One of 100,000 children is born with Menkes disease, a genetic disorder that affects the body’s ability to properly absorb copper from food and leads to neurodegeneration, seizures, impaired movement, stunted growth and, often, death before age 3. Now, a team of biochemistry researchers at the University of Missouri has published conclusive scientific evidence that the gene ATP7A is essential for the dietary absorption of the nutrient copper. Their work with laboratory mice also provides a greater understanding of how this gene impacts Menkes disease as scientists search for a treatment.

Humans cannot survive if their bodies are lacking the ATP7A gene, yet children can develop Menkes disease when the gene is mutated or missing. Previously, scientists did not have a good model to test the gene’s function or develop an understanding of the underlying causes of the disease symptoms. In his new study, Michael Petris, associate professor of biochemistry, was able to modify mice so that they were missing the ATP7A gene in certain areas of the body, specifically the intestinal track where nutrient absorption takes place.

‘These findings help us to understand where in the body the function of this gene is vital and how the loss of the gene in certain tissues can give rise to Menke’s disease,’ said Petris, who is a researcher in the Bond Life Sciences Center and holds an appointment in the Department of Nutrition and Exercise Physiology. ‘We want to continue to explore the underlying biology of Menke’s disease to determine where we should focus our research efforts in the future. If we know which organs or tissues are most responsible for transporting copper throughout the body, we can focus on making sure the gene is expressed in those areas. This disease is ideal for gene therapy down the road.’

Petris found that young mice missing the ATP7A gene in their intestinal cells were unable to absorb copper from food, resulting in an overall copper deficiency that mimics symptoms of Menkes disease in children. Petris says it’s vital to ensure that the developing newborns absorb enough copper during the neonatal period when the demand for the mineral is highest.

‘Copper is a little-appreciated but essential trace mineral in all body tissues,’ Petris said. ‘Cells cannot properly use oxygen without copper; it helps in the formation of red blood cells, and it helps keep the blood vessels, nerves, skin, immune system and bones healthy. Normally, people absorb enough copper through their food. However, in the bodies of those with Menkes disease, copper begins to accumulate at abnormally low levels in the liver and brain and at higher than normal levels in the kidney and intestinal lining.’

Newborn screening for this disorder is not routine, and early detection is infrequent because it can arise spontaneously in families, Petris said. Many times, the disease is not detected until the symptoms are noticed, and by that time, it can be too late for any aggressive treatments.

‘The clinical signs of Menkes disease are subtle in the beginning, so the disease is rarely treated early enough to make a significant difference,’ he said. ‘However, a single dose of copper injected into mice within a few days of birth restored normal growth and life expectancy. Early intervention was critical because treatment that began after symptoms developed wasn’t successful.’

Petris says that understanding the roles of copper in biology may have far-reaching health implications for the general population because copper underpins many facets of biology, including the growth of cancer tumours and the formation of toxic proteins in Alzheimer’s disease.

The development of these mice provides a novel experimental system in which to test treatments for patients with this disease. The early-stage results of this research are promising, but additional studies are needed. University of Missouri

A Johns Hopkins Children’s Center study in mice may help explain why women are more prone than men to a form of liver damage by implicating the female sex hormone oestrogen in the development of autoimmune hepatitis.
A life-threatening condition that often requires transplantation and accounts for half of all acute liver failures, autoimmune hepatitis is often precipitated by certain anaesthetics and antibiotics. Researchers say these drugs contain tiny molecules called haptens that ever so slightly change normal liver proteins, causing the body to mistake its own liver cells for foreign invaders and to attack them. The phenomenon disproportionately occurs in women, even when they take the same drugs at the same doses as men.
Results of the new study reveal that oestrogen and a signalling molecule called interleukin-6 collude to form a powerful duo that leads to immune cell misconduct and fuels autoimmune liver damage.
The findings, the research team says, also suggest therapeutic strategies to curb damage in people who develop drug-induced liver inflammation.
‘Our study shows that oestrogen is not alone in its mischief but working with an accomplice to set off a cascade of events that leads to immune cell dysregulation and culminates in liver damage,’ says Dolores Njoku, M.D., a pediatric anaesthesiologist and critical care expert at Johns Hopkins Children’s Center.
In the study, led by Njoku, researchers induced liver inflammation in mice by injecting them with drug-derived haptens. Female mice developed worse liver damage than male mice, and castrated male mice fared worse than their intact brethren, likely due to loss of testosterone and altered oestrogen-to-testosterone ratio, the researchers say. Female mice with missing ovaries — the chief oestrogen-secreting organs — suffered milder forms of hepatitis than mice with intact ovaries.
Female mice produced more liver-damaging antibodies and more inflammation-triggering chemicals, specifically the inflammatory molecule interleukin-6, known to fuel autoimmunity. Liver damage was notably milder in female mice whose interleukin-6 receptors were blocked or missing compared with normal female mice. On the other hand, male mice and female mice with missing ovaries had nearly undetectable levels of interleukin-6, while castrated male mice showed simultaneous upticks in both oestrogen and interleukin-6.
The research team further zeroed in on a class of cells known as regulatory T cells, whose main function is keeping tabs on other immune cells to ensure they don’t turn against the body’s own tissues. When researchers compared the number of regulatory T cells present in the spleens of male and female mice, they noticed far fewer regulatory T cells in the spleens of female mice. The spleen, the researchers explain, is the primary residence of mature immune cells.
‘Deficiency of regulatory T cells effectively takes the reins off other immune cells, leading to overactive immunity,’ Njoku says.
In a final, dot-connecting move, the researchers immersed spleen-derived immune cells in oestrogen. What they observed proved beyond doubt that oestrogen, interleukin and regulatory T cells form a powerful triangle. Oestrogen induced the immune cells of female mice to express more interleukin-6, which in turn diminished the expression of inflammation-taming regulatory T cells.
When the researchers injected sick female mice with a booster dose of regulatory T cells, their liver inflammation subsided to levels seen in male mice.
This powerful response, the researchers say, suggests that therapy with regulatory T cells may reduce
estrogen-related liver damage in patients with autoimmune hepatitis. Such treatment, however, remains years away from human application.
One reason, the researchers say, is that regulatory T cells maintain the fine equilibrium between overactive and underactive immunity. Because an overactive immune system can lead to autoimmune diseases and an underactive one can promote tumour growth, any therapy with regulatory T cells must be precisely calibrated to avoid tipping this precarious balance.
‘We first must figure out where the golden mean lies,’ Njoku says. John Hopkins Medicine

