Researchers identify genetic evidence for single bacteria cause of sepsis

An international team of academics, including Professor Marco Oggioni from the University of Leicester’s Department of Genetics, has studied how localised infections can turn into the dangerous systematic disease sepsis – and has identified for the first time through genetic evidence that a single bacteria could be the cause.

The study, published in PLOS Pathogens (20 March 2014), examines the events that lead to sepsis by Streptococcus pneumoniae (pneumococcus), a major human pathogen, in mice.

They found that in most cases the bacteria causing sepsis was started by a single pneumococcal cell. The study was an interdisciplinary collaboration between the Departments of Genetics, Infection Immunity and Inflammation and Mathematics at the University of Leicester, Professor Richard Moxon at the University of Oxford and scientists from overseas including the University of Siena.

Professor Oggioni said: “Our data in experimental infection models indicate that we do not need only strategies which target many bacteria when it is too late, but that early intervention schemes which prevent the one-single cell that starts the disease process might provide substantial benefit to the patient.

“In this work we have for the first time provided genetic evidence for a single cell origin of bacterial invasive infection. The scenario was hypothesised over 50 years ago, but so far only phenotypic and statistical evidence could be obtained for this event.”

Under normal circumstances, when different bacteria are used in models of experimental infection of hosts who have not previously encountered the same pathogen, the vast majority is destroyed rapidly by the host’s innate immune system.

In the researcher’s model, a dose of one million bacteria is needed to induce systemic disease in about half of the hosts in the study. This is in stark contrast to a much lower number of bacteria thought to make up the starting “seed” that leads to the development of systemic infection – and the assumption is that there must be one or more “bottlenecks” in the development of the disease.

To investigate these bottlenecks, the researchers injected mice with a mix of three different variants of S. pneumoniae. About half of the mice developed sepsis and in almost all cases the bacteria causing sepsis were derived from only one of the three variants used in the initial challenge.

Using statistical analysis as well as direct DNA sequencing, the researchers could show that in most cases the bacterial population causing sepsis was started by a single pneumococcal cell. When the researchers looked closer at how the immune system resists most injected bacteria, they found that macrophages, a type of immune cell that can gobble up bacteria, and specifically macrophages in the spleen, are the main contributors to an efficient immune response to this pathogen.

Their findings suggest that if bacteria survive this initial counter-attack, a single ‘founder’ bacterium multiplies and re-enters the bloodstream, where its descendants come under strong selective pressure that dynamically shapes the subsequent bacterial population – resulting in the sepsis. The data also suggests that the single bacterium leading to sepsis has no obvious characteristics that give it an advantage over the 999,999 others, but that random events determine which of the injected bacteria survives and multiplies to cause disease.

It is believed that the findings, suggesting that the development of sepsis starting from a single founding cell which survives the immune system’s initial counter-attack in mice, could also potentially apply to human systemic infections. This information could prove vital to understanding sepsis, as the causes of the disease are still largely unknown to the scientific community.

Stem cells created from a drop of blood

Scientists at A*STAR’s Institute of Molecular and Cell Biology (IMCB) have developed a method to generate human induced pluripotent stem cells (hiPSCs) from a single drop of finger-pricked blood.

The method also enables donors to collect their own blood samples, which they can then send to a laboratory for further processing. The easy access to blood samples using the new technique could potentially boost the recruitment of greater numbers and diversities of donors, and could lead to the establishment of large-scale hiPSC banks.

By genetic reprogramming, matured human cells, usually blood cells, can be transformed into hiPSCs. As hiPSCs exhibit properties remarkably similar to human embryonic stem cells, they are invaluable resources for basic research, drug discovery and cell therapy.

In countries like Japan, USA and UK, a number of hiPSC bank initiatives have sprung up to make hiPSCs available for stem cell research and medical studies. Current sample collection for reprogramming into hiPSCs include invasive measures such as collecting cells from the bone marrow or skin, which may put off many potential donors.

Although hiPSCs may also be generated from blood cells, large quantities of blood are usually required.

