038/nature10135 (2011).
    2. Pera, M. F. Nature 471, 46-47 (2011).
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WHO to decide fate of smallpox stocks

Heated debate expected next week over when to destroy lab samples of deadly virus.
Declan Butler
Child with smallpoxDeveloping countries fear that remaining stocks of smallpox raise the risk of the disease returning.NYPL/SCIENCE SOURCE/SCIENCE PHOTO LIBRARY
Health ministers from the World Health Organization's (WHO's) 193 member states will next week debate when to destroy the two last known remaining stocks of the virus that causes smallpox, a scourge that was eradicated in 1980.
Many scientists argue, however, that the variola stocks should be maintained, perhaps indefinitely. The stocks are helping the development of new countermeasures such as drugs, vaccines and diagnostics in case smallpox should reappear, and may also allow researchers to explore the impact of smallpox on the human immune system, providing insights into other diseases such as AIDS.
After smallpox was eradicated, the WHO quickly reached a consensus that existing lab stocks should be destroyed to eliminate the risk of accidental release, and a deadline of 1993 was set. By 1984, stocks from 74 laboratories had been either destroyed or transferred to the two WHO-sanctioned repositories – the US Centers for Disease Control and Prevention in Atlanta, Georgia, and the Russian State Research Center of Virology and Biotechnology in Koltsovo, near Novosibirsk.
Many developing countries that would probably bear the brunt of any accidental release have long backed the destruction plan. But the WHO has repeatedly pushed back the deadline under pressure from developed countries, including the United States, who want to continue research on the virus. At a meeting in Geneva, Switzerland, next week, the United States will seek a further reprieve for the stocks.
The disagreement stems from big differences in the perception of risks and benefits among many developed and developing countries, says David Heymann, chairman of the board of UK Health Protection Agency, and former Assistant Director-General for Health Security and Environment at the WHO, where he also oversaw efforts to eradicate polio.
Many poorer countries view smallpox research as a potentially dangerous luxury, he explains, whereas developed countries' main concern is to continue research that would protect against the consequences of a deliberate release by rogue states or terrorists, who may have access to undeclared stocks.
"There are adequate, if not overwhelming, reasons to be concerned that the two repositories that are under debate right now are not the only places in the world where live smallpox virus still exists," says Nils Daulaire, director of the Office of Global Health Affairs at the US Department of Health and Human Services, and head of the US delegation to the Geneva meeting. "As long as that possibility exists, I think the world is a much safer place with the development of these countermeasures." The United States remains committed to ultimate destruction of the stocks, he adds: "If we knew for a fact when we would have these smallpox vaccines and antivirals fully developed and fully licensed, we would have no problem with a clear timetable for destruction."
Many scientists also argue that destroying the stocks would do little to protect the world from smallpox, because it is now possible to recreate the virus from its genome, which was sequenced in 1994. Only a handful of labs have the skills and resources to do so, but it is likely to become easier over time as DNA-synthesis machines become faster and cheaper.

Pox progress

The WHO last considered destroying the lab stocks in 2007, when it postponed the decision pending an assessment of whether more research on the live variola virus was needed. The WHO has emphasized that science alone cannot justify retention, and that any research must have tangible public-health benefits.
The review panel convened to address the matter was not asked to assess whether the stocks should be destroyed, although its report, published last December, made a strong case for continued research. Perhaps reflecting the debate's complex politics, the WHO asked a separate expert panel (AGIES), made up largely of public-health experts from developing countries, to review the scientific report and recommend whether further research was warranted. They argued that it is largely not.
The AGIES panel's "perception was flavoured by its public-health perspective on scientific arguments", says Grant McFadden, a poxvirus researcher at the University of Florida in Gainesville and a member of the scientific review panel. He believes that the risks of accidental release are low compared with the scientific and public-health benefits.
McFadden points out that the past decade has seen much progress in smallpox research, in particular the development of two new drugs (which have not yet been licensed). Current vaccines are effective, but are unsuitable for people with compromised immune systems, including people with HIV.
The key problem for research on smallpox is that there is no good animal model for the disease. "Human smallpox doesn't cause disease in non-human primates that resembles the human disease," says McFadden. Infections of other poxviruses in their natural hosts provide more realistic, yet still imperfect, models.
Because clinical trials of new drugs and vaccines for the disease are impossible, the US Food and Drug Administration and other regulatory agencies currently demand that vaccines and drugs show efficacy in animals challenged with live smallpox. The WHO's AGIES panel argued, however, that the regulatory system should be changed to allow licensing of smallpox drugs and vaccines on the basis of multiple animal surrogates, using various poxviruses in their natural hosts. The only remaining need for the live virus was to test the efficacy of drugs in vitro, it said.
But the entire debate over regulatory needs may be moot, says Jonathan Tucker, a biosecurity researcher at the Federation of American Scientists in Washington DC. Any likely use of drugs or vaccines against smallpox would come under emergency regulatory provisions that allow the use of treatments that have not been completely tested.
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The outcome of next week's meeting is difficult to predict. WHO resolutions are not legally binding under international law, so the body usually seeks to pass them by consensus, with very few going to a vote. "I'm very confident that at the end of the day there will be a resolution accepted by consensus that will maintain a programme of research and that will call for a review of progress in a number of years," says Daulaire.
If a vote did result in a decision to destroy the stocks, the United States and Russia would be left in a difficult position, says Tucker. They could comply with the WHO's request or defy it – or perhaps, more worryingly, they could be tempted to maintain their stocks covertly. 