Researchers have uncovered mutations in the phosphatase Wip1 that enable cancer cells to foil the tumour suppressor p53. The results could provide a new target for the treatment of certain cancers.
Like a battlefield surgeon who has to decide which casualties can be saved, p53 performs triage on cells with injured DNA. If the damage is serious, p53 spurs the cells to die or stop proliferating. But after milder hits, p53 activates a DNA damage response (DDR) mechanism, which instigates repairs, and temporarily prevent cells from advancing any farther in the cell cycle. Once cells have mended their DNA, the phosphatase Wip1 enables them to re-enter the cell cycle by shutting down p53 and DDR proteins. Because p53 and the DDR stymie cancer cells, it’s no surprise that the rogue cells find ways to circumvent this protection. More than half of all cancers accrue mutations in the p53 gene, for example. Now, researchers from the Czech Republic and the Netherlands tested whether some cells instead carry mutations in the PPM1D gene, which encodes Wip1, to shut down p53.
The team analysed human tumour cell lines that harbour functional p53. Two of the lines displayed mutations in exon 6 of the PPM1D gene that resulted in a shortened version of Wip1. The truncated Wip1 was more stable than the full-length version of the protein, allowing cells to switch off p53 and continue the cell cycle in the presence of DNA damage. Depleting the truncated Wip1, however, halted the cell cycle until the DNA was repaired.
The researchers then looked for PPM1D mutations in 1,000 patients who had colorectal or breast and ovarian cancer. Four of the patients carried mutations, whereas none of the 450 cancer-free individuals did. All of these DNA alterations fell in exon 6 and caused production of shortened Wip1. To the researchers’ surprise, the mutations occurred in the cancer patients’ non-tumour cells as well. That suggests that the patients were born with PPM1D mutations, which set them up for cancer later in life but apparently caused no other illnesses.
‘We’ve identified a new mechanism that could lead to inactivation of p53 in cells and inactivation of the DNA damage response,’ says senior author Libor Macurek. The team suspects that PPM1D mutations could turn up in a variety of tumours. If so, targeting the short but overactive form of Wip1 could provide a new way to treat these cancers. EurekAlert