In the paper published online on the Stem Cell Translational Medicine journal, scientists at IMCB showed for the first time that single-drop volumes of blood are sufficient for reprogramming into hiPSCs. The finger-prick technique is the world’s first to use only a drop of finger-pricked blood to yield hiPSCs with high efficiency.

A patent has been filed for the innovation. The accessibility of the new technique is further enhanced with a DIY sample collection approach.

Donors may collect their own fingerpricked blood, which they can then store and send it to a laboratory for reprogramming. The blood sample remains stable for 48 hours and can be expanded for 12 days in culture, which therefore extends the fingerprick technique to a wide range of geographical regions for recruitment of donors with varied ethnicities, genotypes and diseases.

By integrating it with the hiPSC bank initiatives, the finger-prick technique paves the way for establishing diverse and fully characterised hiPSC banking for stem cell research. The potential access to a wide range of hiPSCs could also replace the use of embryonic stem cells, which are less accessible.

It could also facilitate the set-up of a small hiPSC bank in Singapore to study targeted local diseases. Dr Loh Yuin Han Jonathan, Principal Investigator at IMCB and lead scientist for the finger-prick hiPSC technique, said: “It all began when we wondered if we could reduce the volume of blood used for reprogramming.

We then tested if donors could collect their own blood sample in a normal room environment and store it. Our finger-prick technique, in fact, utilised less than a drop of finger-pricked blood. The remaining blood could even be used for DNA sequencing and other blood tests.”

Dr Stuart Alexander Cook, Senior Consultant at the National Heart Centre Singapore and co-author of the paper, said: “We were able to differentiate the hiPSCs reprogrammed from Jonathan’s finger-prick technique, into functional heart cells. This is a welldesigned, applicable technique that can unlock unrealized potential of biobanks around the world for hiPSC studies at a scale that was previously not possible.”

Prof Hong Wanjin, Executive Director at IMCB, said: “Research on hiPSCs is now highly sought-after, given its potential to be used as a model for studying human diseases and for regenerative medicine. Translational research and technology innovations are constantly encouraged at IMCB and this new technique is very timely.

We hope to eventually help the scientific community gain greater accessibility to hiPSCs for stem cell research through this innovation.”

Researchers generate new neurons in brains, spinal cords of mammals

University of Texas Southwestern Medical Center researchers have created new nerve cells in the brains and spinal cords of living mammals without the need for stem cell transplants to replenish lost cells.

Although the research indicates it may someday be possible to regenerate neurons from the body’s own cells to repair traumatic brain injury or spinal cord damage or to treat conditions such as Alzheimer’s disease, the researchers stressed that it is too soon to know whether the neurons created in these initial studies resulted in any functional improvements, a goal for future research. Spinal cord injuries can lead to an irreversible loss of neurons, and along with scarring, can ultimately lead to impaired motor and sensory functions.

Scientists are hopeful that regenerating cells can be an avenue to repair damage, but adult spinal cords have limited ability to produce new neurons. Biomedical scientists have transplanted stem cells to replace neurons, but have faced other hurdles, underscoring the need for new methods of replenishing lost cells.

Scientists in UT Southwestern’s Department of Molecular Biology first successfully turned astrocytes – the most common non-neuronal brain cells – into neurons that formed networks in mice. They now successfully turned scar-forming astrocytes in the spinal cords of adult mice into neurons.

The latest findings are published in Nature Communications and follow previous findings published in Nature Cell Biology. “Our earlier work was the first to clearly show in vivo (in a living animal) that mature astrocytes can be reprogrammed to become functional neurons without the need of cell transplantation.

The current study did something similar in the spine, turning scar-forming astrocytes into progenitor cells called neuroblasts that regenerated into neurons,” said Dr Chun-Li Zhang, assistant professor of molecular biology at UT Southwestern and senior author of both studies. “Astrocytes are abundant and widely distributed both in the brain and in the spinal cord.

In response to injury, these cells proliferate and contribute to scar formation. Once a scar has formed, it seals the injured area and creates a mechanical and biochemical barrier to neural regeneration,” Dr Zhang explained.

