Bone marrow stem cells

New research, published in BioMed Central’s open access journal Stem Cell Research & Therapy, investigates the therapeutic use of human stem cells from bone marrow against acute lung injury and identifies TNF-a-induced protein 6 as a major molecular component of stem cell action.

Acute lung injury is a major complication of critically ill patients resulting in pulmonary edema, hypoxia and, in the worst cases, organ failure. Consequently up to 40% of all sufferers die because their bodies’ immune systems overreact in an attempt to repair the original lung damage.

Researchers based in Louisiana showed that therapy with human multipotent stromal cells (hMSC) isolated from bone marrow was able to significantly reduce acute lung injury in mice after 48 hours.

At 24 hours the therapy could be seen to be working, reducing the amount of pulmonary edema and protein in the lungs. While injury increased the number of white blood cells (neutrophils) tenfold, this could be reduced by treatment with hMSC.

When the researchers looked in more detail, they found that hMSC treatment reduced over half of the inflammatory proteins tested, including IL-1a, Il-1b, IL-6 and RANTES. hMSC treatment also increased the amount of the anti-inflammatory proteins TNF-a-induced protein 6 (TSG6) and Interleukin 1 receptor antagonist (IL-1RN).

Dr Sullivan, from the Center for Stem Cell Research and Regenerative Medicine at Tulane University School of Medicine, said: “Our research reveals an important mechanism behind the anti-inflammatory activity of stem cells in lung injury because blocking the activity of TSG6 using siRNA prevented most of the antiinflammatory effects of hMSC.

“Stem cell therapy shows great promise in the treatment of acute and life threatening conditions, such as acute lung injury and acute respiratory distress syndrome. Understanding the mechanisms by which hMSC dampen inflammation will likely provide strategies to improve the therapeutic potential of hMSCs for treatment of lung injury. Since it is a simple procedure to collect stem cells from bone marrow, we hope that our research paves the way forward into clinical trials.”

Cystic Fibrosis genomes

Johns Hopkins Institute for Genetic Medicine researchers working as part of the North American Cystic Fibrosis Consortium have discovered two regions of the genome that affect the severity of cystic fibrosis, a genetic condition that causes scarring throughout the body, affecting most notably the pancreas and lungs. Reporting online in Nature Genetics, the team describes the first-ever study to identify genetic variations that are associated with more severe cases of CF.

“We already know which gene causes CF, but to a large extent that gene does not by itself explain how severe the condition will be,” says Garry Cutting, M.D., a professor of pediatrics and member of the McKusick- Nathans Institute for Genetic Medicine at Johns Hopkins. “Now we’ve discovered new genes that influence the course of disease and may enable prediction of disease severity and, most importantly, the customisation of treatments for patients with unfavourable genetic modifiers – this is the realisation of individualised medicine.”

This study used samples from 3,467 patients that included unrelated patients from the Genetic Modifier Study out of University of North Carolina at Chapel Hill, unrelated patients from the Canadian Consortium for Genetic Studies out of University of Toronto, and related patients and their parents from the CF Twin and Sibling Study at Johns Hopkins. “Most CF patients born today live to their mid-30s, but that’s an average. Some succumb to the disease before their tenth birthday while others live into their 50s and we wanted to know why,” says Cutting.

“Of course we want to continue to push the median life expectancy up so that hopefully patients with more severe cases of CF will, with multimodal therapy, survive longer,” says Cutting. “And this is the first step toward developing such therapies for these patients.

Gene variation linked to infertility in women

A variation in a gene involved in regulating cholesterol in the bloodstream also appears to affect progesterone production in women, making it a likely culprit in a substantial number of cases of their infertility, a new study from Johns Hopkins researchers suggests. The Hopkins group has also developed a simple blood test for this variation of the scavenger receptor class B type 1 gene (SCARB1) but emphasised there is no approved therapy yet to address the problem in infertile women. Following up studies in female mice that first linked a deficiency in these receptors for HDL – the so-called “good” or “healthy” cholesterol – and infertility, researchers report finding the same link in studies of women with a history of infertility. If the new study’s findings hold up on further investigation, the John Hopkins team says they not only will offer clues into a genetic cause of some infertility, but could also lead to a treatment already shown to work in mice.

Between November 2007 and March 2010, Rodriguez and her colleagues analysed ovarian cells and fluid collected from 274 women unable to become pregnant for various reasons and undergoing in vitro fertilisation (IVF). Some 207 of them went on to have their eggs collected, fertilised in a test tube and implanted in their wombs. The scientists then measured whether there was evidence of a gestational sac or a fetal heartbeat 42 days after embryo transfer. None of the nine women in the group found to have the mutated SCARB1 had such evidence, meaning none were pregnant.

The researchers also showed that the nine women with the altered gene had low levels of progesterone, a hormone critical to sustaining pregnancy in its earliest stages, despite being supplemented with progesterone as part of the IVF process. Rodriguez says she believes the genetic variation could be present in 8% to 13% of the population.