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Cancer: The flipside of Notch

Nature
 
473,
 
159–160
 
(12 May 2011)
 
doi:10.1038/473159a
Published online
 
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Structure and function of a membrane component SecDF that enhances protein export

Nature
 
(2011)
 
doi:10.1038/nature09980
Received
 
 
Accepted
 
 
Published online
 
Protein translocation across the bacterial membrane, mediated by the secretory translocon SecYEG and the SecA ATPase1, 2, 3, 4, is enhanced by proton motive force5, 6 and membrane-integrated SecDF7, 8, 9, which associates with SecYEG. The role of SecDF has remained unclear, although it is proposed to function in later stages of translocation as well as in membrane protein biogenesis4, 10, 11, 12, 13. Here, we determined the crystal structure of Thermus thermophilus SecDF at 3.3 Å resolution, revealing a pseudo-symmetrical, 12-helix transmembrane domain belonging to the RND superfamily and two major periplasmic domains, P1 and P4. Higher-resolution analysis of the periplasmic domains suggested that P1, which binds an unfolded protein, undergoes functionally important conformational changes. In vitro analyses identified an ATP-independent step of protein translocation that requires both SecDF and proton motive force. Electrophysiological analyses revealed that SecDF conducts protons in a manner dependent on pH and the presence of an unfolded protein, with conserved Asp and Arg residues at the transmembrane interface between SecD and SecF playing essential roles in the movements of protons and preproteins. Therefore, we propose that SecDF functions as a membrane-integrated chaperone, powered by proton motive force, to achieve ATP-independent protein translocation.
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Accession codes

Primary accessions

Biological Magnetic Resonance Data Bank

Protein Data Bank

Author information

  1. These authors contributed equally to this work.

    • Tomoya Tsukazaki & 
    • Hiroyuki Mori

Affiliations

  1. Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan

    • Tomoya Tsukazaki,
      •  
    • Yuka Echizen,
      •  
    • Ryuichiro Ishitani &
      •  
    • Osamu Nureki

  2. Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan

    • Hiroyuki Mori

  3. Structural Biology Laboratory, Life Science Division, Synchrotron Radiation Research Organization, The University of Tokyo, Tokyo 113-0032, Japan

    • Shuya Fukai

  4. Laboratory of Macromolecular Complexes, Center for Structural Biology of Challenging Proteins, The University of Tokyo, Tokyo 113-0032, Japan

    • Shuya Fukai

  5. Mitsubishi Kagaku Institute of Life Sciences, Machida-shi, Tokyo 194-8511, Japan

    • Takeshi Tanaka &
      •  
    • Toshiyuki Kohno

  6. Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA

    • Anna Perederina &
      •  
    • Dmitry G. Vassylyev

  7. Bioengineering Department, Nagaoka University of Technology, Niigata 940-2188, Japan

    • Andrés D. Maturana

  8. Kyoto Sangyo University, Kita-ku, Kyoto 603-8555, Japan

    • Koreaki Ito

Contributions

T.Tsukazaki performed the structural determination and the biochemical experiments with SecDF. H.M. performed the functional analyses of SecDF. Y.E. solved the crystal structure of the SecDF P1 domain and assisted with the functional analysis of SecDF. R.I., S.F., D.G.V. and O.N. assisted with the structural determination. A.D.M. performed patch clamp and pH fluorescence experiments. T.Tanaka and T.K. solved the structure of the P4 domain by NMR. A.P. and D.G.V. assisted with the crystallization and data collection of SecDF. All authors discussed the results and commented on the manuscript. O.N. and K.I. supervised the work and wrote and edited the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to: 
The coordinates and structure factors have been deposited in the Protein Data Bank under the accession codes 3AQP for the entire TtSecDF protein and 3AQO for the P1 domain. The PDB and BMRB codes for the deposited P4 domain are 2RRN and 11426 respectively.

Supplementary 

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