“Our results indicate that the astrocytes may be ideal targets for in vivo reprogramming.” The scientists’ two-step approach first introduces a biological substance that regulates the expression of genes, called a transcription factor, into areas of the brain or spinal cord where that factor is not highly expressed in adult mice.

Of 12 transcription factors tested, only SOX2 switched fully differentiated, adult astrocytes to an earlier neuronal precursor, or neuroblast, stage of development, Dr Zhang said. In the second step, the researchers gave the mice a drug called valproic acid (VPA) that encouraged the survival of the neuroblasts and their maturation (differentiation) into neurons.

VPA has been used to treat epilepsy for more than half a century and also is prescribed to treat bipolar disorder and to prevent migraine headaches, he said. The current study reports neurogenesis (neuron creation) occurred in the spinal cords of both adult and aged (over oneyear old) mice of both sexes, although the response was much weaker in the aged mice, Dr Zhang said.

Researchers now are searching for ways to boost the number and speed of neuron creation. Neuroblasts took four weeks to form and eight weeks to mature into neurons, slower than neurogenesis reported in lab dish experiments, so researchers plan to conduct experiments to determine if the slower pace helps the newly generated neurons properly integrate into their environment.

In the spinal cord study, SOX2-induced mature neurons created from reprogramming of astrocytes persisted for 210 days after the start of the experiment, the longest time the researchers examined, he added.

Obesity and diabetes have adverse effects on outcomes across different tumour types

Both obesity and diabetes have adverse effects on outcomes in breast cancer patients who receive chemotherapy as primary treatment before surgery (neoadjuvant chemotherapy), according to research presented at the 9th European Breast Cancer Conference (EBCC-9) 21 March 2014.

Although a high body mass index (BMI) is known to have a negative impact on cancer development and prognosis, until now there has been uncertainty as to whether having a high BMI had an equal effect on patients with different types of breast tumours. Dr Caterina Fontanella, MD, a trainee in medical oncology from the University of Udine (Italy) and a research fellow with the German Breast Group, based in Neu- Isenburg, Germany, presented an analysis based on nearly 11,000 patients with early breast cancer treated with neoadjuvant chemotherapy.

She showed that a high BMI adversely affects the chances of surviving without the breast cancer recurring or spreading to other parts of the body, , although this detriment was not seen in those women had been diagnosed with HER2-positive disease. “Although the overall survival of patients with metastatic breast cancer has increased over the past few decades, it remains an incurable disease,” Dr Fontanella said.

“So preventing disease relapse after primary treatment of early breast cancer is fundamentally important in oncology daily practice. Considering that about one-third of the worldwide population has a body mass index higher than 25 kg/m², investigating the possible higher risk of relapse that affects overweight and obese patients compared with normal weight patients should be a priority.”

The researchers studied data from 8,872 early breast cancer patients from the German Breast Group, and 1,855 from a joint EORTC/BIG[1] trial. All had received a modern treatment consisting of an anthracycline/ taxane-based neoadjuvant chemotherapy, anti- HER-2 drugs, or hormone therapy according to tumour type and national guidelines.

The vast majority of the patients in this study received chemotherapy doses capped at a body surface area (BSA) of 2.0m², which is often the limit when calculating doses.

“Obese patients may have a BSA of more 2.0m², but the chemotherapy dose they receive will not reflect this. It is a very common practice in these patients for fear of overdosing, but of course it means that they will often receive a relatively lower quantity of chemotherapy,” Dr Fontanella said.

“In my opinion, a deeper understanding of chemotherapy metabolism and distribution in patients with high BMI and with increased adipose tissue is needed.” “We already know that obese hormone receptor-positive tumour patients respond less well to aromatase inhibitors as adjuvant treatment, and this underlines a key role of higher aromatase activity in patients with increased adipose tissue.”

Aromatase is an enzyme that synthesises oestrogen, and blocking it is important in cancers where oestrogen encourages tumours to grow. Final analysis of outcomes from the two groups in the joint study showed a significant decrease in survival without the cancer spreading (metastasising) – distant disease-free survival (DDFS) – or the cance