Rodriguez, who is also director of the Johns Hopkins Diabetes and Cholesterol Metabolism Center, based her work on research with mice genetically engineered without the receptor for good cholesterol. Without the receptor, the mice had abnormally high levels of HDL in the blood since their bodies were unable to uptake the cholesterol. They were also at increased risk for heart disease, and the female mice were infertile.

The Massachusetts Institute of Technology researchers who studied the genetically engineered mice also found a treatment for their infertility in a cholesterol medication developed decades ago. Called probucol, it lowered levels of cholesterol circulating in the blood and restored the rodents’ fertility. The drug is no longer approved for use in the United States, partly because of concerns that it unsafely lowers HDL, but that very “side effect” seemed a good fit for mice with missing HDL receptors.

Rodriguez hopes to conduct a clinical trial to see if probucol can help infertile women with the gene variation get pregnant. She is also planning to collect data on HDL levels in infertile women with the genetic variation to see if that would prove to be an early clue to a genetic cause of their infertility.

Zebrafish genes turned off and on

Researchers have designed a new tool for identifying protein function from genetic code. A team led by Stephen Ekker, Ph.D., succeeded in switching individual genes off and on in zebrafish, then observing embryonic and juvenile development. The study appears in the journal Nature Methods.

The work could help shed light on health-related problems such as how cancerous cells spread, what makes some people more prone to heart attacks or how genes factor in addiction. More complicated issues, like the genetics of behaviour, plasticity and cellular memory, stress, learning and epigenetics, could also be studied with this method.

The research at Mayo Clinic’s Zebrafish Core Facility, that houses over 50,000 zebrafish could help further unify biology and genomics by describing the complex interrelations of DNA, gene function and gene-protein expression and migration.

The study includes several technical firsts in genetic research. Those include a highly effective and reversible insertional transposon mutagen. In nearly all loci tested, endogenous expression knockdown topped 99%.

The research yielded the first collection of conditional mutant alleles outside the mouse; unlike popular mouse conditional alleles that are switched from “on” to “off,” zebrafish mutants conditionally go from “off” to “on,” offering new insight into localised gene requirements. The transposon system results in fluorescence-tagged mutant chromosomes, opening the door to an array of new genetic screens that are difficult or impossible to conduct using more traditional mutagenesis methods, such as chemical or retroviral insertion.

The project also marks the first in vivo mutant protein trap in a vertebrate. Leveraging the natural transparency of the zebrafish larvae lets researchers document gene function and protein dynamics and trafficking for each protein-trapped locus. The research also ties gene/protein expression to function in a single system, providing a direct link among sequence, expression and function for each genetic locus.

Researchers plan to integrate information from this study into a gene codex that could serve as a reference for information stored on the vertebrate genome.

Researchers also exposed translucent zebrafish to transposons, “jumping genes” that move around inside the genome of a cell. The transposons instructed zebrafish cells to mark mutated proteins with a fluorescent protein ‘tag.’

New gene technology for IVF embryos

Researchers at the Johns Hopkins University School of Medicine have devised a new technique, which helps couples that are affected by or are carriers of genetic diseases have in vitro fertilised babies free of both the disease in question and other chromosomal abnormalities. The results were reported in the April issue of Fertility and Sterility.

Because embryos are so small and cells contain too little DNA to do extensive testing, researchers have in the past had to limit genetic testing of IVF embryos to either looking for a specific gene mutation that is known to exist in either parent or for other types of chromosomal abnormalities such as the existence of too many or too few chromosomes (aneuploidy) or other structural chromosomal aberrations. By a method of trial and error that lasted approximately one year, Paul Brezina, M.D., M.B.A., a clinical fellow in obstetrics and gynecology and William G. Kearns, Ph.D., associate professor of obstetrics and gynecology optimised a technique they call “modified multiple displacement amplification” that allows them to amplify or make carbon copies of the DNA they obtain from an embryo obtained by in vitro fertilisation, enough to do multiple tests.

Couples who already have a child affected by a genetic disease such as Cystic Fibrosis or Tay-Sachs have been turning to in vitro fertilisation (IVF) coupled with preimplantation genetic diagnosis (PGD), genetic testing prior to implanting the embryos into the mother’s uterus, to become pregnant again. In PGD, which is also called single-gene testing, doctors remove either one cell from an IVFconceived three-day old embryo, which contains only eight cells total, or a few cells from a five-day old embryo, which contains about 150 cells total. Removing more cells from the embryo is also an unviable option as it can compromise its health and development. They then test the DNA from these cells for the diseasecausing genetic alteration. They then implant back into the mother only those embryos that will give rise to a baby free of the disease.

However, as much of a boon as PGD is, babies conceived in this manner are still exposed to other genetic risks, says Brezina, the most common being the gain or loss of chromosomes, a condition called aneuploidy. Aneuploidy can cause several diseases, the most commonly known of which is Down syndrome.

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