A handheld diagnostic device that Massachusetts General Hospital (MGH) investigators first developed to diagnose cancer has been adapted to rapidly diagnose tuberculosis (TB) and other important infectious bacteria. Two papers describe portable devices that combine microfluidic technology with nuclear magnetic resonance (NMR) to not only diagnose these important infections but also determine the presence of antibiotic-resistant bacterial strains.
‘Rapidly identifying the pathogen responsible for an infection and testing for the presence of resistance are critical not only for diagnosis but also for deciding which antibiotics to give a patient,’ says Ralph Weissleder, MD, PhD, director of the MGH Center for Systems Biology (CSB) and co-senior author of both papers. ‘These described methods allow us to do this in two to three hours, a vast improvement over standard culturing practice, which can take as much as two weeks to provide a diagnosis.’
Investigators at the MGH CSB previously developed portable devices capable of detecting cancer biomarkers in the blood or in very small tissue samples. Target cells or molecules are first labelled with magnetic nanoparticles, and the sample is then passed through a micro NMR system capable of detecting and quantifying levels of the target. But initial efforts to adapt the system to bacterial diagnosis had trouble finding antibodies – the detection method used in the earlier studies – that would accurately detect the specific bacteria. Instead the team switched to targeting specific nucleic acid sequences.
The system detects DNA from the tuberculosis bacteria in small sputum samples. After DNA is extracted from the sample, any of the target sequence that is present is amplified using a standard procedure, then captured by polymer beads containing complementary nucleic acid sequences and labelled with magnetic nanoparticles with sequences that bind to other portions of the target DNA. The miniature NMR coil incorporated into the device – which is about the size of a standard laboratory slide – detects any TB bacterial DNA present in the sample.
Tests of the device on samples from patients known to have TB and from healthy controls identified all positive samples with no false positives in less than three hours. Existing diagnostic procedures can take weeks to provide results and can miss up to 40 percent of infected patients. Results were even stronger for patients infected with both TB and HIV – probably because infection with both pathogens leads to high levels of the TB bacteria – and specialized nucleic acid probes developed by the research team were able to distinguish treatment-resistant bacterial strains.
Another paper describes a similar system using ribosomal RNA (rRNA) – already in use as a bacterial biomarker – as a target for nanoparticle labelling. The investigators developed both a universal nucleic acid probe that detects an rRNA region common to many bacterial species and a set of probes that target sequences specific to 13 clinically important pathogens, including Streptococcus pneumoniae, Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA).
The device was sensitive enough to detect as few as one or two bacteria in a 10 ml blood sample and to accurately estimate bacterial load. Testing the system on blood samples from patients with known infections accurately identified the particular bacterial species in less than two hours and also detected two species that had not been identified with standard culture techniques.
While both systems require further development to incorporate all steps into sealed, stand-alone devices, reducing the risk of contamination, Weissleder notes that the small size and ease of use of these devices make them ideal for use in developing countries. ‘The magnetic interactions that pathogen detection is based on are very reliable, regardless of the quality of the sample, meaning that extensive purification – which would be difficult in resource-limited setting – is not necessary. The ability to diagnose TB in a matter of hours could allow testing and treatment decisions within the same clinic visit, which can be crucial to controlling the spread of TB in developing countries.’ Massachusetts General Hospital

In the summer of 1968, a new strain of influenza appeared in Hong Kong. This strain, known as H3N2, spread around the globe and eventually killed an estimated 1 million people.
A new study from MIT reveals that there are many strains of H3N2 circulating in birds and pigs that are genetically similar to the 1968 strain and have the potential to generate a pandemic if they leap to humans. The researchers, led by Ram Sasisekharan, the Alfred H. Caspary Professor of Biological Engineering at MIT, also found that current flu vaccines might not offer protection against these strains.
‘There are indeed examples of H3N2 that we need to be concerned about,’ says Sasisekharan, who is also a member of MIT’s Koch Institute for Integrative Cancer Research. ‘From a pandemic-preparedness point of view, we should potentially start including some of these H3 strains as part of influenza vaccines.’
The study also offers the World Health Organization and public-health agencies’ insight into viral strains that should raise red flags if detected.
In the past 100 years, influenza viruses that emerged from pigs or birds have caused several notable flu pandemics. When one of these avian or swine viruses gains the ability to infect humans, it can often evade the immune system, which is primed to recognise only strains that commonly infect humans.
Strains of H3N2 have been circulating in humans since the 1968 pandemic, but they have evolved to a less dangerous form that produces a nasty seasonal flu. However, H3N2 strains are also circulating in pigs and birds.
Sasisekharan and his colleagues wanted to determine the risk of H3N2 strains re-emerging in humans, whose immune systems would no longer recognise the more dangerous forms of H3N2. This type of event has a recent precedent: In 2009, a strain of H1N1 emerged that was very similar to the virus that caused a 1918 pandemic that killed 50 million to 100 million people.
‘We asked if that could happen with H3,’ Sasisekharan says. ‘You would think it’s more readily possible with H3 because we observe that there seems to be a lot more mixing of H3 between humans and swine.’
In the new study, the researchers compared the 1968 H3N2 strain and about 1,100 H3 strains now circulating in pigs and birds, focusing on the gene that codes for the viral haemagglutinin (HA) protein.
After comparing HA genetic sequences in five key locations that control the viruses’ interactions with infected hosts, the researchers calculated an ‘antigenic index’ for each strain. This value indicates the percentage of these genetic regions identical to those of the 1968 pandemic strain and helps determine how well an influenza virus can evade a host’s immune response.
The researchers also took into account the patterns of attachment of the HA protein to sugar molecules called glycans. The virus’ ability to attach to glycan receptors found on human respiratory-tract cells is key to infecting humans.
Seeking viruses with an antigenic index of at least 49 percent and glycan-attachment patterns identical to those of the 1968 virus, the research team identified 581 H3 viruses isolated since 2000 that could potentially cause a pandemic. Of these, 549 came from birds and 32 from pigs.
The researchers then exposed some of these strains to antibodies provoked by the current H3 seasonal-flu vaccines. As they predicted, these antibodies were unable to recognise or attack these H3 strains. Of the 581 HA sequences, six swine strains already contain the standard HA mutations necessary for human adaptation, and are thus capable of entering the human population either directly or via genetic reassortment, Sasisekharan says.
‘One of the amazing things about the influenza virus is its ability to grab genes from different pools,’ he says. ‘There could be viral genes that mix among pigs, or between birds and pigs.’
Sasisekharan and colleagues are now doing a similar genetic study of H5 influenza strains. The H3 study was funded by the National Institutes of Health and the National Science Foundation. MIT